# Patent

## Title
Methods for inhibiting cell proliferation in ALK-driven cancers

## Abstract

The invention features a method for treating patients who have an ALK-driven cancer, which is, or has become, refractory to one or more of crizotinib, CH5424802 and ASP3026, or which bears an ALK mutation identified herein, by administering a compound of formula (I) to the patient. The invention also features methods, kits, and compositions for characterizing ALK-driven cancers to determine whether they express an ALK mutant.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit from U.S. Provisional Application No. 61/810,554, filed Apr. 10, 2013.


BACKGROUND OF THE INVENTION
The present invention relates to methods, kits, and pharmaceutical compositions for inhibiting the proliferation of cells.
Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase in the insulin receptor superfamily, was first identified as a chromosomal rearrangement (NPM-ALK fusion gene) in anaplastic large cell lymphoma (ALCL) (Morris et al., Science 263:1281 (1994)). Alternate EML4-ALK gene translocations have been detected in a subset of non-small cell lung cancers (NSCLC), translocations with over a dozen other fusion partners have been observed, and single point mutations in ALK have been identified in a subset of neuroblastomas (Soda et al., Nature 448:561 (2007); Mossé et al., Nature 455:930 (2008); Janoueix-Lerosey et al., Nature 455:967 (2008); George et al., Nature 455:975 (2008); Webb et al., Expert Rev. Anticancer Ther. 9:331 (2009); and Chen et al., Nature 455:930 (2008)). Whereas expression of ALK is limited in normal adult human tissues, genetic studies indicate that aberrant expression of ALK or ALK fusions is a key driver in various tumor types, which highlights ALK as an important target in treating human tumors. The high sequence homology of ALK with other members of the insulin receptor superfamily presents a significant challenge in the design of ALK-selective inhibitors.
Other challenges to developing a successful therapy based on inhibiting a kinase like ALK include the existence or development of compensatory biological mechanisms, such as alternative signaling pathways that bypass or supercede ALK, ALK gene amplification or overexpression, increased drug efflux, or the development of mutations which impair binding of an inhibitor to ALK. If such mutations occur, it would be important to learn of their existence, be able to identify them in patients, identify effective inhibitors for such mutants, and treat a patient who has such a mutation with an inhibitor to target that mutant.
New diagnostic methods and therapies are needed for the detection and treatment of ALK-driven cancers, especially for those in which ALK mutations confer resistance to inhibitors of “wild-type” ALK kinases.
SUMMARY OF THE INVENTION
The invention involves the discovery of new mutations in ALK, including mutations which are of potentially profound medical significance because they confer resistance to one or more of CH5424802, ASP3026 and crizotinib (also known as PF1066, and herein as “compound 2”, recently approved for the treatment of NSCLC). The invention further involves the discovery that compounds of formula (I), such as compound 1, disclosed in detail below, potently inhibit ALK as well as ALK mutants that are resistant to one or more of crizotinib, CH5424802 and ASP3026.
The invention thus provides a method for treating patients who have an ALK-driven cancer which is refractory or resistant to one or more of crizotinib, CH5424802 and ASP3026; who are intolerant to one or more of crizotinib, CH5424802 and ASP3026; or whose cancer is characterized by cells containing at least one of the ALK mutations disclosed herein, in each case, by administering to the patient a treatment-effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. The invention also features a method for characterizing ALK-driven cancers to determine whether the cancer cells contain an ALK gene bearing a given mutation and for identifying cells expressing ALK mutants.
The discoveries underlying this invention also represent the first identification of mutations in the ALK kinase domain present in ALK fusions, which are of increasing medical interest for their role in a variety of devastating cancers.
More specifically, the invention features inter alia a method of characterizing an ALK-driven cancer in a subject by identifying the presence of a mutation in anaplastic lymphoma kinase (ALK) in a sample obtained from the subject, where the mutation encodes an amino acid substitution at a position corresponding to one of the following amino acid positions in SEQ ID NO: 2: (i) threonine at amino acid position 1151; (ii) leucine at amino acid position 1152; (iii) cysteine at amino acid position 1156; (iv) isoleucine at amino acid position 1171; (v) phenylalanine at amino acid position 1174; (vi) valine at amino acid position 1180; (vii) arginine at amino acid position 1181; (viii) leucine at amino acid position 1196; (ix) leucine at amino acid position 1198; (x) glycine at amino acid position 1202; (xi) serine at amino acid position 1206; (xii) glutamic acid at amino acid position 1210; (xiii) glutamic acid at amino acid position 1241; (xiv) isoleucine at amino acid position 1268; and (xv) glycine at amino acid position 1269.
For example, in certain cases, the mutation encodes serine, threonine, or histidine in place of isoleucine at amino acid position 1171; serine in place of phenylalanine at amino acid position 1174; or glutamine in place of leucine at amino acid position 1196.
In certain embodiments, the mutation encodes lysine in place of threonine at amino acid position 1151; valine in place of leucine at amino acid position 1152; tyrosine in place of cysteine at amino acid position 1156; leucine or methionine in place of valine at amino acid position 1180; glycine in place of arginine at amino acid position 1181; methionine in place of leucine at amino acid position 1198; serine in place of glycine at amino acid position 1202; arginine, isoleucine, glycine, or alanine in place of serine at amino acid position 1206; lysine in place of glutamic acid at amino acid position 1210; lysine in place of glutamic acid at amino acid position 1241; valine in place of isoleucine at amino acid position 1268; or cysteine, serine, or alanine in place of glycine at amino acid position 1269.
In particular embodiments, the ALK is full-length ALK or an ALK-fusion product. In another embodiment, the one or more mutations are identified in an ALK protein or an ALK-fusion protein.
The invention also features a method of characterizing an ALK-driven cancer expressing an ALK-fusion protein in a subject including identifying the presence of a mutation in the ALK-fusion protein in a sample from the subject, where the mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for isoleucine with asparagine at amino acid position 1171; (ii) a substitution for phenylalanine with isoleucine, leucine, cysteine, or valine at amino acid position 1174; (iii) a substitution for phenylalanine with isoleucine, cysteine, or valine at amino acid position 1245; and (iv) a substitution for tyrosine with serine at amino acid position 1278. In one particular embodiment, the ALK-fusion protein is EML4-ALK.
The invention further features a method of characterizing an ALK-driven cancer expressing EML4-ALK fusion protein in a subject including identifying the presence of a mutation in the EML4-ALK fusion protein in a sample from the subject, where the mutation corresponds to a substitution for leucine with methionine at amino acid position 1196 of SEQ ID NO: 2.
In one embodiment of any of the methods described herein, the presence of the mutation in the sample indicates that the subject has or is at increased risk for developing an ALK-driven cancer. In other embodiments, the presence of the mutation in the sample indicates that the subject has or is at increased risk for developing an ALK-driven cancer refractory to treatment with a tyrosine kinase inhibitor. In particular embodiments, the presence of the mutation in the sample indicates that the subject has or is at increased risk for developing an ALK-driven cancer refractory to treatment with to one or more of crizotinib, CH5424802 and ASP3026. In yet other embodiments, where the ALK protein or ALK-fusion protein includes a contiguous sequence of between 30 and 1620 amino acids that is at least 95% identical to a corresponding sequence in SEQ ID NO: 2 or SEQ ID NO: 4.
In another embodiment of any of the methods described herein, the one or more mutations are identified by immunoblotting or by an ELISA assay. In certain embodiments, the one or more mutations are identified in a nucleic acid encoding the ALK. In other embodiments, the nucleic acid includes a contiguous sequence of between 60 and 6226 nucleotides that is at least 95% identical to a corresponding sequence in SEQ ID NO: 1 or SEQ ID NO: 3. In yet other embodiments, the nucleic acid is isolated prior to the identification of the one or more mutations in ALK. In particular embodiments, the nucleic acid is an mRNA. In further embodiments, the one or more mutations are identified by PCR, RT-PCR, quantitative real time PCR, allele-specific quantitative real time PCR, in situ hybridization, or single-strand conformational polymorphism.
In further embodiments of any of the methods described herein, the ALK-driven cancer is selected from anaplastic large cell lymphoma, non-small cell lung cancer, and neuroblastoma.
The invention also features a method for treating an ALK-driven cancer in a subject including the steps of: a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK), where the mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for isoleucine to threonine at amino acid position 1171; (ii) a substitution for phenylalanine to cysteine at amino acid position 1174; (iii) a substitution for leucine to methionine at amino acid position 1196; (iv) a substitution for serine with arginine at amino acid position 1206; (v) a substitution for glutamic acid to lysine at amino acid position 1210; (vi) a substitution for phenylalanine to cysteine at amino acid position 1245; (vii) a substitution for glycine to serine at amino acid position 1269; and (viii) a substitution for valine to leucine at amino acid position 1180; and b) administering to the subject a therapeutically effective amount of compound of formula (I), or a pharmaceutically acceptable salt thereof. The compound of formula (I), or a pharmaceutically acceptable salt thereof, includes any compound of formula (I) as described herein. The mutation in ALK that can be treated includes any ALK mutant described herein. In one embodiment, the ALK is full-length ALK. In another embodiment, the ALK is an ALK-fusion product.
The invention further features a method for treating an ALK-driven cancer expressing an ALK-fusion protein in a subject including the steps of a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in the ALK-fusion protein, where the mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for phenylalanine with valine at amino acid position 1174; and (ii) a substitution for tyrosine with serine at amino acid position 1278; and b) administering to the subject a therapeutically effective amount of compound of formula (I), or a pharmaceutically acceptable salt thereof. The compound of formula (I), or a pharmaceutically acceptable salt thereof, includes any compound of formula (I) as described herein. In one embodiment, the ALK-fusion protein is EML4-ALK.
In particular embodiments of any of the methods described herein, such as the method of treating an ALK-driven cancer, the ALK protein or ALK-fusion protein includes a contiguous sequence of between 30 and 1620 amino acids that is at least 95% identical to a corresponding sequence in SEQ ID NO: 2 or SEQ ID NO: 4. In other embodiments, the ALK-driven cancer is selected from anaplastic large cell lymphoma, non-small cell lung cancer, and neuroblastoma. In further embodiments, the method further includes administering to the subject a first agent selected from one or more of crizotinib, CH5424802 and ASP3026, or a pharmaceutically acceptable salt thereof, within 6 days of administering the compound of formula (I), where each of the compound of formula (I) and the first agent are administered in an amount that together is sufficient to treat the ALK-driven cancer. The compound of formula (I), or a pharmaceutically acceptable salt thereof, includes any compound of formula (I) as described herein.
The invention also features a method of inhibiting the proliferation of a cell expressing an ALK mutant, where the ALK mutant is characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK) selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for threonine to lysine at amino acid position 1151; (ii) a substitution for leucine to valine at amino acid position 1152; (iii) a substitution for cysteine to tyrosine at amino acid position 1156; (iv) a substitution for isoleucine with serine at amino acid position 1171; and (v) a substitution for glycine to cysteine, serine, or alanine at amino acid position 1269, the method including contacting the cell with a compound of formula (I), or a pharmaceutically acceptable salt thereof, in an amount sufficient to inhibit the proliferation. The compound of formula (I), or a pharmaceutically acceptable salt thereof, includes any compound of formula (I) as described herein. The ALK mutant characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK) can include any ALK mutant described herein.
The invention further features a method of inhibiting the proliferation of a cell expressing a mutant ALK-fusion protein, where the mutant ALK-fusion protein is characterized by the presence of a mutation in the ALK-fusion protein selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for phenylalanine with valine at amino acid position 1174; and (ii) a substitution for tyrosine with serine at amino acid position 1278, the method including contacting the cell with a compound of formula (I), or a pharmaceutically acceptable salt thereof, in an amount sufficient to inhibit the proliferation. The compound of formula (I), or a pharmaceutically acceptable salt thereof, includes any compound of formula (I) as described herein. The mutant ALK-fusion protein that can be characterized by the presence of a mutation in the ALK-fusion protein can be any ALK mutant or ALK-fusion protein described herein. In one particular embodiment, the ALK-fusion protein is EML4-ALK.
In particular embodiments of any of the methods described herein, the cells are cancer cells. In certain embodiments, the cancer is a neuroblastoma, a glioblastoma, a soft tissue tumor, an ALK expressing lymphoma, an ALK expressing lung, breast, colon, or prostate carcinoma.
The invention also features method of treating an ALK-driven cancer refractory to one or more of crizotinib, CH5424802 and ASP3026 in a subject, the method including administering to the subject an ALK inhibitor of an ALK mutant described herein. The method can include the step of identifying an ALK mutation in the ALK-driven cancer.
The invention also features method of treating an ALK-driven cancer refractory to one or more of crizotinib, CH5424802 and ASP3026 in a subject, the method including administering to the subject a compound of formula I, or a pharmaceutically acceptable salt thereof, in an amount sufficient to treat the cancer. The compound of formula (I), or a pharmaceutically acceptable salt thereof, includes any compound of formula (I) as described herein.
The invention features a method of treating an ALK-driven cancer in a subject intolerant to one or more of crizotinib, CH5424802 and ASP3026, the method including administering to the subject a compound of formula I, or a pharmaceutically acceptable salt thereof, in an amount sufficient to treat the cancer.
The invention further features a method of treating an ALK-driven cancer refractory to an inhibitor of wild-type ALK in a subject, the method including administering to the subject a compound of formula I, or a pharmaceutically acceptable salt thereof, in an amount sufficient to treat the cancer.
In a related aspect the invention features a method for treating an ALK-driven cancer in a subject including the steps of (a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK), and (b) administering to the subject a therapeutically effective amount of compound of compound 1, or a pharmaceutically acceptable salt thereof. In particular embodiments the ALK is full-length ALK. In still other embodiments, the ALK is an ALK-fusion product. The mutation can be selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for threonine at amino acid position 1151; (ii) a substitution for leucine at amino acid position 1152; (iii) a substitution for cysteine at amino acid position 1156; (iv) a substitution for isoleucine at amino acid position 1171; (v) a substitution for phenylalanine at amino acid position 1174; (vi) a substitution for valine at amino acid position 1180; (vii) a substitution for arginine at amino acid position 1181; (viii) a substitution for leucine at amino acid position 1196; (ix) a substitution for leucine at amino acid position 1198; (x) a substitution for glycine at amino acid position 1202; (xi) a substitution for aspartic acid at amino acid position 1203; (xii) a substitution for serine at amino acid position 1206; (xiii) a substitution for glutamic acid at amino acid position 1210; (xiv) a substitution for glutamic acid at amino acid position 1241; (xv) a substitution for phenylalanine at amino acid position 1245; (xvi) a substitution for isoleucine at amino acid position 1268; (xvii) a substitution for glycine at amino acid position 1269; and (xviii) insertion of an amino acid following amino acid position 1151. In particular embodiments, the mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for leucine to arginine at amino acid position 1152; (ii) a substitution for cysteine to tyrosine at amino acid position 1156; (iii) a substitution for isoleucine to serine at amino acid position 1171; (iv) a substitution for isoleucine to asparagine at amino acid position 1171; (v) a substitution for isoleucine to threonine at amino acid position 1171; (vi) a substitution for phenylalanine to leucine at amino acid position 1174; (vii) a substitution for phenylalanine to cysteine at amino acid position 1174; (viii) a substitution for valine to leucine at amino acid position 1180; (ix) a substitution for leucine to methionine at amino acid position 1196; (x) a substitution for glycine to arginine at amino acid position 1202; (xi) a substitution for aspartic acid to asparagine at amino acid position 1203; (xii) a substitution for serine to tyrosine at amino acid position 1206; (xiii) a substitution for serine with arginine at amino acid position 1206; (xiv) a substitution for glutamic acid to lysine at amino acid position 1210; (xv) a substitution for phenylalanine to cysteine at amino acid position 1245; (xvi) a substitution for glycine to alanine at amino acid position 1269; (xvii) a substitution for glycine to serine at amino acid position 1269; and (xviii) insertion of threonine following position 1151 (T1151T insertion).
The invention also features a method for treating an ALK-driven cancer in a subject including the steps of: a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK), wherein the mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: ((i) a substitution for leucine to arginine at amino acid position 1152; (ii) a substitution for cysteine to tyrosine at amino acid position 1156; (iii) a substitution for isoleucine to serine at amino acid position 1171; (iv) a substitution for isoleucine to asparagine at amino acid position 1171; (v) a substitution for isoleucine to threonine at amino acid position 1171; (vi) a substitution for phenylalanine to leucine at amino acid position 1174; (vii) a substitution for phenylalanine to cysteine at amino acid position 1174; (viii) a substitution for valine to leucine at amino acid position 1180; (ix) a substitution for leucine to methionine at amino acid position 1196; (x) a substitution for aspartic acid to asparagine at amino acid position 1203; (xi) a substitution for serine with arginine at amino acid position 1206; (xii) a substitution for glutamic acid to lysine at amino acid position 1210; (xiii) a substitution for phenylalanine to cysteine at amino acid position 1245; (xiv) a substitution for glycine to alanine at amino acid position 1269; and (xv) a substitution for glycine to serine at amino acid position 1269; and b) administering to the subject a therapeutically effective amount of compound of formula I, or a pharmaceutically acceptable salt thereof. In particular embodiments the ALK is full-length ALK. In still other embodiments, the ALK is an ALK-fusion product.
The invention features an isolated probe including 8 to 50 nucleotides that hybridizes a contiguous sequence of nucleotides within a nucleic acid encoding an ALK or ALK-fusion protein that contains the sequence of one or more codons corresponding to the following codons: nucleotides 4362 to 4364 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3599-3601 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4365 to 4367 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3602 to 3604 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4377 to 4379 of SEQ ID NO: 1 (e.g., TAT or TAC); nucleotides 3614 to 3616 of SEQ ID NO: 3 (e.g., TAT or TAC); nucleotides 4422 to 4424 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 3659 to 3661 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 4431 to 4433 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3668 to 3670 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 4449 to 4451 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3686 to 3688 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4452 to 4454 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, or GGG); nucleotides 3689 to 3691 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, or GGG); nucleotides 4497 to 4499 of SEQ ID NO: 1 (e.g., CAA or CAG); nucleotides 3734-3736 of SEQ ID NO: 3 (e.g., CAA or CAG); nucleotides 4503 to 4505 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3740 to 3742 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4515 to 4517 of SEQ ID NO: 1 (e.g, TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3752 to 3754 of SEQ ID NO: 3 (e.g, TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 4527 to 4529 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 3764 to 3766 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 4539 to 4541 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3776 to 3778 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4632-4634 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3869 to 3871 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4713 to 4715 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3950 to 3952 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4716 to 4718 of SEQ ID NO: 1 (e.g, TGT, TGC, TCT, TCC, TCA, TCG, AGT, or AGC); and nucleotides 3953-3955 of SEQ ID NO: 3 (e.g, TGT, TGC, TCT, TCC, TCA, TCG, AGT, or AGC); or a complementary sequence thereof.
In particular embodiments, the probe further includes one or more ligands or labels. In one embodiment, the ligand or label is covalently attached to the 5′ or 3′ terminus of the probe. In another embodiment, ligand is biotin or digoxigenin. In yet another embodiment, the label is detectable by fluorescence, radioactivity, UV/visible light absorption, X-ray diffraction or absorption, magnetism, or enzymatic activity.
In certain embodiments, the label is a fluorophore (e.g., (5-dimethylamino)-1naphthalenesulfonyl; N-oxyl-4,4-dimethyloxazolidine; N-oxyl-2,2,5,5-tetramethylpyrrolidone, N-oxyl-2,2,6,6-tetramethylpiperidine, dinitrophenyl, acridines, coumarins, fluoresein, Cy3, or Cy5). In one embodiment, the fluorophore is covalently attached to one terminus of the probe and a quencher is covalently attached to the other terminus of the probe.
In some embodiments, the probe includes a sequence selected from the group of: ggttcatcctgatggagctcatg (SEQ ID NO: 9); gttcatcctgatggagctcatgg (SEQ ID NO: 11); ttcatcctgatggagctcatggc (SEQ ID NO: 13); cggttcatcctgatggagctcat (SEQ ID NO: 15); ccggttcatcctgatggagctca (SEQ ID NO: 17); ggttcatcctgatggagctcatg (SEQ ID NO: 19); and cggttcatcctgatggagctcat (SEQ ID NO: 21).
The invention also features a kit including: i) one or more probes described herein; and ii) instructions for the use of the probe in the identification of a mutation in a nucleic acid encoding an ALK protein or an ALK-fusion protein.
The invention features a composition including: i) a first isolated probe including 8 to 50 nucleotides, including a sequence that hybridizes a contiguous sequence of nucleotides within a nucleic acid encoding an ALK or ALK-fusion protein that contains the sequence of one or more codons corresponding to the following codons: nucleotides 4362 to 4364 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3599-3601 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4365 to 4367 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3602 to 3604 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4377 to 4379 of SEQ ID NO: 1 (e.g., TAT or TAC); nucleotides 3614 to 3616 of SEQ ID NO: 3 (e.g., TAT or TAC); nucleotides 4422 to 4424 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 3659 to 3661 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 4431 to 4433 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3668 to 3670 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 4449 to 4451 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3686 to 3688 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4452 to 4454 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, or GGG); nucleotides 3689 to 3691 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, or GGG); nucleotides 4497 to 4499 of SEQ ID NO: 1 (e.g., CAA or CAG); nucleotides 3734-3736 of SEQ ID NO: 3 (e.g., CAA or CAG); nucleotides 4503 to 4505 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3740 to 3742 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4515 to 4517 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3752 to 3754 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 4527 to 4529 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 3764 to 3766 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 4539 to 4541 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3776 to 3778 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4632-4634 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3869 to 3871 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4713 to 4715 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3950 to 3952 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4716 to 4718 of SEQ ID NO: 1 (e.g., TGT, TGC, TCT, TCC, TCA, TCG, AGT, or AGC); and nucleotides 3953-3955 of SEQ ID NO: 3 (e.g., TGT, TGC, TCT, TCC, TCA, TCG, AGT, or AGC), or a complementary sequence thereof; and further including a first fluorophore; and ii) a second isolated probe including 8 to 50 nucleotides, including a sequence that is at least 95% identical to a corresponding continguous nucleic acid sequence in SEQ ID NO: 1 or SEQ ID NO: 3, or a complementary sequence thereof, where the second probe hybridizes to a different contiguous nucleic acid sequence within the nucleic acid encoding an ALK or ALK-fusion protein than the first isolated probe, and where the second probe includes a second fluorophore.
In some embodiments, the first fluorophore includes an acceptor fluorophore and the second fluorophore includes a donor fluorophore. In a further embodiment, (1) the acceptor fluorophore is attached to a 3′ end of the first probe and the donor fluorophore is attached to the 5′ end of the second probe, or (2) the acceptor fluorophore is attached to the 5′ end of the first probe and the donor fluorophore is attached to the 3′ end of the second probe.
In other embodiments, the first probe and the second probe are selected from the following pairs: ggttcatcctgatggagctcatg (SEQ ID NO: 9)/cggggggagacctcaagtccttcctcc (SEQ ID NO: 10); gttcatcctgatggagctcatgg (SEQ ID NO: 11)/ggggggagacctcaagtccttcctccg (SEQ ID NO: 12); ttcatcctgatggagctcatggc (SEQ ID NO: 13)/gggggagacctcaagtccttcctccga (SEQ ID NO: 14); cggttcatcctgatggagctcat (SEQ ID NO: 15)/gcggggggagacctcaagtccttcctc (SEQ ID NO: 16); ccggttcatcctgatggagctca (SEQ ID NO: 17)/ggcggggggagacctcaagtccttcct (SEQ ID NO: 18); ggttcatcctgatggagctcatg (SEQ ID NO: 19)/ggggggagacctcaagtccttcctccg (SEQ ID NO: 20); and cggttcatcctgatggagctcat (SEQ ID NO: 21)/cggggggagacctcaagtccttcctcc (SEQ ID NO: 22).
The invention also features a kit including: i) a first isolated probe including 8 to 50 nucleotides, including a sequence that hybridizes a contiguous sequence of nucleotides within a nucleic acid encoding an ALK or ALK-fusion protein that contains the sequence of one or more codons corresponding to the following codons: nucleotides 4362 to 4364 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3599-3601 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4365 to 4367 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3602 to 3604 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4377 to 4379 of SEQ ID NO: 1 (e.g., TAT or TAC); nucleotides 3614 to 3616 of SEQ ID NO: 3 (e.g., TAT or TAC); nucleotides 4422 to 4424 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 3659 to 3661 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 4431 to 4433 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3668 to 3670 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 4449 to 4451 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3686 to 3688 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4452 to 4454 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, or GGG); nucleotides 3689 to 3691 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, or GGG); nucleotides 4497 to 4499 of SEQ ID NO: 1 (e.g., CAA or CAG); nucleotides 3734-3736 of SEQ ID NO: 3 (e.g., CAA or CAG); nucleotides 4503 to 4505 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3740 to 3742 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4515 to 4517 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3752 to 3754 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 4527 to 4529 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 3764 to 3766 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 4539 to 4541 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3776 to 3778 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4632-4634 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3869 to 3871 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4713 to 4715 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3950 to 3952 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4716 to 4718 of SEQ ID NO: 1 (e.g., TGT, TGC, TCT, TCC, TCA, TCG, AGT, or AGC); and nucleotides 3953-3955 of SEQ ID NO: 3 (e.g., TGT, TGC, TCT, TCC, TCA, TCG, AGT, or AGC), or a complementary sequence thereof; and further including a first fluorophore; ii) a second isolated probe including 8 to 50 nucleotides, including a sequence that is at least 95% identical to a corresponding continguous nucleic acid sequence in SEQ ID NO: 1 or SEQ ID NO: 3, where the second probe hybridizes to a different contiguous nucleic acid sequence within the nucleic acid encoding an ALK or ALK-fusion protein than the first isolated probe, and where the second probe includes a second fluorophore; and iii) instructions for the use of the probe in the identification of a mutation in a nucleic acid encoding an ALK protein or an ALK-fusion protein.
In some embodiments, the first fluorophore includes an acceptor fluorophore and the second fluorophore includes a donor fluorophore. In a further embodiment, (1) the acceptor fluorophore is attached to a 3′ end of the first probe and the donor fluorophore is attached to the 5′ end of the second probe, or (2) the acceptor fluorophore is attached to the 5′ end of the first probe and the donor fluorophore is attached to the 3′ end of the second probe.
In other embodiments, the first probe and the second probe are selected from the following pairs: ggttcatcctgatggagctcatg (SEQ ID NO: 9)/cggggggagacctcaagtccttcctcc (SEQ ID NO: 10); gttcatcctgatggagctcatgg (SEQ ID NO: 11)/ggggggagacctcaagtccttcctccg (SEQ ID NO: 10); ttcatcctgatggagctcatggc (SEQ ID NO: 13)/gggggagacctcaagtccttcctccga (SEQ ID NO: 14); cggttcatcctgatggagctcat (SEQ ID NO: 15)/gcggggggagacctcaagtccttcctc (SEQ ID NO: 16); ccggttcatcctgatggagctca (SEQ ID NO: 17)/ggcggggggagacctcaagtccttcct (SEQ ID NO: 18); ggttcatcctgatggagctcatg (SEQ ID NO: 19)/ggggggagacctcaagtccttcctccg (SEQ ID NO: 20); and cggttcatcctgatggagctcat (SEQ ID NO: 21)/cggggggagacctcaagtccttcctcc (SEQ ID NO: 22).
In any of the above methods for inhibiting cell proliferation or treating a subject with an ALK-driven cancer, the compound of formula (I) is described by the formula:




