# Patent

## Title
Use of ERBB4 as a prognostic and therapeutic marker for melanoma

## Abstract

It is disclosed herein that members of the protein tyrosine kinase (PTK) family are highly mutated in patients with melanoma. Described herein are novel somatic mutations in the ERBB4 gene that result in increased kinase activity, transformation ability and anchorage-independent growth. These ERBB4 mutations contribute to the tumorogenicity of melanoma. Thus, provided herein is a method of predicting the prognosis of a patient with melanoma by detecting the presence or absence of a mutation in the ERBB4 gene. In some examples, the ERBB4 mutation is selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbering based on SEQ ID NO: 1). Also provided are methods of selecting a patient as a candidate for treatment with an ERBB4 and/or PI3K/AKT pathway inhibitor, and a method of identifying a therapeutic agent for the treatment of a subject diagnosed with melanoma. Oligonucleotides that specifically hybridize with an ERBB4 nucleic acid molecule comprising a novel mutation, and arrays comprising such oligonucleotides, are also provided.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage of International Application No. PCT/US2009/053005, filed Aug. 6, 2009, published in English under PCT Article 21(2), which claims the benefit of U.S. Provisional Application No. 61/199,156, filed Nov. 12, 2008, which is herein incorporated by reference in its entirety.


FIELD
This disclosure concerns the identification of novel mutations in members of the protein tyrosine (PTK) family, including ERBB4, and methods of use.
BACKGROUND
The protein tyrosine kinases (PTKs) are a family of proteins that catalyze phosphorylation of tyrosine residues in target proteins; PTKs play important roles in cellular signaling. Within this large family of proteins is the ERBB PTK family, which consists of four receptor kinases, ERBB1 (EGFR1, HER1), ERBB2 (c-Neu, HER2), ERBB3 (HER3) and ERBB4 (HER4). The ERBB kinases regulate a wide range of cellular responses, including cell proliferation, survival, migration and differentiation. ERBB signaling pathways are known to be altered in a wide variety of cancers, which has led to the development of drugs to specifically inhibit activity of members of this family (Junttila et al., Cancer Res. 65(4):1384-1393, 2005).
ERBB4 is a protein of approximately 180 kD and is expressed as four alternatively spliced isoforms. Previous studies of the role of ERBB4 in cancer development and prognosis have produced differing and sometimes contradictory results. For example, clinical studies of breast cancer have linked ERBB4 expression to either a favorable or adverse clinical outcome, and in vitro studies have suggested that in breast cancer cells, ERBB4 mediates either differentiation or tumorigenic growth (Junttila et al., Cancer Res. 65(4):1384-1393, 2005).
Cutaneous malignant melanoma is the most common fatal skin cancer (Jermal et al., CA Cancer J. Clin. 156(2):106-130, 2006; Tsao et al., N. Engl. J. of Med. 351:998-1012, 2004), and the incidence of this disease increases each year. Patients diagnosed with malignant melanoma have an average survival time of less than 10 months. PTKs are frequently mutated in cancer, and since they are amenable to pharmacologic inhibition (Futreal et al., Nat. Rev. Cancer 4:177-183, 2004; Sawyers, Nature 432:294-297, 2004), further analysis of the PTK gene family is needed to provide insight into melanoma pathogenesis and to identify new therapeutic strategies. Given the known role of PTKs in human cancer, and the disparate findings of studies of ERBB4 in cancer development, it is desirable to further evaluate ERBB4 in patients with malignant melanoma.
SUMMARY
It is disclosed herein that members of the protein tyrosine kinase family, including ERBB4, are highly mutated in melanoma tumors. Analysis of several ERBB4 mutants revealed that the mutations result in increased kinase activity of ERBB4 protein, increased transformation ability and increased anchorage-independent growth.
Thus, provided herein is a method of predicting the prognosis of a subject diagnosed with melanoma, comprising detecting the presence or absence of a mutation in the ERBB4 gene, wherein the presence of the mutation in the ERBB4 gene predicts a poor prognosis. In some embodiments, the ERBB4 mutation is selected from one or more of G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbering based on SEQ ID NO: 1).