In formula (I), Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two, or three heteroatoms, independently selected from N, S and O, wherein the 5- or 6-membered ring is substituted by Rh; Rh is H, C1-4 alkyl, or halo; Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy; Rg is —P(O)(R3A)(R3B), —S(O)N(R3C)(R3D), —S(O)2R3E, —OC(O)N(R3F)(R3G), —C(O)N(R3F)(R3G), —NR3HC(O)OR3I, a 5 or 6 member heterocyclic ring comprising 1, 2, 3 or 4 N atoms, or combined with Rg2 forms a 5- to 7-member heterocyclic ring; each of R3A, R3B, R3C, R3D, R3E, R3F, R3G, R3H, and R3I is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, or R3C and R3D, or R3F and R3G, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted; Rg2 is H, halo (e.g., F, Br, Cl, I), halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, (e.g., C1-6 alkyl, C3-6 cycloalkyl, C1-6 alkoxyl, C3-6 cycloalkoxyl, CF3) or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted; Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted; Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted; Rb4 is H, halo (e.g., F, Br, Cl, I), halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, (e.g., C1-6 alkyl, C3-6 cycloalkyl, C1-6 alkoxyl, C3-6 cycloalkoxyl, CF3), C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy, —OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted; each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted; Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—; each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl; each of X1 and X2 is, independently, selected from CH and N; and R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl.

In formula (I), for any of Ra2, Rd, Rh, R1, R2, R4, R3A, R3B, R3C, R3D, R3E, R3F, R3G, R3H, and R3I selected from C1-6 alkoxy, C3-6 alkenyloxy, C3-6 cycloalkyloxy, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic, and heteroaryl, the substituent is substituted or unsubstituted.
In particular embodiments, the compound of formula (I) is further described by formula (IIa) or formula (IIb):




In formulas (IIa) and (IIb), Ra1; Ra2; Rb2; Rb4; Rg; Rg1; Rg2; Rd; and Rh are as defined above.

In certain embodiments of the compounds of formula (I), (IIa), or (IIb), Rg1, Rg2, Rb2 and Rb4 are H or F.
In an embodiment of the compounds of formula (IIa), Rd is Cl, F or CF3.
In certain embodiments of the compounds of formula (I), (IIa), or (IIb), Ra1 is methoxy.
In one embodiment of the compounds of formula (I), (IIa), or (IIb), Rg is —P(O)(R3A)(R3B) or —S(O)2R3E.
In another embodiment of the compounds of formula (I), (IIa), or (IIb), Ra1 is a 5 or 6 member heterocyclic ring comprising 1 or 2 N or O atoms which is unsubstituted or substituted with an alkyl group.
In some embodiments, the compound of formula (I) can further be described by formula (III):




In formula (III), Ra2 is alkoxy; Rg is —P(O)(R3A)(R3B); —S(O)N(R3c)(R3D); or —S(O)2R3E; each of R3A, R3B, R3C, R3D, and R3E is, independently, selected from H and C1-7 alkyl, or R3A and R3B, or R3C and R3D, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted; Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one or two heteroatoms, independently selected from N, S and O, wherein the 5- or 6-membered ring is substituted by Rh; Rh is H, C1-4 alkyl, or halo; Ra1 is halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—; each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl; each of X1 and X2 is, independently, selected from CH and N; and R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl.

In particular embodiments, the compound of formula (III) is further described by formula (IVa) or formula (IVb):




In formulas (IVa) and (IVb), Ra2; Rg; Rd; Rh; and Ra1 are as defined above for formula (III).

In certain embodiments of the compounds of any of formulas (III), (IVa) and (IVb), Ra2 is a methoxy, ethoxy, or propoxy group.
In some embodiments of the compounds of formula (III) and (IVa), Rd is selected from Cl, F, CF3, and cyclopropyl;
In still other embodiments, the compound of formula (III) is further described by any of formulas (Va)-(Vd):




In formulas (Va)-(Vd), Rg; Rd; Rh; and Ra1 are as defined above for formula (III).

In particular embodiments of the compounds of any of the above formulas, Rg is —P(O)(CH3)2 or —S(O)2(CH(CH3)2).
In particular embodiments of the compounds of any of the above formulas, Ra1 is:




wherein X1, X2, and R4 are as defined above for formula (III). For example, Ra1 can be selected from any of the following groups:





In particular embodiments, the compound of formula (I) is further described by one of formulas (VIa)-(VIf):