Also provided is a method of selecting a subject diagnosed with melanoma as a candidate for treatment with an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both, comprising detecting the presence or absence of a mutation in the ERBB4 gene of the subject, wherein the presence of a mutation in the ERBB4 gene indicates that the subject is a candidate for treatment with an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both. In some embodiments, the method further includes administering to the subject an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both. Further provided is a method of identifying a therapeutic agent for the treatment of a subject diagnosed with melanoma, comprising screening candidate agents to select an agent that decreases activity of ERBB4, or decreases activity of the PI3K/AKT pathway, thereby identifying a therapeutic agent for the treatment of a subject with melanoma. In some embodiments of the methods, the ERBB4 mutation is selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbering based on SEQ ID NO: 1).
Further provided are oligonucleotides that specifically hybridize with an ERBB4 nucleic acid molecule, wherein the ERBB4 nucleic acid molecule comprises at least one mutation selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbering based on SEQ ID NO: 1). Also provided are arrays comprising one or more of such ERBB4 mutant-specific oligonucleotides.
The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Distribution of mutations in ERBB4 and increased basal activation of ERBB4 mutants. (A) Arrows indicate the location of ERBB4 somatic mutations found in this screen. Numbering of the ERBB4 amino acid residues is based on SEQ ID NO: 2. Stars indicate ERBB4 mutants evaluated for increased tyrosine kinase activity. Boxes represent functional domains (I, extracellular domain subregion I; II, extracellular domain subregion II; III, extracellular domain subregion III; IV, extracellular domain subregion IV; PTK, tyrosine kinase domain). (B) ERBB4 mutants have increased tyrosine phosphorylation. HEK 293T cells were transiently transfected with the indicated constructs. Twenty-four hours after transfection, cells were serum starved and lysed. Shown are immunoblots of immunoprecipitated ERBB4 probed with the indicated antibodies. Lysates were immunoprobed with an anti α-tubulin antibody. (C and D) ERBB4 mutants exhibit increased in vitro kinase activity. (C) HEK 293T cells were transiently transfected as in (B). Twenty-four hours after transfection, cells were either grown in 10% serum or serum starved and then lysed. Protein lysates were immunoprecipitated and used in a kinase assay. (D) The same samples that were used in the kinase assay were immunoblotted with ERBB4 antibody and lysates were blotted with α-tubulin (ns=non specific; KD=kinase dead). (E) ERBB4 mutants exhibit increased in vitro kinase activity. HEK 293T cells were transiently transfected as in (B). Equivalent amounts of protein from cell lysates were immunoprecipitated and used in a kinase assay to measure receptor autophosphorylation. The same samples that were used in the kinase assay were immunoblotted with ERBB4 antibody and lysates were blotted with α-tubulin. KD=kinase dead. (F) Increased basal activation of endogenous mutant ERBB4. Melanoma lines that harbor either WT or mutant ERBB4 were serum starved and then lysed, immunoprecipitated for ERBB4, then immunoblotted with α-PY20 or α-ERBB4. (G) Mutant ERBB4 has increased basal activity. Melanoma lines harboring either WT or mutant ERBB4 were serum deprived, lysed, immunoprecipitated for ERBB4, and analyzed by immunoblotting with α-P-ERBB4 (P-Y1162) or α-ERBB4.
FIG. 2: Mutant ERBB4 induces transformation and anchorage independent growth in NIH 3T3 and SK-Mel-2 cells. (A) NIH 3T3 cells were transfected with the indicated ERBB4 mutant or control constructs. The graph indicates the average number of transformed foci after 10 days. (B) Growth in soft agar of melanoma SK-Mel-2 cells stably expressing either vector, WT ERBB4 or various ERBB4 missense mutants. The graph indicates the number of colonies after 14 days.
FIG. 3: Expression of mutant ERBB4 provides an essential cell survival signal in melanoma. (A) HEK 293 cells were transiently co-transfected with either vector or WT ERBB4 together with either control vector or shRNAs that target ERBB4. Cell lysates were analyzed by immunoblotting using α-ERBB4. For normalization, lysates were analyzed in parallel by α-tubulin immunoblotting. (B) Cells transduced with shRNA targeting ERBB4 were lysed and immunoprecipitated using α-ERBB4 beads Immunoprecipitates were blotted with specific antibodies, as indicated. (C-G) shRNA-mediated ERBB4 knockdown in melanoma lines containing ERBB4 mutations results in reduced cell growth. Cells were seeded in 96-well plates and incubated for 13-17 days. Plates were analyzed every other day for cell proliferation, where the average cell number at each time point was measured by determining DNA content using SYBR Green I. Melanoma cells harboring ERBB4 mutations stably transduced with shRNA constructs targeting ERBB4, but not those stably transduced with the control vector only, showed decreased growth relative to control. This did not occur in melanoma cells harboring WT ERBB4.