In formulas (VIa)-(VIf), Ra2 is a methoxy, ethoxy, or propoxy group; and Rg is —P(O)(CH3)2 or —S(O)2(CH(CH3)2).
In any of the above formulas, the compound can be either in its free base form, or a pharmaceutically acceptable salt.
The compounds of formula (I) include those described in PCT Publication Nos. WO2009/143389, WO 2006/021454, WO 2006/021457, and WO2009/126515, each of which is incorporated herein by reference.
The response criteria for the methods of the invention can be graded according to the response evaluation criteria in solid tumors (RECIST) guidelines (see Eur. J. Cancer 45:228 (2009)) that define when cancer patients improve (“respond”), stay the same (“stabilize”), or worsen (“progression”) during treatments. A complete response is characterized by: (i) disappearance of all target lesions; and (ii) any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. A partial response is characterized by: (i) at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. A progressive disease is characterized by (i) at least a 5%, 10%, or 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study); or (ii) the appearance of one or more new lesions.
By “acceptor fluorophore” is meant a compound which absorbs energy from a donor fluorophore (e.g., in the range of about 400 to 900 nm, such as in the range of about 500 to 800 nm). Acceptor fluorophores generally absorb energy at a wavelength that is usually at least 10 nm higher (such as at least 20 nm higher), than the maximum absorbance wavelength of the donor fluorophore, and have a fluorescence emission maximum at a wavelength ranging from about 400 to 900 nm. Acceptor fluorophores have an extinction spectrum which overlaps with the emission of the donor fluorophore, such that energy emitted by the donor can excite the acceptor. Ideally, an acceptor fluorophore is capable of being attached to a nucleic acid molecule, such as an isolated probe or primer of the invention. Exemplary acceptor fluorophores include, but are not limited to, rhodamine and its derivatives (such as N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX)), fluorescein derivatives (such as 5-carboxyfluorescein (FAM) and 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE)), green fluorescent protein (GFP), BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), LightCycler Red (LC-Red) 640, LC-Red 705, and cyanine dyes (such as Cy5 and Cy5.5). In particular examples, an acceptor fluorophore is capable of being attached to a nucleotide, such as the base, sugar, or phosphate (α, β, or γ) of the nucleotide. For example, an acceptor fluorophore can be attached to a nucleotide that is part of a probe disclosed herein, such as the 3′- or 5′-end of the probe. In a particular example, an acceptor fluorophore is a dark quencher, such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACK HOLE QUENCHERTM (Glen Research), ECLIPSETM Dark Quencher (Epoch Biosciences), IOWA BLACKTM (Integrated DNA Technologies). A quencher can reduce or quench the emission of a donor fluorophore.
In such an example, instead of detecting an increase in emission signal from the acceptor fluorophore when in sufficient proximity to the donor fluorophore (or detecting a decrease in emission signal from the acceptor fluorophore when a significant distance from the donor fluorophore), an increase in the emission signal from the donor fluorophore can be detected when the quencher is a significant distance from the donor fluorophore (or a decrease in emission signal from the donor fluorophore when in sufficient proximity to the quencher acceptor fluorophore).
The term “administration” or “administering” refers to a method of giving a dosage of a pharmaceutical composition to a mammal, where the method is, e.g., oral, intravenous, intraperitoneal, intraarterial, or intramuscular. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, site of the potential or actual disease and severity of disease. While compounds of formula I will generally be administered perorally, other routes of administration can be useful in carrying out the methods of the invention.
By “ALK-driven cancer” is meant a cancer characterized by cells which inappropriately overexpress an ALK gene, which contain an ALK fusion gene, or which contain an ALK gene (or ALK fusion gene) bearing a mutation in an ALK gene that alters the biological activity of the ALK nucleic acid or its encoded polypeptide. In certain cases of particular interest, the ALK driven cancer is a cancer characterized by cells which have an ALK fusion gene containing at least one of the mutations disclosed herein. ALK-driven cancers can arise in any tissue, including brain, blood, connective tissue, liver, mouth, muscle, spleen, stomach, testis, and trachea. ALK-driven cancers can include cancers of the breast, non small cell lung cancer (NSCLS), neural tumors such as glioblastomas and neuroblastomas; adenocarcinomas; inflammatory myofibroblastic tumors; esophageal carcinomas, soft tissue cancers such as rhabdomyosarcomas, among others); various forms of lymphoma such as a non-Hodgkin's lymphoma (NHL) known as anaplastic large-cell lymphoma (ALCL), various forms of leukemia; and including cancers which are ALK mediated.
As used herein, the term “ALK fusion product” refers to ALK fusion proteins and/or ALK fusion genes, as described herein.
As used herein, the terms “ALK fusion protein” and “ALK fusion gene” denote a protein or nucleotide sequence that comprises a portion of the amino acid of ALK or a fragment of the nucleotide sequence of ALK, where the portions may have one or more deletions, substitutions, or additions relative to human ALK, and the portion is fused to a fusion partner. ALK fusion genes of particular interest in the practice of this invention encode an ALK fusion protein that comprises all or nearly all of the ALK intracellular portion, which encompasses the kinase domain of ALK, as discussed below. Such ALK fusion proteins retain phosphorylation activity. Exemplary fragments of ALK include the kinase domain, which includes Tyr1278, Tyr1282, and Tyr1283 of SEQ ID NOs: 2 or 4; the intracytoplasmic portion, which includes amino acids 1058 to 1620 of full length ALK (SEQ ID NOs: 2 or 4); portions of the intracytoplasmic portion, such as amino acids 1065 to 1620 of SEQ ID NOs: 2 or 4 or amino acids 1075 to 1620 of SEQ ID NOs: 2 or 4; exon 20 of ALK (SEQ ID NOs: 1 or 3); or exon 20 to exon 29 of SEQ ID NOs: 1 or 3.
As used herein, the term “ALK mutant” or “mutant” comprises one or more deletions, substitutions, or additions in the amino acid or nucleotide sequences of ALK, or fragments thereof. ALK mutants include ALK fusion proteins and ALK fusion genes. The ALK mutant can also include one or more deletions, substitutions, or additions, or a fragment thereof, as long as the mutant retains kinase phosphorylation activity. In particular ALK mutations, phosphorylation activity can be increased (e.g., by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%), as compared to wild type ALK. ALK mutants comprise an amino acid sequence corresponding to the amino acids 1058 to 1620 of SEQ ID NOs: 2 or 4, or a fragment thereof, where said amino acid sequence has at least 85-90%, 91-93%, 94-96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NOs: 2 or 4. The position of mutations in ALK and ALK-fusion products are described relative to the position of an amino acid in human ALK (SEQ ID NO: 2 or SEQ ID NO: 4). ALK mutants of the invention include the one or more of the following: a substitution for threonine at amino acid position 1151 (e.g., a threonine to lysine substitution); a substitution for leucine at amino acid position 1152 (e.g., a leucine to valine substitution); a substitution for cysteine at amino acid position 1156 (e.g., a cysteine to tyrosine substitution); a substitution for isoleucine with serine, threonine, or histidine at amino acid position 1171; a substitution for phenylalanine with serine at amino acid position 1174; a substitution for valine at amino acid position 1180 (e.g., a valine to leucine or methionine substitution); a substitution for arginine at amino acid position 1181 (e.g., an arginine to glycine substitution); a substitution for leucine with glutamine at amino acid position 1196; a substitution for leucine at amino acid position 1198 (e.g., a leucine to methionine substitution); a substitution for glycine at amino acid position 1202 (e.g., a glycine to serine substitution); a substitution for serine at amino acid position 1206 (e.g., a serine to glycine, arginine, or isoleucine substitution); a substitution for glutamic acid at amino acid position 1210 (e.g., a glutamic acid to lysine substitution); a substitution for glutamic acid at amino acid position 1241 (e.g., a glutamic acid to lysine substitution); a substitution for isoleucine at amino acid position 1268 (e.g., an isoleucine to valine substitution); and a substitution for glycine at amino acid position 1269 (e.g., a glycine to cysteine, serine, or alanine substitution) of SEQ ID NO: 2 or SEQ ID NO: 4. At the nucleic acid level the ALK mutants can comprise an amino acid sequence corresponding to the nucleotides 4083 to 5795 of SEQ ID NO: 1 or nucleotides 3320 to 5032 of SEQ ID NO: 3, or a fragment thereof, where the nucleic acid sequence has at least 85-90%, 91-93%, 94-96%, 97%, 98%, or 99% identity to the corresponding nucleic acid sequence in SEQ ID NO: 1 or SEQ ID NO: 3. For example a nucleic acid encoding a mutant ALK protein or ALK-fusion protein may contain one or more codons for the amino acid corresponding to the amino acid at position 1151 (e.g., nucleotides 4362 to 4364 of SEQ ID NO: 1 or nucleotides 3599-3601 of SEQ ID NO: 3), the amino acid at position 1152 (e.g., nucleotides 4365 to 4367 of SEQ ID NO: 1 or nucleotides 3602 to 3604 of SEQ ID NO: 3), the amino acid at position 1156 (e.g., nucleotides 4377 to 4379 of SEQ ID NO: 1 or nucleotides 3614 to 3616 of SEQ ID NO: 3), the amino acid at position 1171 (e.g., nucleotides 4422 to 4424 of SEQ ID NO: 1 or nucleotides 3659 to 3661 of SEQ ID NO: 3), the amino acid at position 1198 (e.g., nucleotides 4503 to 4505 of SEQ ID NO: 1 or nucleotides 3740 to 3742 of SEQ ID NO: 3), the amino acid at position 1202 (e.g., nucleotides 4515 to 4517 of SEQ ID NO: 1 or nucleotides 3752 to 3754 of SEQ ID NO: 3), the amino acid at position 1206 (e.g., nucleotides 4527 to 4529 of SEQ ID NO: 1 or nucleotides 3764 to 3766 of SEQ ID NO: 3), the amino acid at position 1210 (e.g., nucleotides 4539 to 4541 of SEQ ID NO: 1 or nucleotides 3776 to 3778 of SEQ ID NO: 3), the amino acid at position 1268 (e.g., nucleotides 4713 to 4715 of SEQ ID NO: 1 or nucleotides 3950 to 3952 of SEQ ID NO: 3), and the amino acid at position 1269 (e.g., nucleotides 4716 to 4718 of SEQ ID NO: 1 or nucleotides 3953-3955 of SEQ ID NO: 3) of SEQ ID NO: 2 or SEQ ID NO: 4. Non-limiting examples of nucleic acids encoding mutant ALK proteins and ALK-fusion proteins contain a nucleotide sequence corresponding to one or more of the following sequences: AAA or AAG at nucleotides 4362 to 4364 of SEQ ID NO: 1; AAA or AAG at nucleotides 3599-3601 of SEQ ID NO: 3; GTT, GTC, GTA, or GTG at nucleotides 4365 to 4367 of SEQ ID NO: 1; GTT, GTC, GTA, or GTG at nucleotides 3602 to 3604 of SEQ ID NO: 3; TAT or TAC at nucleotides 4377 to 4379 of SEQ ID NO: 1; TAT or TAC at nucleotides 3614 to 3616 of SEQ ID NO: 3; TCT, TCC, TCA, TCG, ACT, ACC, ACA, ACG, CAT, or CAC at nucleotides 4422 to 4424 of SEQ ID NO: 1; TCT, TCC, TCA, TCG, ACT, ACC, ACA, ACG, CAT, or CAC at nucleotides 3659 to 3661 of SEQ ID NO: 3; ATG at nucleotides 4503 to 4505 of SEQ ID NO: 1; ATG at nucleotides 3740 to 3742 of SEQ ID NO: 3; TCT, TCC, TCA, or TCG at nucleotides 4515 to 4517 of SEQ ID NO: 1; TCT, TCC, TCA, or TCG at nucleotides 3752 to 3754 of SEQ ID NO: 3; GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, or AGG at nucleotides 4527 to 4529 of SEQ ID NO: 1; GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, or AGG at nucleotides 3764 to 3766 of SEQ ID NO: 3; AAA or AAG at nucleotides 4539 to 4541 of SEQ ID NO: 1; AAA or AAG at nucleotides 3776 to 3778 of SEQ ID NO: 3; GTT, GTC, GTA, or GTG at nucleotides 4713 to 4715 of SEQ ID NO: 1; GTT, GTC, GTA, or GTG at nucleotides 3950 to 3952 of SEQ ID NO: 3; TGT, TGC, TCT, TCC, TCA, or TCG at nucleotides 4716 to 4718 of SEQ ID NO: 1; and TGT, TGC, TCT, TCC, TCA, or TCG at nucleotides 3953-3955 of SEQ ID NO: 3.
As used herein, the term “compound 1” refers to the following compound and pharmaceutically acceptable salts thereof.




As used herein, the term “crizotinib” refers to (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine], and pharmaceutically acceptable salts thereof (also referred to herein as “PF1066”).
As used herein, the term “CH5424802” refers to a compound of the formula:




and pharmaceutically acceptable salts thereof.

As used herein, the term “ASP3026” refers to a compound of the formula:




and pharmaceutically acceptable salts thereof.

By the term “donor fluorophore” is meant fluorophores or luminescent molecules capable of transferring energy to an acceptor fluorophore, thereby generating a detectable fluorescent signal from the acceptor. Donor fluorophores are generally compounds that absorb in the range of about 300 to 900 nm, for example about 350 to 800 nm. Donor fluorophores have a strong molar absorbance coefficient at the desired excitation wavelength, for example greater than about 103M−1 cm−1. A variety of compounds can be employed as donor fluorescent components, for example, in conjunction with the probes and primers of the invention, including fluorescein (and derivatives thereof), rhodamine (and derivatives thereof), GFP, pycoerythrin, BODIPY, DAPI (4′6-diamidino-2-phenylindole), Indo-1, coumarin, dansyl, and cyanine dyes. In particular examples, a donor fluorophore is a chemiluminescent molecule, such as aequorin.
As used herein, the term “fluorophore” refers to a chemical compound, which when excited by exposure to a particular stimulus such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light). Fluorophores are part of the larger class of luminescent compounds. Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy. Therefore, the use of chemiluminescence molecules (such as aequorin) eliminates the need for an external source of electromagnetic radiation, such as a laser. Examples of particular fluorophores that can be used in the probes described herein are provided in U.S. Pat. No. 5,866,366 (herein incorporated by reference). Other suitable fluorophores are known in the art, for example, those available from Molecular Probes (Eugene, Oreg.). In particular examples, the fluorophore is used as a donor fluorophore or an acceptor fluorophore. Ideally, fluorophores have the ability to be attached to a nucleic acid probe without sufficiently interfering with the ability of the probe to interact with the target nucleic acid molecule, are stable against photobleaching, and have high quantum efficiency.
By “fluorescence reasonance energy transfer” or “FRET” is meant a process in which an excited fluorophore (the donor) transfers its excited state energy to a lower-energy light absorbing molecule (the acceptor). This energy transfer is non-radiative, and due primarily to a dipole-dipole interaction between the donor and acceptor fluorophores. This energy can be passed over a distance, for example a limited distance such as 10-100 Å. FRET efficiency drops off according to 1/(1+(R/R0)6), where R0 is the distance at which the FRET efficiency is 50%.
By “FRET pairs” is meant a set of fluorophores that can engage in fluorescence resonance energy transfer (FRET). Examples of FRET pairs that can be used as listed below. However, one skilled in the art will recognize that numerous other combinations of fluorophores can be used. In particular examples, the probes disclosed herein include a FRET pair, wherein one probe of the pair includes a donor fluorophore and the other probe of the pair includes an acceptor fluorophore that can be excited by the wavelength of light emitted from the donor fluorophore. For example, an anchor probe can include a donor fluorphore and a mutation-specific probe can include the appropriate acceptor fluorophore (or vice versa). FAM is most efficiently excited by light with a wavelength of 488 nm, emits light with a spectrum of 500 to 650 nm, and has an emission maximum of 525 nm. FAM is a suitable donor fluorophore for use with JOE, TAMRA, and ROX (all of which have their excitation maxima at 514 nm, and will not be significantly stimulated by the light that stimulates FAM), as well as Cy5, Cy5.5, and LC-Red 640. The fluorophore 3-(ε-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CYA) is maximally excited at 488 nm and can therefore serve as a donor fluorophore for rhodamine derivatives (e.g., R6G, TAMRA, and ROX), which can be used as acceptor fluorophores (see Hung et al., Anal. Biochem. 243:15 (1996)). However, CYA and FAM are not examples of a good FRET pair, because both are excited maximally at the same wavelength (488 nm). Particular examples of FRET pairs are (donor/acceptor): fluorescein/rhodamine; phycoerythrin/Cy7; fluorescein/Cy5; fluorescein/Cy5.5; fluorescein/LC-Red 640; fluorescein/LC-Red 705; and fluorescein/JA286. Suitable donor-acceptor FRET pairs are described in Grant et al. Biosens. Bioelectron. 16:231 (2001). Such FRET pairs can be used in the probes of the invention.
As used herein, the term “fusion partner” means an amino acid or a nucleotide sequence of a protein or gene other than ALK. Exemplary fusion partners include 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), ALK lymphoma oligomerization partner on chromosome 17 (ALOK), clathrin light chain (CLTC), cysteinyl-tRNA synthetase (CARS), echinoderm microtubule-associated protein-like 4(EML4), kinesin-1 heavy chain KIF5B), moesin (MSN), myosin (MYH), nucleophosmin (NPM), Ran-binding protein (RanBP2), TRK-fused gene (TFG), and tropomyosin (TPM).
As used herein, the term “inhibiting the proliferation of ALK-expressing cells” or cells that express an ALK mutant described herein refers to measurably slowing, stopping, or reversing the growth rate of the ALK-expressing cells in vitro or in vivo. Desirably, a slowing of the growth rate is by at least 10%, 20%, 30%, 50%, or even 70%, as determined using a suitable assay for determination of cell growth rates (e.g., a cell growth assay described herein).
By the term “primer” is meant a short nucleic acid molecule, such as a DNA oligonucleotide, for example sequences of at least 8 nucleotides, which can be annealed to a complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand. A primer can be extended along the target nucleic acid molecule by a polymerase enzyme. Therefore, primers can be used to amplify a target nucleic acid molecule (such as a portion of an ALK or an ALK-fusion nucleic acid), wherein the sequence of the primer is specific for the target nucleic acid molecule, for example, so that the primer will hybridize to the target nucleic acid molecule under high stringency hybridization conditions. The specificity of the primer increases with length. Thus, for example, a primer that includes 30 consecutive nucleotides will anneal to a target sequence with a higher specificity than a corresponding primer of only 8 or 15 nucleotides. Thus, to obtain greater specificity, probes and primers can be selected that include at least 8, 15, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides. In particular examples, a primer is at least 8 or 15 nucleotides in length, such as at least 15 contiguous nucleotides complementary to a target nucleic acid molecule. Particular lengths of primers that can be used to practice the methods of the present disclosure (for example, to amplify a region in an ALK or an ALK-fusion nucleic acid) include primers having at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, or more continguous nucleotides complementary to the target nucleic acid to be amplified, such as a primer of 8-60 nucleotides, 8-50 nucleotides, or 15-30 nucleotides. Primer pairs can be used for amplification of nucleic acid sequence, for example, by PCR, real-time PCR, reverse transcription PCR, or other nucleic-acid amplification methods known in the art. An “upstream” or “forward” primer is a primer 5′ to a reference point in a nucleic acid sequence. A “downstream” or “reverse” primer is a primer 3′ to a reference point in a nucleic acid sequence. In general, at least one forward and one reverse primer are included in an amplification reaction. PCR primer pairs can be derived from a known sequence (such as the SEQ ID NO: 1 or SEQ ID NO: 3), for example, using computer programs intended for that purpose such as Primer (Version 0.5, ©1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.) or PRIMER EXPRESS Software® (Applied Biosystems, AB, Foster City, Calif.). Methods for preparing and suing primers are described in, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.; Ausubel et al., (1987), Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences. In one example, a primer includes a label.
The term “probe” as used herein refers to an isolated nucleic acid molecule that may include a detectable label or ligand, such as a primer that includes a label. Typical labels include without limitation radioactive isotopes, ligands, chemiluminescent agents, fluorophores, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987). In a particular example, a probe includes at least on fluorophore, such as an acceptor fluorophore or donor fluorophore. For example, a fluorophore can be covalently attached at the 5′- or 3′-end of the probe. In specific examples, a fluorophore is attached to the base of the 5′-end of the probe, the base at its 3′-end, or the phosphate group at its 5′-end. Probes are generally at least 8 nucleotides in length, such as at least 12 nucleotides, at least 15 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, or at least 25 nucleotides, such as 8-50 nucleotides, 12-50 nucleotides, 12-40 nucleotides, or 12-30 nucleotides.
As used herein, the term “refractory” refers to a cancer which is progressive in response to a given particular therapy. The cancer can be refractory either from the initial administration of the therapy; or become refractory over time in response to the therapy.
As used herein, the term “intolerant to” a drug refers to a patient who must discontinue or suspend treatment with a drug because of unacceptable side effects. The patient can be intolerant either from the initial administration of the therapy; or become intolerant over time in response to the therapy.
The term “sequence identity” is meant the shared identity between two or more nucleic acid sequence, or two or more amino acid sequences, expressed in the terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Watermann, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988); Corpet et al., Nuc. Acid Res. 16:10881 (1988); Huang et al., Computer Appls. in the Biosciences 8:155 (1992); and Pearson et al., Meth. Mol. Biol. 24:307 (1994). Altschul et al. (J. Mol. Biol. 215:403 (1990)) presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403 (1990)) is available from several sources, including the National Center for Biological Information (NCBI) website, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI website. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the option can be set as follows: −i is set to a file containing the first nucleic acid sequence to be compared; −j is set to a file containing the second nucleic acid sequence to be compared; −p is set to blastn; −o is set to any desired file name; −q is set to −1; −r is set to 2; and all other options are left at their default setting. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 400 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence-dependent and are different under different environmental parameters. Nucleic acid molecules that hybridize under stringent conditions to an ALK or ALK-fusion gene sequence typically hybridize to a probe based on either an entire ALK or ALK-fusion gene or selected portions of the gene (e.g., the kinase domain or a segment of the gene that contains the mutated codons described herein), under conditions described above.
As used herein, the term “treating” refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. To “prevent disease” refers to prophylactic treatment of a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease. To “treat disease” or use for “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease to improve or stabilize the subject's condition. Thus, in the claims and embodiments, treating is the administration to a subject either for therapeutic or prophylactic purposes.
The term “alkyl” refers to linear, branched, cyclic, and polycyclic non aromatic hydrocarbon groups, which may be substituted or unsubstituted. Unless otherwise specified, “alkyl” groups contain one to eight, and preferably one to six carbon atoms. Lower alkyl refers to alkyl groups containing 1 to 6 carbon atoms. Examples of alkyl include, without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl tert-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl, and n-heptyl, among others. Exemplary substituted alkyl groups include, without limitation, haloalkyl groups (e.g., fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl), hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substituted benzyl, and phenethyl, among others.
The term “alkoxy” refers to a subset of alkyl in which an alkyl group as defined above with the indicated number of carbons attached through an oxygen bridge, —O-alkyl, wherein the alkyl group contains 1 to 8 carbons atoms and is substituted or unsubstituted.
Exemplary alkoxy groups include, without limitation, methoxy, ethoxy, n-propoxy, i-propoxy, t-butoxy, n-butoxy, s-pentoxy, —OCF3, and —O-cyclopropyl.
The term “haloalkyl” refers to a subset of alkyl in which an alkyl group as defined above having one or more hydrogen atoms of the alkyl substituted with a halogen atom. Exemplary haloalkyl groups include, without limitation, trifluoromethyl, trichloromethyl, pentafluoroethyl and the like.
The term “alkenyl” refers to a branched or unbranched hydrocarbon group containing one or more double bonds and having from 2 to 8 carbon atoms. An alkenyl may optionally include monocyclic or polycyclic rings, in which each ring desirably has from three to six members. The alkenyl group may be substituted or unsubstituted. Alkenyl groups include, without limitation, vinyl, allyl, 2-cyclopropyl-1-ethenyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, and 2-methyl-2-propenyl.
The term “alkynyl” refers to a branched or unbranched hydrocarbon group containing one or more triple bonds and having from 2 to 8 carbon atoms. The alkynyl group may be substituted or unsubstituted. Alkynyls include, without limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
The term “cycloalkyl” refers to cyclic or polycyclic hydrocarbon groups of from 3 to 13 carbon atoms, any of which is saturated. Cycloalkyl groups may be substituted or unsubstituted. Exemplary cycloalkyl groups include, without limitation, cyclopropyl, norbornyl, [2.2.2]bicyclooctane, and [4.4.0]bicyclodecane, and the like, which, as in the case of other alkyl moieties, may optionally be substituted.
The term “cycloalkenyl” refers to cyclic or polycyclic hydrocarbon groups of from 3 to 13 carbon atoms, preferably from 5 to 8 carbon atoms, containing one or more double bonds. Cycloalkenyl groups may be substituted or unsubstituted. Exemplary cycloalkenyl groups include, without limitation, cyclopentenyl, cyclohexenyl, and cyclooctenyl.
The term “cycloalkynyl” refers to cyclic or polycyclic hydrocarbon groups of from 5 to 13 carbon atoms containing one or more triple bonds. Cycloalkynyl groups may be substituted or unsubstituted.
The term “heteroalkyl” is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 14 carbon atoms in addition to 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include, without limitation, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. The heteroalkyl group may be substituted or unsubstituted. Examples of heteroalkyls include, without limitation, polyethers, such as methoxymethyl and ethoxyethyl.
As used herein, “heterocyclic ring” and “heterocyclyl” refer to non-aromatic ring systems having five to fourteen ring atoms in which one or more ring carbons, preferably one to four, are each replaced by a heteroatom such as N, O, S, or P. Heterocyclic rings may be substituted or unsubstituted and may include one, two, or three fused or unfused ring systems. Desirably, the heterocyclic ring is a 5- to 7-membered monocyclic or 7- to 14-membered bicyclic heterocyclic ring consisting of 2 to 6 carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. Exemplary heterocyclic rings include, without limitation, 3-1H-benzimidazol-2-one, (1-substituted)-2-oxo-benzimidazol-3-yl, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholinyl, 3-morpholinyl, 4-morpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl, diazolonyl, N-substituted diazolonyl, 1-phthalimidinyl, benzoxanyl, benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl, and benzothianyl. A heterocyclyl group can include two or more of the ring systems listed above. Heterocyclic rings include those in which a non-aromatic heteroatom-containing ring is fused to one or more aromatic or non-aromatic rings, such as in an indolinyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the non-aromatic heteroatom-containing ring.
The term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to aromatic monocyclic or polycyclic ring groups having six to fourteen ring atoms, such as phenyl, 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. An “aryl” ring may be substituted or unsubstituted. The term “aryl” includes fused polycyclic aromatic ring systems in which an aromatic ring is fused to one or more rings. Non-limiting examples of aryl groups include phenyl, hydroxyphenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro, 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. Also included within the scope of the term “aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as in a indanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.
The term “heteroaryl” as used herein refers to stable heterocyclic, and polyheterocyclic aromatic moieties having 5-14 ring atoms. Heteroaryl groups may be substituted or unsubstituted and include both monocyclic and polycyclic ring systems. Examples of typical heteroaryl rings include 5-membered monocyclic rings, such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, and thiazolyl; 6-membered monocyclic rings, such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl; and polycyclic heterocyclic rings, such as benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole, tetrahydroquinoline cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, and phenoxazinyl (see e.g. Katritzky, Handbook of Heterocyclic Chemistry). Exemplary heteroaryl rings include, without limitation, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, indolyl, isoindolyl, acridinyl, and benzoisoxazolyl. Heteroaryl groups further include a group in which a heteroaromatic ring is fused to one or more aromatic or nonaromatic rings where the radical or point of attachment is on the heteroaromatic ring, such as tetrahydroquinoline, tetrahydroisoquinoline, and pyrido[3,4-d]pyrimidinyl, imidazo[1,2-a]pyrimidyl, imidazo[1,2-a]pyrazinyl, imidazo[1,2-a]pyiridinyl, imidazo[1,2-c]pyrimidyl, pyrazolo[1,5-a][1,3,5]triazinyl, pyrazolo[1,5-c]pyrimidyl, imidazo[1,2-b]pyridazinyl, imidazo[1,5-a]pyrimidyl, pyrazolo[1,5-b][1,2,4]triazine, quinolyl, isoquinolyl, quinoxalyl, imidazotriazinyl, pyrrolo[2,3-d]pyrimidyl, triazolopyrimidyl, and pyridopyrazinyl.
An aryl group or heteroaryl group may contain one or more substituents. Exemplary substituents for aryl or heteroaryl group include halogen (F, Cl, Br or I), alkyl, alkenyl, alkynyl, heteroalkyl, —NO2, —CN, —RA, —ORB, —S(O)rRB, (wherein r is 0, 1 or 2), —SO2NRARB, —NRARB, —O—NRARB, —NRA—NRARB, —(CO)YRB, —O(CO)YRB, —NRA(CO)YRB, —S(CO)YRB, —NRAC(═S)YRB, —OC(═S)YRB, —C(═S)YRB, —YC(═NRA)YRB, —YC(═N—ORA)YRB, —YC(═N—NRARB)YRB, —COCORB, —COMCORB (where M is a C1-6 alkyl group), —YP(O)(YRC)(YRC), —P(O)(RC)2, —Si(RC)3, —NRASO2RB, and —NRASO2NRARB, wherein each occurrence of Y is, independently, —O—, —S—, —NRA—, or a chemical bond (i.e., —(CO)YRB thus encompasses —C(═O)RB, —C(═O)ORB, and —C(═O)NRARB).
RC is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and heterocyclyl. At each occurrence, each of RA and RB is, independently, selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and heterocyclyl.
Each of RA, RB and RC optionally bears one or more substituents selected from amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, aryl, heteroalkyl, heteroaryl, carbocycle, heterocycle, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, alkoxy, haloalkoxy groups, hydroxy, protected hydroxyl groups (e.g., —O—X, where X is acyl, phenyl, substituted phenyl, benzyl, substituted benzyl, phenethyl, or substituted phenethyl), -M-heteroaryl, -M-heterocycle, -M-aryl, -M-ORB, -M-SRB, -M-NRARB, -M-OC(O)NRARB, -M-C(═NRB)NRARB, -M-C(═NRA)ORB, -M-P(═O)(RC)2, Si(RC)3, -M-NRAC(O)RB, -M-NRAC(O)ORB, -M-C(O)RB, -M-C(═S)RB, -M-C(═S)NRARB, -M-C(O)NRARB, -M-C(O)NRB-M-NRARB, -M-NRBC(NRA)NRARB, -M-NRAC(S)NRARB, -M-S(O)2RA, -M-C(O) RA, -M-OC(O) RA, -MC(O)SRB, -M-S(O)2NRARB, —C(O)-M-C(O)RB, -MCO2RB, -MC(═O)NRARB, -M-C(═NH)NRARB, and -M-OC(═NH)NRARB, wherein M is a C1-6 alkyl group. Non-limiting illustrations of a substituted RA, RB or RC group include haloalkyl and trihaloalkyl, alkoxyalkyl, halophenyl, chloromethyl, trichloromethyl, trifluoromethyl, methoxyethyl, alkoxyphenyl, halophenyl, —CH2-aryl, —CH2-heterocycle, —CH2C(O)NH2, —C(O)CH2N(CH3)2, —CH2CH2OH, —CH2OC(O)NH2, —CH2CH2NH2, —CH2CH2CH2NEt2, —CH2OCH3, —C(O)NH2, —CH2CH2-heterocycle, —C(═S)CH3, —C(═S)NH2, —C(═NH)NH2, —C(═NH)OEt, —C(O)NH-cyclopropyl, —C(O)NHCH2CH2-heterocycle, —C(O)NHCH2CH2OCH3, —C(O)CH2CH2NHCH3, —CH2CH2F, —C(O)CH2-heterocycle, —CH2C(O)NHCH3, —CH2CH2P(═O)(CH3)2, and —Si(CH3)3.
An alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, or heterocyclic group may contain one or more substituents selected from those listed above for aryl and heteroaryl groups, in addition to ═O, ═S, ═NH, ═NNRARB, ═NNHC(O)RB, ═NNHCO2RB, or ═NNHSO2RB, wherein RA and RB are as defined above.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of an exemplary protocol for accelerated in vitro mutagenesis.
FIGS. 2A and 2B show an in vitro mutagenesis screen with frequency among recovered clones. FIG. 2A shows data for exposure to crizotinib at 500, 720, 1000, or 1440 nM. FIG. 2B shows data for exposure to compound 1 at 100, 200, 500, or 1000 nM.
FIG. 3 is a homology model of crizotinib bound to ALK.
FIGS. 4A and 4B show the effect of compound 1 and crizotinib in a cellular proliferation assay with different EML-4-ALK mutants. FIG. 4A shows a representative proliferation curve for crizotinib. FIG. 4B shows a representative proliferation curve for compound 1.
FIGS. 5A and 5B show the effect of compound 1 and crizotinib for ALK phosphorylation in EML4-ALK mutant tumors in vivo. FIG. 5A is a graph showing ALK phosphorylation (% p-ALK) and plasma levels for a dosage of crizotinib at 200 mg/kg. FIG. 5B is a graph showing ALK phosphorylation (% p-ALK) and plasma levels for a dosage of compound 1 at 50 mg/kg.
FIGS. 6A-6D show the effect of various dosages of crizotinib on a xenograft model. Data is provided for native EML4-ALK (FIG. 6A) and for the L1196M (FIG. 6B), S1206R (FIG. 6C), and G1269S (FIG. 6D) mutants.
FIGS. 7A-7D show the effect of various dosages of compound 1 on a xenograft model. Data is provided for native EML4-ALK (FIG. 7A) and for the L1196M (FIG. 7B), S1206R (FIG. 7C), and G1269S (FIG. 7D) mutants.
FIG. 8 shows the mRNA sequence for human ALK (SEQ ID NOs: 1 and 3). The codons in bold and underlined represent the codons that are mutated in the mutant ALK nucleic acids described herein.
FIG. 9 shows the protein sequence for human ALK (SEQ ID NOs: 2 and 4). The amino acids in bold and underlined represent the amino acids that are mutated in the mutant ALK proteins described herein.
FIG. 10 shows the protein and mRNA sequence of the NPM/ALK fusion (SEQ ID NOs: 5 and 6).
FIGS. 11A and 11B are graphs depicting the efficacy of compound 1 (FIG. 11A) and crizotinib (FIG. 11B) to L1196M mutant-driven tumors in a xenograft model. The tumors respond to treatment with compound 1, but crizotinib has no effect.
FIG. 12 is a series of bar graphs depicting the emergence of cells resistant to CH5424802 (“AF-802”) and ASP3026. Selection of BaF3 cells expressing EML4-ALK using various concentrations of AF-802 and ASP3026 led to the identification of the mutations identified in the bar graphs. These compounds are susceptible to mutational weaknesses.



DETAILED DESCRIPTION
Culturing EML4-ALK-expressing cells in the presence of certain ALK-inhibitors (e.g. to one or more of crizotinib, CH5424802 and ASP3026) led to the selection and identification of cells expressing ALK mutations. The nucleotide sequences were analyzed and a set of seven mutations were identified that conferred varying degrees of resistance to one or more of crizotinib, CH5424802 and ASP3026.
We have observed that the ALK mutants that are resistant to one or more of crizotinib, CH5424802 and ASP3026 can still be inhibited potently by compounds of formula (I), such as compound 1.
The invention features a method for treating patients who have an ALK-driven cancer, which is, or has become, refractory to one or more of crizotinib, CH5424802 and ASP3026, or which bears an ALK mutation identified herein, by administering a compound of formula (I) to the patient. The invention also features a method for characterizing ALK-driven cancers to determine whether they express an ALK mutant.
ALK Mutants
ALK mutants include one or more deletions, substitutions, or additions in the amino acid or nucleotide sequences of ALK, or fragments thereof, and can include ALK fusion proteins and ALK fusion genes. Thus, an ALK mutant includes ALK fusion proteins and ALK genes with one or more deletions, substitutions, or additions, or a fragment thereof.
An ALK mutant also includes amino acid and nucleotide sequences of ALK with one or more deletions, substitutions, or additions, such as point mutations. Where the mutant is a protein or polypeptide, preferable substitutions are conservative substitutions, which are substitutions between amino acids similar in properties such as structural, electric, polar, or hydrophobic properties. For example, the substitution can be conducted between basic amino acids (e.g., Lys, Arg, and His), or between acidic amino acids (e.g., Asp and Glu), or between amino acids having non-charged polar side chains (e.g., Gly, Asn, Gln, Ser, Thr, Tyr, and Cys), or between amino acids having hydrophobic side chains (e.g., Ala, Val, Leu, Ile, Pro, Phe, and Met), or between amino acids having branched side chains (e.g., Thr, Val, Leu, and Ile), or between amino acids having aromatic side chains (e.g., Tyr, Trp, Phe, and His). ALK mutants of particular interest are those identified by culturing EML4-ALK-expressing cells in the presence of the ALK-inhibitor PF 1066.
Where the mutant is a nucleic acid, the DNA encoding an ALK mutant protein may comprise a nucleotide sequence capable of hybridizing to a complement sequence of the nucleotide sequence encoding an ALK mutant as defined above, under stringent conditions. As used herein, the stringent conditions include low, medium or high stringent conditions. An example of the stringent conditions includes hybridization at approximately 42-55° C. in approximately 2-6×SSC, followed by wash at approximately 50-65° C. in approximately 0.1-1×SSC containing approximately 0.1-0.2% SDS, where 1×SSC is a solution containing 0.15 M NaCl and 0.015 M Na citrate, pH 7.0. Wash can be performed once or more. In general, stringent conditions may be set at a temperature approximately 5° C. lower than a melting temperature (Tm) of a specific nucleotide sequence at defined ionic strength and pH.
Many ALK fusion proteins and ALK fusion genes include the entire intracytoplasmic portion or most of the intracytoplasmic portions of ALK. The amino acid and nucleotide sequences of ALK and DNAs encoding them are available from known databases such as NCBI GenBank (USA), EMBL (Europe), etc. For example, GenBank accession numbers are ALK [Homo sapiens] (AB209477, U62540, NG_009445.1, and GeneID: 238). Exemplary ALK fusion proteins and fusion genes having the entire intracytoplasmic portion include TRK-fused gene/anaplastic lymphoma kinase (Ki-1) fusion protein long form [Homo sapiens] (AAF27292.1), TRK-fused gene-anaplastic lymphoma kinase fusion protein [Homo sapiens] (AAF42734.1), fusion protein EML4-ALK variant 5 splicing isoform a [Homo sapiens] (BAG75148.1), fusion protein KIF5B-ALK [Homo sapiens] (BAH57337.1), fusion protein EML4-ALK variant 5 splicing isoform b [Homo sapiens] (BAG75149.1), fusion protein EML4-ALK variant 4 [Homo sapiens] (BAG75147.1), fusion protein EML4-ALK variant 2 [Homo sapiens] (BAF73612.1), fusion protein EML4-ALK variant 1 [Homo sapiens] (BAF73611.1), fusion protein EML4-ALK variant 7 [Homo sapiens] (BAH57336.1), fusion protein EML4-ALK variant 6 [Homo sapiens] (BAH57335.1), fusion protein EML4-ALK variant 3 splicing isoform b [Homo sapiens] (BAG55004.1), fusion protein EML4-ALK variant 3 splicing isoform a [Homo sapiens] (BAG55003.1), nucleophosmin-anaplastic lymphoma kinase fusion protein [Homo sapiens] (AAA58698.1), tropomyosin 4-anaplastic lymphoma kinase fusion protein minor isoform [Homo sapiens] (AAK51964.1), tropomyosin 4-anaplastic lymphoma kinase fusion protein major isoform [Homo sapiens] (AAK51963.1), EML4/ALK fusion protein variant 3 [Homo sapiens] (ABX59674.1), moesin/anaplastic lymphoma kinase fusion protein [Homo sapiens] (AAK71522.1), TPM4-ALK fusion oncoprotein type 1 [Homo sapiens] (AAG17015.1), TRK-fused gene/anaplastic large cell lymphoma kinase extra long form [Homo sapiens] (AAM17922.1), EML4/ALK fusion protein variant 3 (EML4/ALK fusion) mRNA [Homo sapiens] (EU236948.1), TRK-fused gene (TFG), transcript variant 1, mRNA [Homo sapiens] (NM_006070.4 GI:56090655), TRK-fused gene (TFG), transcript variant 2, mRNA [Homo sapiens] (NM_001007565.1), TRK-fused gene-anaplastic lymphoma kinase fusion protein (TFG/ALK) mRNA, complete cds [Homo sapiens] (AF125093.1), and TRK-fused gene/anaplastic lymphoma kinase (Ki-1) fusion protein long form (TFG/ALK fusion) mRNA, complete cds [Homo sapiens] (AF143407.1). Exemplary ALK fusion proteins having a portion of the entire intracytoplasmic portion include moesin/anaplastic lymphoma kinase fusion protein and fusion gene (AAK71522.1 and AF295356.1), which contain the final 557 amino acids of ALK; and MYH-9-ALK (see Lamant et al., Genes Chromosomes Cancer. 37:427-432, 2003), which contains the final 556 residues of ALK.
Other exemplary ALK fusion proteins are ALO17-ALK, ATIC-ALK (e.g., inv(2(p23;q35)), CARS-ALK, CLTC-ALK (e.g., t(2;17)(p23;q21)), EML4-ALK (e.g., inv(2)(p21;p23)), KIF5B-ALK, MSN-ALK (e.g., t(2;X)(p23;q11-12)), MYH9-ALK, NPM-ALK (e.g., t(2;5)(p23;q35)), RanBP2-ALK, SEC31L1-ALK, forms of TFG-ALK (including TFG-ALK (e.g., t(2;3)(p23;q21)), TFGL-ALK (e.g., t(2;3)(p23;q21)), and TFGXL-ALK), TPM3-ALK (e.g., t(1;2)(q25;p23)), and TPM4-ALK (e.g., t(2;19)(p23;p13.1)). ALK fusion proteins also include variants of any fusion. Exemplary variants of an EML4-ALK fusion protein include that arise from variable exon regions of EML4 fused to exon 20 of ALK, such as variant 1 with fused exon 13 of EML4 to exon 20 of ALK (see, e.g., Koivunen et al., Clin. Cancer Res. 14:4275 (2008)); variant 2 with fused exon 20 of EML-4 (see, e.g., Horn et al., J. Clin. Oncol. 27:4232 (2009)); variant 3 with fused exon 6 of EML-4 (see, e.g, Koivunen et al., supra, and Choi et al., Cancer Res. 68:4971 (2008)); variant 4 with fused exon 14 of EML-4 (see, e.g., Horn et al., supra); variant 5 with fused exon 18 of EML-4 (see, e.g., Wong et al., Cancer 115:1723 (2009)); variant 6 with fused exon 13 of EML-4; variant 7 with fused exon of 14; and other variants that include additional nucleotides from the adjacent intron or that exclude nucleotides from the recited exon (e.g., variant 3a with fused exon 6 of EML-4 and variant 3b with fused exon 6 and an addition 33 base pairs from intron 6 of EML-4).
Characterization of ALK-Driven Cancers
ALK mutant expression or overexpression is determined in a diagnostic or prognostic assay by evaluating levels of ALK mutants in biological sample, or secreted by the cell (e.g., via an immunohistochemistry assay using anti-ALK antibodies or anti-p-ALK antibodies; FACS analysis, etc.). Alternatively, or additionally, one can measure levels of ALK mutant-encoding nucleic acid or mRNA in the cell, e.g., via fluorescent in situ hybridization using a nucleic acid based probe corresponding to an ALK mutant-encoding nucleic acid or the complement thereof; (FISH; see WO98/45479, published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One can also study ALK mutant expression by measuring shed antigen in a biological sample, such as serum, e.g., using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294, issued Jun. 12, 1990; WO91/05264, published Apr. 18, 1991; U.S. Pat. No. 5,401,638, issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods 132:73 (1990)). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one can expose cells within the body of the mammal to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the mammal can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a mammal previously exposed to the antibody.
Examples of biological properties that can be measured in isolated cells include mRNA expression, protein expression, and DNA quantification. Additionally, the DNA of cells isolated by the methods of the invention can be sequenced, or certain sequence characteristics (e.g., polymorphisms and chromosomal abnormalities) can be identified using standard techniques, e.g., FISH or PCR. The chemical components of cells, and other analytes, may also be assayed after isolation. Cells may also be assayed without lysis, e.g., using extracellular or intracellular stains or by other observation, e.g., morphology or growth characteristics in various media.
While any hybridization technique can be used to detect the gene rearrangements, one preferred technique is fluorescent in situ hybridization (FISH). FISH is a cytogenetic technique which can be used to detect and localize the presence or absence of specific DNA or RNA sequences on chromosomes. FISH incorporates the use of fluorescently labeled nucleic acid probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome. The basic steps of FISH are outlined below. Exemplary FISH probes include Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe (Abbott, Downers Grove, Ill.), which hybridizes to band 2p23 on either side of the ALK gene breakpoint; and Split Signal ALK FISH DNA Probe (Dako), which hybridizes to a 289 kb segment telomeric to the ALK breakpoint and to a 557 kb segment centromeric to the ALK breakpoint.
For FISH, a probe is constructed that is long enough to hybridize specifically to its target (and not to similar sequences in the genome), but not too large to impede the hybridization process. Probes are generally labeled with fluorophores, with targets for antibodies, with biotin, or any combination thereof. This can be done in various ways, for example using random priming, nick translation, and PCR using tagged nucleotides.
Generally, a sample or aliquot of a population of cells is used for FISH analysis. For example, in one method of preparation, cells are trypsinized to disperse into single cells, cytospun onto glass slides, and then fixed with paraformaldehyde before storing in 70% ethanol. For preparation of the chromosomes for FISH, the chromosomes are firmly attached to a substrate, usually glass. After preparation, the probe is applied to the chromosome RNA and starts to hybridize. In several wash steps all unhybridized or partially hybridized probes are washed away. If signal amplification is necessary to exceed the detection threshold of the microscope (which depends on many factors such as probe labeling efficiency, the kind of probe, and the fluorescent dye), fluorescent tagged antibodies or strepavidin are bound to the tag molecules, thus amplifying the fluorescence.
An epifluorescence microscope is used for observation of the hybridized sequences. The white light of the source lamp is filtered so that only the relevant wavelengths for excitation of the fluorescent molecules arrive onto the sample. Emission of the fluorochromes happens, in general, at larger wavelengths, which allows one to distinguish between excitation and emission light by mean of another optical filter. With a more sophisticated filter set, it is possible to distinguish between several excitation and emission bands, and thus between several fluorochromes, which allows observation of many different probes on the same strand.
Depending on the probes used, FISH can have resolution ranging from huge chromosomes or tiny ( ̃100 kilobase) sequences. The probes can be quantified simply by counting dots or comparing color.
Methods for the identification of a mutation in an ALK or an ALK-fusion product are provided that include contacting a target ALK or ALK-fusion nucleic acid (e.g., mRNA or amplified product) with a first and second probes (as described herein), under conditions that permit hybridization between the target nucleic acid molecule and the first and second probes. The resulting hybridization complex is heated to increase the temperature and permit melting of the hybridization complex. The resulting melting curve is detected, for example, by detecting a fluorescent signal from a donor or acceptor fluorophore on the probes, wherein a melting point temperature shift compared to a melting curve expected for the wild-type target sequence indicates the presence of a mutation in the target ALK or ALK-fusion nucleic acid. Is some examples, the test melting curve is compared to a melting curve for a control, such as a wild type or mutant ALK or mutant ALK-fusion nucleic acid. The methods described herein may used to determine a treatment protocol.
An additional method that may be used to identify a nucleic acid encoding a mutant ALK or mutant ALK-fusion protein (described herein) is allele-specific quantitative real time-PCR (see, for e.g., Diagnostic Innovations DxS BCR-ABL T3151 Mutation Test Kit, and Singer et al., Methods in Molec. Biol. 181:145 (2001)). This technique utilizes Taq DNA polymerase, which is extremely effective at distinguishing between a match and a mismatch at the 3′-end of the primer (when the 3′-base is mismatched, no efficient amplification occurs). Using this technique, the 3′-end of the primer may be designed to specifically hybridize to a nucleic acid sequence that corresponds to a codon that encodes a mutant amino acid in an ALK or an ALK-fusion protein (as described herein). In this way, the specific mutated sequences can be selectively amplified in a patient sample. This technique further utilizes a Scorpion probe molecule, which is a bifunctional molecule containing a PCR primer, a fluorophore, and a quencher. The fluorophore in the probe interacts with a quencher, which reduces fluorescence. During a PCR reaction, when the Scorpion probe binds to the amplicon, the fluorophore and quencher in the Scorpion probe become separated, which leads to an increase in fluorescence from the reaction tube. Any of the primers described herein may be used in allele-specific quantitative real time PCR.
A biological sample can be analyzed to detect a mutation in an ALK gene, or expression levels of an ALK gene, by methods that are known in the art. For example, methods such as direct nucleic acid sequencing, altered hybridization, aberrant electrophoretic gel migration, binding or cleavage mediated by mismatch binding proteins, single-strand conformational polymorphism (SSCP) analysis, or restriction fragment length polymorphism (RFLP) analysis of PCR products derived from a patient sample can be used to detect a mutation in an ALK gene; ELISA can be used to measure levels of ALK polypeptide; and PCR can be used to measure the level of an ALK nucleic acid molecule.
Any of these techniques may be used to facilitate detection of a mutation in a candidate gene, and each is well known in the art; examples of particular techniques are described, without limitation, in Orita et al. (Proc. Natl. Acad. Sci. USA 86:2766 (1989)) and Sheffield et al. (Proc. Natl. Acad. Sci. USA 86:232 (1989)). Furthermore, expression of the candidate gene in a biological sample (e.g., a biopsy) may be monitored by standard Northern blot analysis or may be aided by PCR (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. (1995); PCR Technology: Principles and Applications for DNA Amplification, H.A. Ehrlich, Ed., Stockton Press, NY; Yap et al., Nucl. Acids. Res. 19:4294 (1991)).
Primers for use in PCR, reverse-transcription PCR, or quantitative real time PCR may be designed to amplify a sequence within an ALK gene, an ALK fusion gene, or an mRNA encoding an ALK protein or an ALK fusion protein, that contains one or more (e.g., 1, 2, 3, 4, or 5) specific mutation(s) associated with an increased risk of developing a proliferative order or associated with increased resistance to treatment with a tyrosine kinase inhibitor (e.g., to one or more of crizotinib, CH5424802 and ASP3026). For example, primers (e.g., 8 to 30 nucleotides) may be designed to amplify a specific contiguous sequence within a nucleic acid (e.g., mRNA) encoding human full-length ALK (human ALK mRNA, Genbank Accession No: U62540 and Genbank Accession No. U66559 (SEQ ID NOS: 1 and 3, respectively) or human ALK-fusion proteins (e.g., ALO17-ALK, ATIC-ALK, CARS-ALK, CLTC-ALK, EML4-ALK, KIF5B-ALK, MSN-ALK (e.g., t(2;X)(p23;q11-12)), MYH9-ALK, NPM-ALK, RanBP2-ALK, SEC31L1-ALK, TFGS-ALK, TFGL-ALK, TFGXL-ALK, TPM3-ALK, or TPM4-ALK) that encodes one or more of the amino acids corresponding to: the amino acid at position 1151, the amino acid at position 1152, the amino acid at position 1156, the amino acid at position 1171, the amino acid at position 1174, the amino acid at position 1180, the amino acid at position 1181, the amino acid at position 1196, the amino acid at position 1198, the amino acid at position 1202, the amino acid at position 1206, the amino acid at position 1210, the amino acid at position 1241, the amino acid at position 1268, and the amino acid at position 1269 of SEQ ID NO: 2 or SEQ ID NO: 4. For example, primers may be designed to amplify the kinase domain of nucleic acid encoding an ALK or ALK-fusion protein (e.g., 5′-AGCGATGCAGATGGAATTGCAGAG-3 (SEQ ID NO: 7) and 5′-CAATAGGCAGCGCCGTGTTGATTA-3′(SEQ ID NO: 8)). The kinase domain in an ALK protein or an ALK-fusion protein corresponds to the sequence of amino acids 1058 to 1620 in SEQ ID NO: 2 or 4 (encoded by a sequence that corresponds to nucleotides 4083 to 5795 in SEQ ID NO: 1 and nucleotides 3320 to 5032 in SEQ ID NO: 3). Desirably, the primers or probes of the invention are designed to amplify or hybridize a contiguous sequence of nucleotides (e.g., 20 to 6226 nucleotides) within a nucleic acid encoding an ALK or ALK-fusion protein sequence that contains one or more codons for the amino acid corresponding to the amino acid at position 1151 (e.g., nucleotides 4362 to 4364 of SEQ ID NO: 1 or nucleotides 3599-3601 of SEQ ID NO: 3), the amino acid at position 1152 (e.g., nucleotides 4365 to 4367 of SEQ ID NO: 1 or nucleotides 3602 to 3604 of SEQ ID NO: 3), the amino acid at position 1156 (e.g., nucleotides 4377 to 4379 of SEQ ID NO: 1 or nucleotides 3614 to 3616 of SEQ ID NO: 3), the amino acid at position 1171 (e.g., nucleotides 4422 to 4424 of SEQ ID NO: 1 or nucleotides 3659 to 3661 of SEQ ID NO: 3), the amino acid at position 1174 (e.g., nucleotides 4431 to 4433 of SEQ ID NO: 1 or nucleotides 3668 to 3670 of SEQ ID NO: 3), the amino acid at position 1180 (e.g., nucleotides 4449 to 4451 of SEQ ID NO: 1 or nucleotides 3686 to 3688 of SEQ ID NO: 3), the amino acid at position 1181 (e.g., nucleotides 4452 to 4454 of SEQ ID NO: 1 or nucleotides 3689 to 3691 of SEQ ID NO: 3), the amino acid at position 1196 (e.g., nucleotides 4497 to 4499 of SEQ ID NO: 1 or nucleotides 3734 to 3736 of SEQ ID NO: 3), the amino acid at position 1198 (e.g., nucleotides 4503 to 4505 of SEQ ID NO: 1 or nucleotides 3740 to 3742 of SEQ ID NO: 3), the amino acid at position 1202 (e.g., nucleotides 4515 to 4517 of SEQ ID NO: 1 or nucleotides 3752 to 3754 of SEQ ID NO: 3), the amino acid at position 1206 (e.g., nucleotides 4527 to 4529 of SEQ ID NO: 1 or nucleotides 3764 to 3766 of SEQ ID NO: 3), the amino acid at position 1210 (e.g., nucleotides 4539 to 4541 of SEQ ID NO: 1 or nucleotides 3776 to 3778 of SEQ ID NO: 3), the amino acid at position 1241 (e.g., nucleotides 4632 to 4634 of SEQ ID NO: 1 or nucleotides 3869 to 3871 of SEQ ID NO: 3), the amino acid at position 1268 (e.g., nucleotides 4713 to 4715 of SEQ ID NO: 1 or nucleotides 3950 to 3952 of SEQ ID NO: 3), and the amino acid at position 1269 (e.g., nucleotides 4716 to 4718 of SEQ ID NO: 1 or nucleotides 3953-3955 of SEQ ID NO: 3) of SEQ ID NO: 2 or SEQ ID NO: 4. The amino acids at positions 1151, 1152, 1156, 1171, 1174, 1180, 1181, 1196, 1198, 1202, 1206, 1210, 1241, 1268, and 1269 in wild-type ALK protein (SEQ ID NOS: 2 or 4) are T1151, L1152, C1156, I1171, F1174, V1180, R1181, L1196, L1198, G1202, S1206, E1210, E1241, I1268, and G1269 (indicated in bold and underlined in FIG. 9).
The methods of the invention provide primers and probes that may be used to amplify or identify (hybridize under stringent conditions) a nucleic acid (e.g., mRNA) encoding a mutant ALK or ALK fusion protein that contains one or more codons corresponding to the following sequences: nucleotides 4362 to 4364 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3599-3601 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4365 to 4367 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3602 to 3604 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4377 to 4379 of SEQ ID NO: 1 (e.g., TAT or TAC); nucleotides 3614 to 3616 of SEQ ID NO: 3 (e.g., TAT or TAC); nucleotides 4422 to 4424 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 3659 to 3661 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, ACT, ACC, ACA, ACG, CAT, or CAC); nucleotides 4431 to 4433 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3668 to 3670 of SEQ ID NO: 3 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 4449 to 4451 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3686 to 3688 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4452 to 4454 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, or GGG); nucleotides 3689 to 3691 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, or GGG); nucleotides 4497 to 4499 of SEQ ID NO: 1 (e.g., CAA or CAG); nucleotides 3734 to 3736 of SEQ ID NO: 3 (e.g., CAA or CAG); nucleotides 4503 to 4505 of SEQ ID NO: 1 (e.g., ATG); nucleotides 3740 to 3742 of SEQ ID NO: 3 (e.g., ATG); nucleotides 4515 to 4517 of SEQ ID NO: 1 (e.g., TCT, TCC, TCA, TCG, AGT, or AGC); nucleotides 3752 to 3754 of SEQ ID NO: 3(e.g., TCT, TCC, TCA, or TCG); nucleotides 4527 to 4529 of SEQ ID NO: 1 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 3764 to 3766 of SEQ ID NO: 3 (e.g., GGT, GGC, GGA, GGG, ATT, ATC, ATA, AGA, AGG, GCT, GCC, GCA, or GCG); nucleotides 4539 to 4541 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3776 to 3778 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4632 to 4634 of SEQ ID NO: 1 (e.g., AAA or AAG); nucleotides 3869 to 3871 of SEQ ID NO: 3 (e.g., AAA or AAG); nucleotides 4713 to 4715 of SEQ ID NO: 1 (e.g., GTT, GTC, GTA, or GTG); nucleotides 3950 to 3952 of SEQ ID NO: 3 (e.g., GTT, GTC, GTA, or GTG); nucleotides 4716 to 4718 of SEQ ID NO: 1 (e.g., TGT, TGC, TCT, TCC, TCA, TCG, AGT, or AGC); and nucleotides 3953-3955 of SEQ ID NO: 3 (e.g., TGT, TGC, TCT, TCC, TCA, or TCG); or a complement sequence thereof. The corresponding codons in the wild-type ALK nucleic acid sequence are: ACG at nucleotides 4362 to 4364 of SEQ ID NO: 1 and nucleotides 3599 to 3601 of SEQ ID NO: 3; CTG at nucleotides 4365 to 4367 of SEQ ID NO: 1 and nucleotides 3602 to 3604 of SEQ ID NO: 3; TGC at nucleotides 4377 to 4379 of SEQ ID NO: 1 and nucleotides 3614 to 3616 of SEQ ID NO: 3; ATC for nucleotides 4422 to 4424 of SEQ ID NO: 1 and nucleotides 3659 to 3661 of SEQ ID NO: 3; TTC for nucleotides 4431 to 4433 of SEQ ID NO: 1 and nucleotides 3668 to 3670 of SEQ ID NO: 3; GTT for nucleotides 4449 to 4451 of SEQ ID NO: 1 and nucleotides 3686 to 3688 of SEQ ID NO: 3; CGC for nucleotides 4452 to 4454 of SEQ ID NO: 1 and nucleotides 3689 to 3691 of SEQ ID NO: 3; CTG for nucleotides 4497 to 4499 of SEQ ID NO: 1 and nucleotides 3734 to 3736 of SEQ ID NO: 3; CTC for nucleotides 4503 to 4505 of SEQ ID NO: 1 and nucleotides 3740 to 3742 of SEQ ID NO: 3; GGA for nucleotides 4515 to 4517 of SEQ ID NO: 1 and nucleotides 3752 to 3754 of SEQ ID NO: 3; TCC for nucleotides 4527 to 4529 of SEQ ID NO: 1 and nucleotides 3764 to 3766 of SEQ ID NO: 3; GAG for nucleotides 4539 to 4541 of SEQ ID NO: 1 and nucleotides 3776 to 3778 of SEQ ID NO: 3; GAG for nucleotides 4632 to 4634 of SEQ ID NO: 1 and nucleotides 3869 to 3871 of SEQ ID NO: 3; ATT for nucleotides 4713 to 4715 of SEQ ID NO: 1 and nucleotides 3950 to 3952 of SEQ ID NO: 3; and GGA for nucleotides 4716 to 4718 of SEQ ID NO: 1 and nucleotides 3953-3955 of SEQ ID NO: 3 (indicated in bold and underlined in FIG. 8).
Non-limiting exemplary probes of the invention that hybridize to the antisense strand of an ALK or ALK-fusion nucleic acid that contains a sequence encoding a mutation corresponding to amino acid position 1196 (leucine to methionine substitution) of SEQ ID NO: 2 or 4 are: ggttcatcctgatggagctcatg (SEQ ID NO: 9); gttcatcctgatggagctcatgg (SEQ ID NO: 11); ttcatcctgatggagctcatggc (SEQ ID NO: 13); cggttcatcctgatggagctcat (SEQ ID NO: 15); ccggttcatcctgatggagctca (SEQ ID NO: 17); ggttcatcctgatggagctcatg (SEQ ID NO: 19); and cggttcatcctgatggagctcat (SEQ ID NO: 21).
One skilled in the art may design a variety or probes or primers to hybridize or amplify a nucleic acid sequence (e.g., mRNA or genomic DNA) that encodes an ALK protein, an ALK-fusion protein, a mutant ALK protein, or a mutant ALK-fusion protein containing a contiguous nucleic acid sequence that encodes any of the above-identified codons corresponding to the indicated codons in SEQ ID NO: 1 or SEQ ID NO: 3.
One skilled in the art may identify in a nucleic acid or protein sequence a residue (e.g., amino acid or nucleotide) or codon that corresponds to a residue or codon in SEQ ID NOS: 1-4 using a number of sequence alignment software programs (e.g., NCBI BLAST website). Such software programs may allow for gaps in the alignment of the compared sequences. Using such software, one skilled in the art may identify a nucleotide, amino acid, or amino acid that corresponding to a specific nucleotide, amino acid, or codon in SEQ ID NOS: 1-4.
Also provided by the present application are probes that include variations to the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3, as long as such variations permit detection of the desired mutations in a nucleic acid that encodes an ALK or ALK fusion protein. For example, a probe can have at least 90%, at least 95%, or at least 98% sequence identity to a contiguous sequence of nucleotides present in SEQ ID NO: 1 or SEQ ID NO: 2.
Likewise, probes (e.g., 8 to 50 nucleotides in length) may be designed for use in situ hybridization (e.g., FISH), Southern blotting, Northern blotting, or gene array analysis, that specifically hybridize to a contiguous sequence within an ALK gene, an ALK fusion gene, or an mRNA encoding an ALK protein or an ALK fusion protein, that encodes one or more of the amino acids corresponding to: the amino acid at position 1151, the amino acid at position 1152, the amino acid at position 1156, the amino acid at position 1171, the amino acid at position 1174, the amino acid at position 1180, the amino acid at position 1181, the amino acid at position 1196, the amino acid at position 1198, the amino acid at position 1202, the amino acid at position 1206, the amino acid at position 1210, the amino acid at position 1241, the amino acid at position 1268, and the amino acid at position 1269 of SEQ ID NO: 2 or SEQ ID NO: 4. For example, the probes may be designed to hybridize to a contiguous sequence within the human full-length ALK (human ALK mRNA, Genbank Accession Nos: U62540 and U66559 (SEQ ID NOS: 1 and 3, respectively)), human ALK fusion proteins, a nucleic acid that contains a contiguous sequence of between 60 and 1626 nucleotides that is at least 95% identical to a corresponding sequence in SEQ ID NO: 1 or SEQ ID NO: 3, or a nucleic acid (e.g., mRNA) encoding a mutant ALK or ALK fusion protein (as described herein), or the complement sequence thereof.
Computer software for the design of primers and probes are publicly available, for example, at the NCBI website. The Gene Infinity website also provides a listing of publicly available software programs for the design of probes and primers. Once the appropriate primer or probe sequences have been selected, the probes and primers may be synthesized using commercially available methods and equipment as described in the art (e.g., Koshkin et al., Tetrahedron 54:3607 (1998)). For example, the solid phase phosphoramidate method can be used to produce short LNA probes (Caruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411 (1982)). Desirable LNA monomers and their methods of synthesis are also disclosed in U.S. Pat. Nos. 6,043,060 and 6,268,490.
Probes of the invention may further include any number of modifications. Probes may be labeled or conjugated to other functional moieties, e.g., through the attachment of duplex-stabilizing agents such as minor-groove-binders (MGB) or intercalating nucleic acids (INA). Additionally, the modifications may also include addition of non-discriminatory bases, e.g., such as 5-nitroindole, which are capable of stabilizing duplex formation regardless of the nucleobase at the opposing position on the target strand. The modifications may also include the addition of naturally and non-naturally occurring nucleobases including those disclosed in U.S. Pat. No. 3,687,808, in Englisch et al., Angewandte Chemie, International Edition 30:613-722, 1991, and in WO 97/12896.
The probes and primers of invention may contain one or more nucleotide analogue monomers (e.g., LNA) in order to increase the sensitivity and specificity of the primers or probes to bind to the target sequence. For example, the probes and primers of the invention may contain LNA, such as oxy-LNA (e.g., beta-D-oxy-LNA). In the probe or the primer, two nucleotide analogues may be spaced 2, 3, or 4 nucleotides apart. Desirably, the probes of the invention may be modified to have stable modified nucleotides at the 5′ and 3′ ends of the primer or the probe with nucleotides having phosphodiester or phosphorothioate bonds in the center of the probe to enhance probe stability in a cellular environment (Kang et al., Nucleic Acids Res. 32:4411 (2004); and U.S. Patent Application No. 2005/0203042).
The primers and probes of the invention may also contain a modified backbone (e.g., a nucleotide backbone structure other than the naturally occurring ribose-phosphate or deoxyribose-phosphate backbone). Exemplary modified backbones include a ribonucleotide moiety that is substituted at the 2′-position. The substituents at the 2′-position may, for example, be a saturated, unsaturated, unbranched, or branched C1 to C4 alkyl group, e.g., 2′-O-methyl ribose. Another suitable example of a substituent at the 2′ position of a modified ribonucleotide moiety is a C1 to C4-alkoxy-C1 to C4 alkyl group. Another modification is a ribonucleotide that is substituted at the 2′-position with a fluoro group.
The probe or primers may also include a further 5′ and/or 3′amino group, a 5′ and/or 3′ ligand, and/or a 5′ and/or 3′ label, e.g., a fluorescent label (e.g., fluorescein), a radioactive label, or a label that is a complex including an enzyme (e.g., a complex containing digoxigenin). The probe or primer is for example 8 to 30 nucleotides long (e.g., 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 30 nucleotide, or 8 to 20 nucleotides). An attached ligand may be a drug or a ligand that can be bound by an antibody. Exemplary ligands include biotin, digoxigenin (DIG), and functional groups such as: aromatic groups, heteroaromatic groups, carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbizides, thiosemicar-bazides, aldehydes, ketones, primary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, C1-C20 alkyl groups optionally interrupted or terminated with one or more heteroatoms such as oxygen atoms, nitrogen atoms, and/or sulphur atoms, optionally containing aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-α-alanine, polyglycine, polylysine, peptides, oligo/polysaccharides, toxins, antibiotics, cell poisons, steroids, and affinity ligands, i.e., functional groups or biomolecules that have a specific affinity for sites on particular proteins, antibodies, poly- and oligosaccharides, and other biomolecules. One or more ligands that increase cell membrane permeability may also be included in the probes. Exemplary ligands that increase cell membrane permeability include lipophilic groups (e.g., sterols such as cholesterols, lanosterol, phytosterols, adamantols, fatty acids), peptides (e.g., cell penetrating peptides), charged moieties, functionalized alkyls, functionalized heteroalkyls, or other moieities assisting cellular uptake of oligonucleotides passively or actively.
Probes or primers of the invention may include single labels or a plurality of labels, e.g., a pair of labels that interact with each other either to produce a signal or to produce a change in a signal when hybridized to a target sequence. The probe or primer may include a fluorophore moiety and a quencher moiety, positioned in such a way that the hybridized state of the probe can be distinguished from the unhybridized state of the probe by an increase in the fluorescent signal from the probe or primer. The probe or primer may have, in addition to a sequence that hybridizes to a sequence in a target molecule, first and second complementary sequences which specifically hybridize to each other when the probe or primer is not hybridized to a sequence in a target molecule, bringing the quencher molecule in sufficient proximity to the report molecule to quench fluorescence of the reporter molecule. Hybridization of the target molecule distances the quencher from the fluorophore moiety and results in a signal proportional to the amount of hybridization.
Labels as used herein refer to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal and which can be attached to the probe or primer of the invention. Labels may provide signals detectable by fluorescence, radioactivity, colorimetric (UV/vis spectroscopy), X-ray diffraction or absorption, magnetism, enzymatic activity, and the like or adhere to complexes that generate such signals. The label may be detectable either by itself or as a part of a detection series. Non-limiting examples of functional parts of labels are biotin, digoxigenin (DIG), fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g., light or X-rays, of a certain wavelength, and which subsequently re-emits the energy absorbed as radiation of longer wavelength, e.g., DANSYL (5-dimethylamino)-1naphthalenesulfonyl), DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidone), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines, coumarins, Cy3, and Cy5 (trademarks for Biological Detection Systems, Inc.), erythrosine, coumaric acid, umbelliferone, Texas red, rhodamine, tetramethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene, fluorescein, Europium, Ruthenium, Samarium, rare earth metals, radioisotopic labels, chemiluminescence labels, and spin labels. Desirable labels include biotin, fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin, Ruthenium, Europium, Cy5, and Cy3.
Many synthetic approaches may be used to add the above moieties, ligands, and labels to probes and primers. The flexibility of the phosphoroamidite synthesis approach facilitates the production of probes and primers carrying all commercially available linkers, fluorophores, and labeling-molecules available for this standard chemistry. Probes and primers may also be labeled by enzymatic reactions, e.g., by phosphorylation using T4 polynucleotide kinase and gamma-32P-ATP or by using terminal deoxynucleotidyl transferase (DTT) and any given digoxigenin-conjugated nucleotide triphosphate (dNTP) or dideoxynucleotide triphosphate (ddNTP).
In a specific example, a mutation-specific probe, as described herein, includes an acceptor fluorophore quencher at one end of the probe and a donor fluorophore at the other end of the probe. In this example, the probe does not emit significant donor emission when hybridized to the target nucleic acid molecule (because the quence is in sufficient proximity to the donor to reduce fluorescence emission by the donor), and the donor emission increases when the hybridization complex is disrupted during melting. Therefore, presence of the target target nucleic acid (containing the ALK mutation) can be detected by forming a hybridization complex between a mutation-specific probe containing both a quencher and a donor fluorophore, then increasing the temperature sufficient to melt or disrupt the hybridization complex, and monitoring the melting curve to determine if a target mutation is present. When using such a mutation-specific probe, an anchor probe is not needed to practice the method of identifying a mutation.
The invention further provides compositions containing a mutation-specific probe and an anchor probe. In particular examples, the composition includes a first nucleic acid probe and a second nucleic acid probe, wherein each probe includes a different fluorophore, such as a FRET pair (for example, when one of the probes includes a FRET donor and the other includes a FRET acceptor). If desired, the end of the probe not including the fluorophore can be blocked to significantly decrease extension (for example, if the probes are included in an amplification reaction). One of the probes is referred to as a mutation-specific probe (first probe), the other is referred to as an anchor probe (second probe), which is generally longer than the mutation-specific probe. The probes recognize adjacent sequences in ALK or ALK-fusion nucleic acids (e.g., adjacent nucleic acid sequences encoding the kinase domain of an ALK or ALK-fusion nucleic acid). The first probe (mutation-specific probe) hybridizes to the nucleic acid sequence encoding one or mutations in the ALK or ALK-fusion protein. The anchor probe (second probe) hybridizes to a sequence downstream of the nucleic acid sequence encoding the one or more mutations recognized by the mutation-specific probe (first probe). For example, the probes can be separated by at least 1 nucleotide, such as 1-2 nucleotides. The mutation-specific probe can be designed so that a single base mismatch (such as a nucleotide substitution encoding for a mutation) will result in a melting temperature (Tm) shift of at least 3° C., such as 4° C., at least 6° C., or at least 8° C., such as 4-10° C.
Particular examples of mutation-specific probes are described herein and include probes having at least 90%, 95%, 98%, or 99% identity to a nucleotide sequence corresponding to a nucleic acid sequence that encodes the kinase domain of an ALK or ALK-fusion gene (nucleotides 4083 to 5795 of SEQ ID NO: 1 or nucleotides 3320 to 5032 of SEQ ID NO: 3), or the complement sequence thereof. The anchor probe (second probe) may contain a donor fluorophore (thereby permitting detection of the change in acceptor fluorophore emission during melting) or may contain an acceptor fluorophore (thereby permitting detection of the change in acceptor fluorophore emission on the anchor probe during melting, wherein a decrease in acceptor fluorophore emission indicates that the mutation-specific probe is no longer hybridized to the target nucleic acid). Non-limiting examples of mutation-specific probes and anchor probes that hybridize to the antisense sequence of a nucleic acid encoding a codon corresponding to nucleotides 4497 to 4499 of SEQ ID NO: 1 or 3734 to 3736 of SEQ ID NO: 3 are: ggttcatcctgatggagctcatg (SEQ ID NO: 9)/cggggggagacctcaagtccttcctcc (SEQ ID NO: 10); gttcatcctgatggagctcatgg (SEQ ID NO: 11)/ggggggagacctcaagtccttcctccg (SEQ ID NO: 12); ttcatcctgatggagctcatggc (SEQ ID NO: 13)/gggggagacctcaagtccttcctccga (SEQ ID NO: 14); cggttcatcctgatggagctcat (SEQ ID NO: 15)/gcggggggagacctcaagtccttcctc (SEQ ID NO: 16); ccggttcatcctgatggagctca (SEQ ID NO: 17)/ggcggggggagacctcaagtccttcct (SEQ ID NO: 18); ggttcatcctgatggagctcatg (SEQ ID NO: 19)/ggggggagacctcaagtccttcctccg (SEQ ID NO: 20); and cggttcatcctgatggagctcat (SEQ ID NO: 21)/cggggggagacctcaagtccttcctcc (SEQ ID NO: 22).
The probe sequence can be varied slightly by moving the probes a few nucleotides upstream or downstream from the nucleotide positions that hybridize in the target ALK or ALK-fusion nucleic acid. However, the mutation-specific probe will target the one or more codons corresponding the mutant amino acids in ALK (SEQ ID NOS: 2 or 4) described above.
For example, the acceptor fluorophore can be attached to the 3′-end of the mutation-specific probe (first probe) and the donor fluorophore can be attached to the 3′-end of the anchor probe. Appropriate donor/acceptor fluorophore pairs can be selected using routine methods. In one example, the donor emission wavelength is one that can significantly excite the acceptor, thereby generating a detectable emission from the acceptor. Non-limiting examples of donor/acceptor FRET pairs include fluorescein as the donor and LightCycler Red 640 (LC-Red 640), LC-Red 705, JA286, Cy5, or Cy5.5 as the acceptor.
The acceptor fluorophore can be a dark quencher that significantly decreases the detectable emission when in sufficient proximity to the donor fluorophore. With this approach, when the mutation-specific probe and the anchor probe are hybridized to the target ALK or ALK-fusion nucleic acid, no significant donor emission signal is detected. However, during melting, when the probes are released from the target ALK or ALK-fusion nucleic acid, the emission signal from the donor increases, because it is no longer quenched by the dark quencher. Exemplary dark quenchers include Dabcyl, QSY7, QSY33, BLACK HOLE QUENCHERSTM, ECLIPSETM, and IOWA BLACKTM.
The probes of the invention may be covalently bonded to a solid support, e.g., by reaction of a nucleoside phorphoramidite with an activated solid support, and subsequent reaction of a nucleoside phosphoramide with an activated nucleotide or nucleic acid bound to the solid support. Preferably, the solid support or the oligonucleotide probe bound to the solid support is activated by illumination, a photogenerated acid, or electric current. The oligonucleotide probe may contain a spacer, e.g., a randomized nucleotide sequence or a non-base sequence, such as hexaethylene glycol, between the reactive group and the rest of the oligonucleotide probe. Such covalently bonded oligonucleotide probes are highly useful for large-scale detection and expression profiling of mRNA expression. The probes may also have a photochemically active group, a thermochemically active group, a chelating group, a reporter group, or a ligand that facilitates the direct or indirect detection of the probe or the immobilization of the probe onto a solid support. The photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand may include a spacer having a chemically cleavable group; or the thermochemically active group, the chelating group, the reporter group, or the ligand may be attached via the biradical of at least one of the nucleotide analogues of the probe.
The solid support may contain a material, e.g., selected from borosilicate glass, soda-lime glass, polysterene, polycarbonate, polypropylene, polyethylene, polyethylene-glycol terephthalate, polyvinylacetate, polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride, preferably polystyrene and polycarbonate. The solid support may be in the form of a specimen tube, a vial, a slide, a sheet, a film, a bead, a disc, a plate, a ring, a rod, a net, a filter, a tray, a microtiter plate, a stick, or a multi-bladed stick.
One or more probes or primers of the invention may be assembled in a kit with packaging and/or labeling to provide instructions for the identification of a mutation in an ALK gene, an ALK-fusion gene, or an mRNA encoding an ALK protein or an ALK-fusion protein. The kits provided by the invention may be used to isolate, purify, amplify, detect, identify, quantify, or capture a nucleic acid encoding an ALK gene, an ALK-fusion gene, or an mRNA encoding an ALK protein or an ALK-fusion protein. Desirably, the kits of the invention contain one or more probes and primers that specifically amplify or identify a contingous sequence in a nucleic acid (e.g., mRNA) encoding an ALK protein or an ALK fusion protein that encodes one or more of the amino acids corresponding to: the amino acid at position 1151, the amino acid at position 1152, the amino acid at position 1156, the amino acid at position 1171, the amino acid at position 1174, the amino acid at position 1180, the amino acid at position 1181, the amino acid at position 1196, the amino acid at position 1198, the amino acid at position 1202, the amino acid at position 1206, the amino acid at position 1210, the amino acid at position 1241, the amino acid at position 1268, and the amino acid at position 1269 of SEQ ID NO: 2 or SEQ ID NO: 4 (as described above), or the complementary sequence thereof.
Kits of the invention can include a pair of probes, such as a first probe (mutation-specific probe) and a second probe (anchor probe) (as described above), wherein the probes hybridize to adjacent regions in an ALK or an ALK-fusion nucleic acid. In particular examples, the probes are provided in separate vessels.
Kits of the invention can further include at least one other component, such as a component that can be used to amplify the target ALK or ALK-fusion nucleic acid or an ALK or ALK-fusion nucleic acid (control). Exemplary components include a pair of nucleic amplification primers (e.g., mutation-specific probe and an anchor probe), a polymerizing agent (e.g., a thermostable DNA polymerase), deoxynucleoside triphosphates, a buffer suitable for use in a nucleic acid amplification reaction, or combinations thereof. Particular examples of primers and probes that hybridize to or amplify an ALK or ALK-fusion nucleic acid encoding a mutant ALK or ALK-fusion protein are described above.
Once a mutation is identified using the methods of the invention (as is described above), the mutation is analyzed for association with an increased risk of developing a proliferative disorder. In this respect, the present invention provides a method of detecting the presence of a mutation in the human ALK gene in a biological sample.
Levels of ALK expression (e.g., DNA, mRNA, or protein) in a biological sample can be determined by using any of a number of standard techniques that are well known in the art or described herein. Exemplary biological samples include plasma, blood, sputum, pleural effusion, bronchoalveolar lavage, or biopsy, such as a lung biopsy and lymph node biopsy. For example, ALK expression in a biological sample (e.g., a blood or tissue sample) from a patient can be monitored by standard northern blot analysis or by quantitative PCR (see, e.g., Ausubel et al., supra; PCR Technology: Principles and Applications for DNA Amplification, H.A. Ehrlich, Ed., Stockton Press, NY; Yap et al., Nucl. Acids. Res. 19:4294 (1991)).
Mismatch detection assays also provide an opportunity to diagnose an ALK-mediated predisposition to a disease before the onset of symptoms. For example, a patient with an ALK mutation that suppresses normal ALK biological activity or expression may show no clinical symptoms of an ALK-related disease, and yet possess a higher than normal probability of developing a cancer. Given such a diagnosis, patients can take precautions to minimize their exposure to adverse environmental factors and to carefully monitor their medical condition (for example, through frequent physical examinations). As mentioned above, this type of diagnostic approach can also be used to detect ALK mutations in mutagenesis screens.
The ALK diagnostic assays described above can be carried out using any biological sample (for example, a blood, sputum, or tissue sample) in which ALK is normally expressed. Identification of a mutant ALK gene can also be assayed using these sources for test samples. Alternatively, an ALK mutation, particularly as part of a diagnosis for predisposition to an ALK-associated disease, can be tested for using a DNA sample from any cell, for example, by mismatch detection techniques. Preferably, the DNA sample is subjected to PCR amplification prior to analysis.
In yet another diagnostic approach of the invention, an immunoassay is used to detect or monitor ALK protein expression in a biological sample. ALK-specific polyclonal or monoclonal antibodies (produced as described below) can be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA; see, e.g., Ausubel et al., supra) to measure ALK or ALK fusion protein polypeptide levels. These levels are compared to wild-type ALK levels. For example, an increase in ALK production may be indicative of a condition or a predisposition to a condition involving increased ALK biological activity. Exemplary antibodies to detect ALK include highly specific monoclonal antibodies (see, e.g., Mino-Kenudson et al., Clin. Cancer Res. 16:1561 (2010), incorporated herein by reference); ALK123, ALKc22, Clone 5A4 anti-ALK antibodies (Thermo Fisher Scientific, Fremont Calif.); and rabbit mAb ALK/p80 (clone SP8; Thermo Fisher Scientific) Immunohistochemical techniques can also be utilized for ALK detection. For example, a tissue sample can be obtained from a patient, sectioned, and stained for the presence of ALK using an anti-ALK antibody (see below) and any standard detection system (e.g., one that includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in, e.g., Bancroft et al., Theory and Practice of Histological Techniques, Churchill Livingstone, 1982, and Ausubel et al., supra.
In a preferred example, a combined diagnostic method can be employed that includes an evaluation of ALK protein production (for example, by immunological techniques or the protein truncation test (Hogerrorst et al., Nat. Genet. 10:208 (1995)), and a nucleic acid molecule-based detection technique designed to identify more subtle ALK mutations (for example, point mutations). As described above, a number of mismatch detection assays are available to those skilled in the art, and any preferred technique can be used. Mutations in ALK can be detected that either result in loss of ALK expression or loss of normal ALK biological activity.
Furthermore, antibodies against a protein produced by the gene included in the mutation, for example the ALK protein. Antibodies may be used to detect altered expression levels of the protein, including a lack of expression, or a change in its mobility on a gel, indicating a change in structure or size. In addition, antibodies may be used for detecting an alteration in the expression pattern or the sub-cellular localization of the protein. Such antibodies include ones that recognize both the wild-type and mutant protein, as well as ones that are specific for either the wild-type or an altered form of the protein, for example, one encoded by a polymorphic ALK gene. Monoclonal antibodies may be prepared using the ALK or ALK fusion proteins described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York, N.Y., 1981; Ausubel et al., supra). Once produced, monoclonal antibodies are also tested for specific ALK protein recognition by Western blot or immunoprecipitation analysis (by the methods described in, for example, Ausubel et al., supra).
Antibodies used in the methods of the invention may be produced using amino acid sequences that do not reside within highly conserved regions, and that appear likely to be antigenic, as analyzed by criteria such as those provided by the Peptide Structure Program (Genetics Computer Group Sequence Analysis Package, Program Manual for the GCG Package, Version 7, 1991) using the algorithm of Jameson and Wolf (CABIOS 4:181 (1988)). These fragments can be generated by standard techniques, e.g., by the PCR, and cloned into the pGEX expression vector (Ausubel et al., supra). GST fusion proteins are expressed in E. coli and purified using a glutathione agarose affinity matrix as described in Ausubel et al. supra).
Synthesis
Compounds of formula I can be prepared using known methods and materials, e.g., as disclosed in detail in International patent applications WO 2004/080980, WO 2005/016894, WO 2006/021454, WO 2006/021457, WO 2009/143389, and WO 2009/126515. For instance, compounds of formula I in which Re is H and Rd is H, Cl, CF3, or CH3, can be synthesized from 2,4-dichloropyrimidine, 2,4,5-trichloropyrimidine, 2,4-dichloro-5-(trifluoromethyl)pyrimidine, or 2,4-dichloro-5-methylpyrimidine, respectively, as described in PCT Publication No. WO/2009/143389 (see, for example, Schemes 1A and 1B below).




Compounds of formula I in which Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one or two heteroatoms can be synthesized as described in PCT Publication No. WO2009/126515. See, for example, Scheme 2, which describes the synthesis of a starting material from which compounds of formula (I) can be synthesized. In Scheme 2, X is CH3 or H.




A detailed description for the synthesis of compound 1 is provided in Scheme 3.




Synthesis of A:




To a solution of 2-iodoaniline (1.0 eq) and dimethylphosphine oxide (1.1 eq) in DMF were added potassium phosphate (1.1 eq), palladium acetate/Xantphos (catalytic). The reaction was stirred at 150° C. for 3 hours and cooled to room temperature. The solvent was evaporated and the residue was worked up with DCM/water. The crude product was purified with a column (EtOAc/MeOH 10:1) to give A as a brown solid (80% yield).
Synthesis of B:




2,4,5-Trichloropyrimidine (1.57 eq), A (1.0 eq), and potassium carbonate (3.14 eq) in DMF were stirred at 60° C. for 5 hours and then cooled to r.t. The mixture was filtered and the filtrate was concentrated. The residue was purified with ISCO (DCM/MeOH 20:1) to give B as a yellow solid (61% yield).
Synthesis of C:




5-Fluoro-2-nitroanisole (1.0 eq), 1-methyl-4-(piperidin-4-yl)piperazine (1.0 eq), and potassium carbonate (2.0 eq) in DMF were stirred at 120° C. for 6 hours and then cooled to r.t. The mixture was filtered and evaporated. The crude product was crystallized from ethanol to give C as a yellow solid (72% yield).
Synthesis of D:




Palladium on activated carbon was added to a solution of C in ethanol under nitrogen. The suspension was then shaken under hydrogen (50 psi) for 3 hours. The mixture was filtered and the filtration was evaporated to give D as a purple solid in a quantitative yield.
Synthesis of compound 1:




A solution of B (1.0 eq), D (1.4 eq), and 2.5 M HCl in ethanol (excess) in 2-methoxyethanol was sealed and heated at 120° C. with stirring for 5.5 hours and then cooled to r.t. The reaction was repeated 5 times and combined. The mixture was filtered and evaporated. Saturated Na2CO3 was added, followed by DCM with stirring strongly. The layers were separated and the aqueous layer was extracted with DCM. The organics were dried, evaporated and chromatographed [EtOAc/MeOH (7M ammonia) 20:1] to give a yellow solid. EtOAc was added and the suspension was refluxed for 30 minutes. After cooled to room temperature, filtration gave a solid, which was dissolved in DCM, filtered, and evaporated to afford compound 1 as an off-white solid (66% yield).
Formulation
Compounds of formula (I) can be formulated for any route of administration (e.g., orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by transdermal patch, powders, ointments, or drops), sublingually, bucally, as an oral or nasal spray) effective for use in the methods of the invention. For use in the methods of the invention, compounds of formula (I) are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. For example, a compound of formula (I) can be formulated for as a capsule for oral administration containing nominally 10 mg, 50 mg, 100 mg, 150 mg, 250 mg, 500 mg, or any dosage amounts described herein as the free base or acid addition salt of compound 1 (e.g., the hydrochloride salt). The unit dosage forms of the invention can include compound 1, or a salt thereof, formulated with fillers, flow enhancers, lubricants, and/or disintegrants as needed. For example, a unit dosage form can include colloidal silicon dioxide (a flow enhancer), lactose anhydrous (a filler), magnesium stearate (a lubricant), microcrystalline cellulose (a filler), and/or sodium starch glycolate (a disintegrant). Compound 1 and the inactive ingredients can be formulated utilizing, for example, conventional blending, and encapsulation processes. Alternatively, compounds of formula (I) are formulated as described in PCT Publication Nos. WO2009/143389 and WO2009/126515.
Therapy
Compounds of formula (I) can be useful for treating ALK-driven cancers. In particular, the compounds can be useful for treating ALK-driven cancers that express ALK mutants and for treating ALK-driven cancers that are refractory to one or more of crizotinib, CH5424802 and ASP3026.
Such cancers can include, among others, cancers of the breast, non small cell lung cancer (NSCLS), neural tumors such as glioblastomas and neuroblastomas; esophaegeal carcinomas, soft tissue cancers such as rhabdomyosarcomas, among others); various forms of lymphoma such as a non-Hodgkin's lymphoma (NHL) known as anaplastic large-cell lymphoma (ALCL), various forms of leukemia; and including cancers which are ALK mediated.
In common with other RTKs, translocations affect the ALK gene, resulting in expression of oncogenic fusion kinases—the most common of which is NPM-ALK. For example, approximately sixty percent of anaplastic large cell lymphomas (ALCL) are associated with a chromosome mutation that generates a fusion protein consisting of nucleophosmin (NMP) and the intracellular domain of ALK. (Armitage et al., Cancer: principle and practice of oncology, 6th Edition, 2001, 2256-2316; Kutok et al., J. Clin. Oncol. 20:3691 (2002); and Wan et al., Blood 107:1617 (2006)). This mutant protein, NMP-ALK, possesses a constitutively active tyrosine kinase domain that is responsible for its oncogenic property through activation of downstream effectors (Falini et al., Blood, 94:3509 (1999); and Morris et al., Brit. J. Haematol. 113:275 (2001)). Experimental data have demonstrated that the aberrant expression of constitutuvely active ALK is directly implicated in the pathogenesis of ALCL and that inhibition of ALK can markedly impair the growth of ALK positive lymphoma cells (Kuefer et al., Blood 90:2901 (1997); Bai et al., Exp. Hematol. 29:1082 (2001); Slupianek et al., Cancer Res. 61:2194 (2001); and Turturro et al., Clin. Cancer Res. 8:240 (2002)). The constitutively activated chimeric ALK has also been demonstrated in about 60% of inflammatory myofibroblastic tumors (IMTs), a slow growing sarcoma that mainly affects children and young adults (Lawrence et al., Am. J. Pathol. 157:377 (2000)). Furthermore, recent reports have also described the occurrence of a variant ALK fusion, TPM4-ALK, in cases of squamous cell carcinoma (SCC) of the esophagus (Jazzi et al., World J. Gastroenterol. 12:7104 (2006); Du et al., J. Mol. Med. 85:863 (2007); and Aklilu M., Semin. Radiat. Oncol. 17:62 (2007)). Thus, ALK is one of the few examples of an RTK implicated in oncogenesis in both non-hematopoietic and hematopoietic malignancies. More recently it has been shown that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells (Soda et al., Nature 448:561 (2007)).
The present invention is based in part upon the discovery that ALK-driven tumors can develop kinase domain mutations, which impair inhibitor binding. The present invention is also based upon the discovery that compounds of formula (I) can be used to treat ALK-driven cancers that express ALK mutants and for treating ALK-driven cancers that are refractory to one or more of crizotinib, CH5424802 and ASP3026. Compounds of formula (I) can also be used in a maintenance role to prevent recurrence of cancer in patients in need of such a treatment.
The effective systemic dose of a compound of formula (I) will typically be in the range of an average daily dose of from 10 mg to 2,000 mg of compound 1 per kg of patient body weight, administered in single or multiple doses. Generally, compound 1 may be administered to patients in need of such treatment in a daily dose range of about 50 to about 2,000 mg per patient. Administration may be once or multiple times daily, weekly (or at some other multiple-day interval) or on an intermittent schedule. For example, the compound may be administered one or more times per day on a weekly basis (e.g. every Monday) indefinitely or for a period of weeks, e.g. 4-10 weeks. Alternatively, it may be administered daily for a period of days (e.g. 2-10 days) followed by a period of days (e.g. 1-30 days) without administration of the compound, with that cycle repeated indefinitely or for a given number of repetitions, e.g. 4-10 cycles. As an example, a compound of the invention may be administered daily for 5 days, then discontinued for 9 days, then administered daily for another 5 day period, then discontinued for 9 days, and so on, repeating the cycle indefinitely, or for a total of 4-10 times.
When one or more of crizotinib, CH5424802 and ASP3026 is used in combination with a compound of formula (I), each component of the combination therapy may be administered at their monotherapy dosing levels and schedules. For example, crizotinib has been administered orally for the treatment of NSCLC at 250 mg BID.
The effective systemic dose of compound 1 will typically be in the range of an average daily dose of from 10 mg to 2,000 mg of compound 1 per kg of patient body weight, administered in single or multiple doses. Generally, compound 1 may be administered to patients in need of such treatment in a daily dose range of about 50 to about 2,000 mg per patient. Administration may be once or multiple times daily, weekly (or at some other multiple-day interval) or on an intermittent schedule. For example, the compound may be administered one or more times per day on a weekly basis (e.g. every Monday) indefinitely or for a period of weeks, e.g. 4-10 weeks. Alternatively, it may be administered daily for a period of days (e.g. 2-10 days) followed by a period of days (e.g. 1-30 days) without administration of the compound, with that cycle repeated indefinitely or for a given number of repetitions, e.g. 4-10 cycles. As an example, a compound of the invention may be administered daily for 5 days, then discontinued for 9 days, then administered daily for another 5 day period, then discontinued for 9 days, and so on, repeating the cycle indefinitely, or for a total of 4-10 times.
Alternatively, one or more of crizotinib, CH5424802 and ASP3026 is used in combination with a compound of formula (I) with a reduced dosing level in one or both components.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.
Experimental
Reagents: Compound 1 was synthesized by ARIAD Pharmaceuticals. Crizotinib was either synthesized by ARIAD Pharmaceuticals or purchased from Wuxi Howfond Biopharma, Co., Ltd., a chemical CRO company.
In vitro mutagenesis screen: Ba/F3 cells expressing native EML4-ALK were treated overnight with 100 μg/ml N-ethyl-N-nitrosourea (ENU, Sigma-Aldrich), pelleted, resuspended in fresh media, and distributed into 96-well plates at a density of 5*104 cells/well in 200 μl media supplemented with graded concentrations of Compound 1 or crizotinib. The wells were observed for media color change and cell growth twice a week throughout the 28-35 day experiment. The content of outgrown wells were transferred to 24-well plates and expanded in media supplemented with Compound 1 or crizotinib at the same concentration as in the initial 96-well plate. At confluence, cells in 24-well plate were collected by centrifuge for DNA extraction and for further characterization. Genomic DNA was extracted from cell pellets using DNEasy 96 Blood & Tissue kit (Qiagen). The ALK kinase region was amplified using primer:

    
    
        5′-AGCGATGCAGATGGAATTGCAGAG-3′ and
        5′-CAATAGGCAGCGCCGTGTTGATTA-3′.

PCR products were purified by QIAquick PCR purification kit (Qiagen) and sequenced by MGH DNA sequencing Core.

    
    


Mapping mutation sites onto model of inhibitor bind ALK: A homology model of ALK was built and optimized based on the crystal structure of activated insulin kinase (PDB code: 3irk) with Prime (Schrodinger software package). Crizotinib was docked into ALK by using the Glide SP procedure with refinement option (Schrodinger) and the top scored pose was chosen for further analysis.
Re-introduce mutation into Ba/F3 cells: pMSCVneo plasmid encoding EML4-ALK mutations were generated with QuickChange site-directed mutagenesis kit (Stratagene) according to manufacturer's instruction using pMSCVneo encoding native EML4-ALK as the template. Each mutation was confirmed by DNA sequencing. Ba/F3 cells expressing EML4-ALK mutants were generated by infecting Ba/F3 parental cells (supplemented with IL-3 in media) with retrovirus encoding EML4-ALK mutations followed by selection with G418 and cultured in the absence of IL-3.
In vitro viability assay: Ba/F3 parental and Ba/F3 cells expressing EML4-ALK either from ENU-isolated clones or from re-introduced lines were plated in 96-well plates and treated with compounds for 72 h. Cell viability (IC50) was assessed using CellTiter 96 AQueous One (Promega) and plotted as percent viable relative to vehicle treated cells using XLfit.
Inhibition of ALK phosphorylation: EML4-ALK expressing Ba/F3 cells either from ENU-isolated clones or from re-introduced lines were treated with increasing concentrations of Compound 1 or crizotinib for 2 hours. Cell lysates were prepared and loaded onto PathScan Sandwich ELISA kit (Cell Signaling) for p-ALK and for total ALK. IC50 was determined as the concentration of compound required to decrease ALK phosphorylation by 50% comparing to vehicle control.
In vivo efficacy: Ba/F3 cells expressing native or mutant EML4-ALK were implanted into the right flank of SCID Beige mice (10×106 cells/mice). When the average tumor volume reached  ̃200 mm3, either vehicle or compound was administered by once daily oral gavage for 8-12 days. Mean tumor volume (±SE) was then calculated for each group.
Pharmacodynamics/Pharmacokinetics: For pharmacodynamic analysis, 6 or 24 hours after treating tumor-bearing mice with a single dose of vehicle or compound, tumors were collected, homogenized, and analyzed by ELISA. Inhibitor concentrations in plasma were determined by an LC/MS/MS method. Calibration standards were prepared in blank mouse plasma. Internal standards were added to all plasma samples and the samples were deproteinized before analysis.
Example 1
EML-4-ALK Mutants Recovered by Accelerated In Vitro Mutagenesis
FIG. 1 provides a schematic illustration of accelerated in vitro mutagenesis. Details of this protocol are provided in the Experimental section above. EML4-ALK mutants were recovered in the presence of inhibitors, either Compound 1 or crizotinib. FIGS. 2A-2B provide the frequency among recovered clones for crizotinib (FIG. 2A) and for Compound 1 (FIG. 2B). Mutagenesis was repeated 2 additional times with similar results. With respect to Compound 1, one additional mutation (D1203N) was observed at 100 nM
Compound 1 completely suppressed the emergence of resistance at 1000 nM, where two mutants were observed for crizotinib at 1440 nM. FIG. 3 is a model of crizotinib bound to ALK. Various groups are provided and mutation sites are provided, based on the interaction of crizotinib with ALK.
Example 2
In Vitro Inhibition of EML-4-ALK Mutants
Compound 1 and crizotinib were assessed by using cellular proliferation assays. Table 1 shows IC50 values for Ba/F3 cellular proliferation assays. FIG. 4A shows a representative proliferation curve for crizotinib with different EML-4-ALK mutants. FIG. 4B shows a representative proliferation curve for Compound 1.







TABLE 1







IC50 values for Ba/F3 cellular proliferation assays










Compound 2
Compound 1














Ba/F3 line
IC50s (nM)
TI**
IC50s (nM)
TI**





Parental*
1210 ± 334 
1
2189 ± 410 
1


Nat veEML4-ALK
139 ± 30  
9
11 ± 4 
205
















EML4-ALK Mutants:
ENU-clone
Re-introduced

ENU-clone
Re-introduced






11171T
395 ± 199
413 ± 62 
3
22 ± 11
18 ± 4 
102


F1 174C
478 ± 31 
319 ± 90  
3
68 ± 31
51 ± 14
32


L1196M
1131 ± 296 
1215 ± 708 
1
45 ± 23
40 ± 19
49


S1206R
711 ± 250
728 ± 362
2
194 ± 84 
158 ± 86 
1


E1210K
315 ± 124
297 ± 92 
4
64 ± 33
86 ± 21
34


F1245C
41 ± 90
269 ± 194
3
44 ± 29
34 ± 28
50


G1269S
1141 ± 527 
1196 ± 649 
1
16 ± 6 
16 ± 5 
141





*Parental Ba/F3 cells lack EML4-ALK and therefore require IL-3 for growth.


**Therapeutic Index (TI) = Parental IC50 / EML4-ALK IC50.


(n = 5)






Compound 1 was also evaluated against crizotinib, ASP3026, and CH5424802 in a similar cellular proliferation assay, in this case using Ba/F3 cells expressing a different set of EML4-ALK mutations. In this study, the mutations are resistance-conferring ALK mutations identified by mutagenesis screening and/or from clinical samples of crizotinib-resistant cancers. The results are provided in Table 2.







TABLE 2







IC50 (nM) values for Ba/F3 cellular proliferation assays











Ba/F3 cell line
Compound 1
crizotinib
ASP3026
CH5424802





Ba/F3 parental
4580 ± 1553
2065 ± 373 
>10000 ± 0    
>10000 ± 0    


EML4-ALK
17 ± 4 
137 ± 49 
129 ± 021
15 ± 8 


1151 T insertion1*
244 ± 16 
1336 ± 4902
5939 ± 2278
455 ± 143


L1152R*
17 ± 5 
911 ± 136
5934 ± 2156
105 ± 35 


C1156Y*
150 ± 9  
2323 ± 117 
2555 ± 86  
194 ± 39 


F1174L*
95 ± 5 
449 ± 117
978 ± 132
81 ± 4 


L1196M*
99 ± 21
870 ± 143
1902 ± 448 
220 ± 49 


G1202R*
379 ± 68 
874 ± 53 
1856 ± 550 
1009 ± 137 


D1203N*
144 ± 8  
693 ± 51 
854 ± 164
85 ± 17


S1206Y*
88 ± 3 
509 ± 85 
518 ± 74 
58 ± 11


G1269A*
24 ± 4 
798 ± 62 
264 ± 65 
103 ± 15 


S1206A
120 ± 29 
391 ± 21 
1679 ± 556 
150 ± 23 


F1174V
107 ± 37 
347 ± 77 
1662 ± 141 
102 ± 60 


11171S
61 ± 8 
348 ± 27 
948 ± 60 
1132 ± 313 


11171N
123 ± 29 
586 ± 63 
1201 ± 19  
>10000


V1180L
19 ± 4 
201 ± 6  
183 ± 13 
>10000





1Insertion of a threonine at position 1151. Mutations that were identified clinically as conferring resistance or were so identified by screening and later confirmed clinically are marked with *.






In addition to the mutations already known to confer crizotinib-resistance clinically, a number of mutations were observed that could also lead to tumors resistant to treatment with ASP3026 or CH5424802 (see FIG. 12). For example, the I1171N, I1171S, and V1180L mutants are particularly resistant to CH5424802. Importantly, in the case of compound 1, no ALK mutation, not even the 1151 T insertion, raises the IC50 value for compound 1 beyond a trough concentrations achievable with clinically relevant doses of compound 1.
Compound 1 also inhibited ALK phosphorylation in Ba/F3 cell lines expressing the EML4-ALK mutations. Table 3 shows ALK phosphorylation in cell lysates from cell lines exposed to Compound 1 or crizotinib.







TABLE 3







ALK phosphorylation in Ba/F3 cell lines










Crizotinib
Compound 1





EML4-ALK
IC50s (nM)
IC50s (nM)





Native
74 ± 48
5.3 ± 3.8














Mutants:
ENU-clone
Re-introduced
ENU-clone
Re-introduced





F1174C
696 ± 514

12 ± 4 



L1196M
1291 ± 704 
817 ± 324
15 ± 8 
7.6 ± 0.2


S1206R
1012 ± 347 
172 ± 71 
170 ± 135
17 ± 2 


E1210K
335 ± 106

76 ± 25



F1245C
364 ± 223

7 ± 4



G1269S
1353 ± 733 
1005 ± 24 
17 ± 5 
8.6 ± 0.1





(n = 2 or 3)






Example 3
In Vivo Inhibition of EML-4-ALK Mutants
In vivo experiments for pharmacodynamics/pharmacokinetics were also conducted. FIG. 5A shows in vivo experiments for ALK phosphorylation in EML4-ALK mutant tumors in vivo, as well as inhibitors concentrations in plasma. As shown in FIG. 5B, Compound 1 inhibited ALK phosphorylation in EML4-ALK mutant tumors in vivo.
Xenograft models were also used to determine efficacy of Compound 1 and crizotinib. FIGS. 6A-6D shows tumor volume after treatment with crizotinib for native EML4-ALK (FIG. 6A) and for the L1196M (FIG. 6B), S1206R (FIG. 6C), and G1269S (FIG. 6D) mutants.
Compound 1 is efficacious in crizotinib-resistant EML4-ALK mutant mouse xenograft models. FIGS. 7A-7D shows tumor volume after treatment with Compound 1 for native EML4-ALK (FIG. 7A) and for the L1196M (FIG. 7B), S1206R (FIG. 7C), and G1269S (FIG. 7D) mutants.
Summary
Eighteen different mutants were recovered in the crizotinib screen. Eight mutants were common to both Compound 1 and crizotinib. Eight were unique for crizotinib.
For crizotinib, resistant mutations were identified at concentrations up to 1440 nM, which inhibited >90% parental Ba/F3 cell proliferation. The most frequently occurring mutations all conferred certain degree of resistance with IC50s of 311-1419 nM in the viability assay. Three mutations conferred strongest resistance: L1196M (gate keeper), S1206R, and G1269S. The results from in vitro experiments were indistinguishable from parental Ba/F3 in cell viability assays (711-1141 nM). In vivo results provided that crizotinib was completely inefficacious in xenograph mouse model, even at a dosing of 200 mg/kg. Other mutations (I1171T, F1174C, G1210K and F1245C) resulted in in vitro IC50 values of 315-478 nM.
For Compound 1, ten mutants were recovered in the screen. Two mutants (G1202S and D1203) were unique for Compound 1. These mutants were rare and only appeared at low concentrations. Four mutations overlapped with ALK mutations detected in neuroblastoma patients: T1151, 11171, F1174, and F1245.
Compound 1 was able to overcome all mutations that confer resistance to crizotinib, ASP3026 or CH542802. Compound 1 completely suppressed the emergence of resistance at 1000 nM, and only one mutation (S1206R) recovered at 500 nM. Compound 1 also remained active against three mutants that conferred complete resistance to crizotinib. The results from in vitro experiments provided IC50s ranging from 23-269 nM in cell viability assays, accompanied by inhibition of ALK phosphorylation. In vitro data for L1196M and G1269S included IC50 values of 45 nM and 16 nM. For other mutations (I1171T, F1174C, G1210K and F1245C), in vitro IC50 values were <70 nM. For 51206R, potent in vitro (IC50: 194 nM) and in vivo (75 mg/kg) activity was observed against mutant that confers greatest resistance to Compound 1.
Pharmacokinetic/pharmacodynamic data revealed that Compound 1 sustained p-ALK inhibition (80-90% for L1196M and G1269S, 40-60% for S1206R) for 24 hrs after single dose of 50 mg/kg. In vivo results provided complete tumor regression at 50 and 75 mg/kg dosing of Compound 1 for the L1196M and G1269S mutants, and tumor growth inhibition at 75 mg/kg for the S1206R mutant.
The crizotinib screen was extended using CH5424802 and ASP-3026 to identify additional mutations, including among others amino acid substitutions for S1136, T1151, I1268, I1171, F1174, V1180, L1196, S1206, F1245, I1268, and G1269. Specific mutations identified included among others, I1171N, I1171S, I1171T, G1269C, G1269S, L1196M, F1174{C, I, V and L}, and others. As described above, compounds of Formula I, and in particular, Compound 1, retain potency against the various mutant ALK proteins, even in cases of profound resistance of the mutant to crizotinib, CH5424802 or ASP-3026.
Conclusion
Based on the results disclosed herein, and the validation of crizotinib-resistant mutations in the clinical setting, a reasonable prediction is that resistance will arise in the clinic based on its modest potency, narrow therapeutic window (or therapeutic index), and reported clinical achievable plasma level (median Ctrough: ̃225 ng/ml at MTD). In contrast, Compound 1 appears to be a more robust ALK inhibitor, may to provide a more complete clinical response and may suppress the emergence of resistance more effectively than inhibitors of wild-type ALK such as crizotinib, CH5424802 or ASP-3026.
Example 4
Inhibition of Crizotinib-Resistant Tumors with Compound 1 In Vivo
Xenograft models were also used to determine efficacy of compound 1 against crizotinib-resistant tumors (L1196M mutant-driven tumors). The results are depicted in FIG. 11A (showing response to therapy with compound 1) and 11B (showing no response to therapy with crizotinib).
Tumor growth regression was observed in animals treated with compound 1.
OTHER EMBODIMENTS
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
Other embodiments are within the claims.

## Claims

1. A method for treating an ALK-driven cancer in a subject comprising the steps of:
a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK), wherein said mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for isoleucine to threonine at amino acid position 1171; (ii) a substitution for phenylalanine to cysteine at amino acid position 1174; (iii) a substitution for leucine to methionine at amino acid position 1196; (iv) a substitution for serine with arginine at amino acid position 1206; (v) a substitution for glutamic acid to lysine at amino acid position 1210; (vi) a substitution for phenylalanine to cysteine at amino acid position 1245; (vii) a substitution for glycine to serine at amino acid position 1269; and (viii) a substitution for valine to leucine at amino acid position 1180; and
b) administering to said subject a therapeutically effective amount of compound of formula (I), or a pharmaceutically acceptable salt thereof:




wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two or three heteroatoms, independently selected from N, S and O, wherein the 5- or 6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2,


—SO2NR1R2, —NR1SO2NR1R2, or



each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl.

2. The method of claim 1, wherein said ALK is full-length ALK.

3. The method of claim 1, wherein said ALK is an ALK-fusion product.

4. A method for treating an ALK-driven cancer expressing an ALK-fusion protein in a subject comprising the steps of:
a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in said ALK-fusion protein, wherein said mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for phenylalanine with valine at amino acid position 1174; and (ii) a substitution for tyrosine with serine at amino acid position 1278; and
b) administering to said subject a therapeutically effective amount of compound of formula (I), or a pharmaceutically acceptable salt thereof:




wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two, or three heteroatoms, independently selected from N, S and O, wherein the 5- or
6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B, is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl.

5. A method of inhibiting the proliferation of a cell expressing an ALK mutant, wherein said ALK mutant is characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK) selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for threonine to lysine at amino acid position 1151; (ii) a substitution for leucine to valine at amino acid position 1152; (iii) a substitution for cysteine to tyrosine at amino acid position 1156; (iv) a substitution for isoleucine with serine at amino acid position 1171; and (v) a substitution for glycine to cysteine, serine, or alanine at amino acid position 1269,
said method comprising contacting said cell with a compound of formula (I), or a pharmaceutically acceptable salt thereof:




wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one or two heteroatoms, independently selected from N, S and O, wherein the 5- or 6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl,
in an amount sufficient to inhibit said proliferation.

6. A method of inhibiting the proliferation of a cell expressing a mutant ALK-fusion protein, wherein said mutant ALK-fusion protein is characterized by the presence of a mutation in said ALK-fusion protein selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for phenylalanine with valine at amino acid position 1174; and (ii) a substitution for tyrosine with serine at amino acid position 1278,
said method comprising contacting said cell with a compound of formula (I), or a pharmaceutically acceptable salt thereof:




wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two, or three heteroatoms, independently selected from N, S and O, wherein the 5- or
6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl,
in an amount sufficient to inhibit said proliferation.

7. A method of treating an ALK-driven cancer refractory to one or more of crizotinib, CH5424802 and ASP3026 in a subject, said method comprising administering to said subject a compound of formula I:



wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two, or three heteroatoms, independently selected from N, S and O, wherein the 5- or
6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl,

or a pharmaceutically acceptable salt thereof, in an amount sufficient to treat said cancer, and wherein the term CH5424802 refers to the compound,




and the term ASP3026 refers to the compound,

8. A method of treating an ALK-driven cancer in a subject intolerant to one or more of crizotinib, CH5424802 and ASP3026, said method comprising administering to said subject a compound of formula I:



wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two, or three heteroatoms, independently selected from N, S and O, wherein the 5- or
6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl,

or a pharmaceutically acceptable salt thereof, in an amount sufficient to treat said cancer, and wherein the term CH5424802 refers to the compound,




and the term ASP3026 refers to the compound,

9. A method of treating an ALK-driven cancer refractory to an inhibitor of wild-type ALK in a subject, said method comprising administering to said subject a compound of formula I:



wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two, or three heteroatoms, independently selected from N, S and O, wherein the 5- or
6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
RA is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl,

or a pharmaceutically acceptable salt thereof, in an amount sufficient to treat said cancer.

10. A method for treating an ALK-driven cancer in a subject comprising the steps of (a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK), and (b) administering to said subject a therapeutically effective amount of compound 1, having the following structure,




or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein said ALK is full-length ALK.

12. The method of claim 10, wherein said ALK is an ALK-fusion product.

13. The method of claim 10, wherein said mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for:
(i) a substitution for threonine at amino acid position 1151; (ii) a substitution for leucine at amino acid position 1152; (iii) a substitution for cysteine at amino acid position 1156; (iv) a substitution for isoleucine at amino acid position 1171; (v) a substitution for phenylalanine at amino acid position 1174; (vi) a substitution for valine at amino acid position 1180; (vii) a substitution for arginine at amino acid position 1181; (viii) a substitution for leucine at amino acid position 1196; (ix) a substitution for leucine at amino acid position 1198; (x) a substitution for glycine at amino acid position 1202; (xi) a substitution for aspartic acid at amino acid position 1203; (xii) a substitution for serine at amino acid position 1206; (xiii) a substitution for glutamic acid at amino acid position 1210; (xiv) a substitution for glutamic acid at amino acid position 1241; (xv) a substitution for phenylalanine at amino acid position 1245; (xvi) a substitution for isoleucine at amino acid position 1268; (xvii) a substitution for glycine at amino acid position 1269; and (xviii) insertion of an amino acid following amino acid position 1151.

14. The method of claim 13, wherein said mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: (i) a substitution for leucine to arginine at amino acid position 1152; (ii) a substitution for cysteine to tyrosine at amino acid position 1156; (iii) a substitution for isoleucine to serine at amino acid position 1171; (iv) a substitution for isoleucine to asparagine at amino acid position 1171; (v) a substitution for isoleucine to threonine at amino acid position 1171; (vi) a substitution for phenylalanine to leucine at amino acid position 1174; (vii) a substitution for phenylalanine to cysteine at amino acid position 1174; (viii) a substitution for valine to leucine at amino acid position 1180; (ix) a substitution for leucine to methionine at amino acid position 1196; (x) a substitution for glycine to arginine at amino acid position 1202; (xi) a substitution for aspartic acid to asparagine at amino acid position 1203; (xii) a substitution for serine to tyrosine at amino acid position 1206; (xiii) a substitution for serine with arginine at amino acid position 1206; (xiv) a substitution for glutamic acid to lysine at amino acid position 1210; (xv) a substitution for phenylalanine to cysteine at amino acid position 1245; (xvi) a substitution for glycine to alanine at amino acid position 1269; (xvii) a substitution for glycine to serine at amino acid position 1269; and (xviii) insertion of threonine following position 1151.

15. A method for treating an ALK-driven cancer in a subject comprising the steps of:
a) providing a subject having an ALK-driven cancer characterized by the presence of a mutation in anaplastic lymphoma kinase (ALK), wherein said mutation is selected from mutations corresponding to the amino acid positions in SEQ ID NO: 2 for: ((i) a substitution for leucine to arginine at amino acid position 1152; (ii) a substitution for cysteine to tyrosine at amino acid position 1156; (iii) a substitution for isoleucine to serine at amino acid position 1171; (iv) a substitution for isoleucine to asparagine at amino acid position 1171; (v) a substitution for isoleucine to threonine at amino acid position 1171; (vi) a substitution for phenylalanine to leucine at amino acid position 1174; (vii) a substitution for phenylalanine to cysteine at amino acid position 1174; (viii) a substitution for valine to leucine at amino acid position 1180; (ix) a substitution for leucine to methionine at amino acid position 1196; (x) a substitution for aspartic acid to asparagine at amino acid position 1203; (xi) a substitution for serine with arginine at amino acid position 1206; (xii) a substitution for glutamic acid to lysine at amino acid position 1210; (xiii) a substitution for phenylalanine to cysteine at amino acid position 1245; (xiv) a substitution for glycine to alanine at amino acid position 1269; and (xv) a substitution for glycine to serine at amino acid position 1269; and
b) administering to said subject a therapeutically effective amount of compound of formula (I), or a pharmaceutically acceptable salt thereof:




wherein
Rd is H, C1-4 alkyl, or halo; and Re is H; or Rd and Re, together with the pyrimidine ring atoms to which they are attached, form a 5- or 6-membered ring containing one, two or three heteroatoms, independently selected from N, S and O, wherein the 5- or
6-membered ring is substituted by Rh;
Rh is H, C1-4 alkyl, or halo;
Ra2 is H, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy;
Rg is —P(O)(R3A)(R3B);
each of R3A and R3B is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, and heteroalkyl, or R3A and R3B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Rg2 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, or, Rg2 and Rg together with the atoms to which they are attached form a 5- to 7-member heterocyclic ring comprising 1-3 hetero atoms independently selected from P, N, O and S, the heterocyclic ring being unsubstituted or substituted;
Rg1 is H, F, or a 5 or 6 member heterocyclic ring comprising 1 or 2 N atoms, the heterocyclic ring being unsubstituted or substituted;
Rb2 is H, F, or is a 5 or 6 member heterocyclic ring containing 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted;
Rb4 is H, halo, CN, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, C1-6 alkoxy, C3-6 alkenyloxy, or C3-6 cycloalkyloxy,

—OC(O)N(R5A)(R5B), —NR5CC(O)OR5D; a 5 or 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms, the heterocyclic ring being unsubstituted or substituted, or, Rb4 and Ra1 together with the atoms to which they are attached form a 6 member heterocyclic ring comprising 1, 2 or 3 N or O atoms which is unsubstituted or substituted;
each of R5A, R5B, R5C, and R5D is, independently, selected from H, alkyl, alkenyl, alkynyl, and heteroalkyl, or R5A and R5B, together with the atoms to which they are attached, combine to form a 5- or 6-membered heterocyclic ring which is unsubstituted or substituted;
Ra1 combines with Rb4 to form a 6 member heterocyclic ring, or is H, halo, —CN, —NO2, —R1, —OR2, —O—NR1R2, —NR1R2, —NR1—NR1R2, —NR1—OR2, —C(O)YR2, —OC(O)YR2, —NR1C(O)YR2, —SC(O)YR2, —NR1C(═S)YR2, —OC(═S)YR2, —C(═S)YR2, —YC(═NR1)YR2, —YC(═N—OR1)YR2, —YC(═N—NR1R2)YR2, —YP(═O)(YR1)(YR2), —NR1SO2R2, —S(O)rR2, —SO2NR1R2, —NR1SO2NR1R2, or





each Y is, independently, a bond, —O—, —S— or —NR1—;
each occurrence of R1 and R2 is, independently, selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl;
each of X1 and X2 is, independently, selected from CH and N; and
R4 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroalkyl, heterocyclic and heteroaryl.

16. The method of claim 15, wherein said ALK is full-length ALK.

17. The method of claim 15, wherein said ALK is an ALK-fusion product.