FIG. 4: Detection of mutations in ERBB4. Shown are eight matched sets of two chromatograms each, illustrating somatic mutations in the ERBB4 gene. In each case, the top sequence chromatogram was obtained from normal tissue and the lower sequence chromatogram from the indicated tumors. Arrows indicate the location of missense mutations. The nucleotide and amino acid alterations are indicated below the tumor chromatograms; numbering of the mutation locations is based on SEQ ID NO: 1 (nucleotide) and SEQ ID NO: 2 (amino acid).
FIG. 5: Distribution of mutations in ERBB4, FL T1, EphB2, EphB6, PTK2B, and TIE1. Shown is a schematic of the domain structure of select PTKs. Arrows indicate positions of nonsynonymous mutations and boxes represent functional domains (Rcpt L, receptor L; GFR, growth factor receptor; PTK, protein tyrosine kinase; IG, immunoglobin; IGc2, immunoglobin C-2 Type; Eph Rcpt, ephrin receptor; FNIII, fibronectin type III; SAM, sterile alpha motif; FERM, protein 4.1, ezrin, radixin, moesin domain; Focal AT, focal adhesion targeting region).
FIG. 6: Mutation spectra of single base pair substitutions. (A) Shown is a Kinome mutation spectrum. The number of each of the six classes of base substitutions resulting in nonsynonymous changes in the kinome screen is shown. (B) Shown is a mutation spectrum of single base pair substitutions in ERBB4. The number of each of the six classes of base substitutions resulting in nonsynonymous changes in ERBB4 is shown.
FIG. 7: Specificity of phosphorylation site-specific antibodies. Shown is a series of immunoblots to detect phosphorylation status of ERBB4 mutants. HEK 293T cells were transiently transfected with either vector or ERBB4 E452K missense mutant. Cells were serum starved and then lysed. Shown are immunoblots of immunoprecipitated ERBB4 probed with several anti-phosphoERBB4 (Y1162; Y1284), or total ERBB4 antibodies in the presence or absence of phosphorylated (pPep) or unphosphorylated (Pep) competitive peptide.
FIG. 8: Increased basal activation of endogenous mutant ERBB4. Shown is a set of two immunoblots demonstrating detection of phosphorylated ERBB4 in melanoma cell lines expressing WT or mutant ERBB4. MM lines that harbor either WT or mutant ERBB4 cells were either grown in 10% serum or serum starved and then lysed. Shown are immunoblots of immunoprecipitated ERBB4 probed with the indicated antibodies.
FIG. 9: Effect of ERBB4 mutations on cell growth in NIH 3T3 and SK− Mel-2 cells. (A) Growth in soft agar of NIH 3T3 cells expressing either vector, WT ERBB4 or various ERBB4 missense mutants. The graph indicates the number of colonies after 14 days. (B and C) Detection of ERBB4 protein expression in stable transfectants of SK-Mel-2 melanoma cells by western blot analysis. Lysates from the different clones stably transfected with an empty vector, human ERBB4 or the indicated ERBB4 mutants were immunoprecipitated and immunoblotted with ERBB4 antibody (B). Anchorage independent proliferation of SK-Mel-2 cell clones expressing the indicated constructs was assessed by measuring colony growth in soft agar (C). The graph indicates the number of colonies observed after 14 days of growth.
FIG. 10: Melanoma lines expressing ERBB4 mutants exhibit increased sensitivity to ERBB inhibition by lapatinib. (A) Representative dose response curves showing lapatinib efficacy against ERBB4 mutant cell lines compared to WT ERBB4 cell lines. Cells were treated for 72 hours in the presence of increasing concentrations (0.01-30 μM) of lapatinib, and relative cell number was estimated by methylene blue protein staining and plotted as percent survival when compared to vehicle-treated control versus Log (lapatinib) nM (where 1 is 10 nM lapatinib). Fitted lines were generated using 4-parameter nonlinear regression via GraphPad Prism. (B) ERBB4 mutant cells lines have increased sensitivity to lapatinib compared to WT ERBB4 cell lines. The IC50 values for inhibition of cell growth by 72 hour treatment with lapatinib of a larger panel of lines harboring WT and mutant ERBB4 were analyzed using GraphPad Prism v.5 (n=3). (C) Immunoprecipitation and western blot analysis of ERBB4 autophosphorylation in cells treated with lapatinib. Cells were treated for 1 hour with lapatinib or vehicle alone as control. Lysates were immunoprecipitated with α-ERBB4 followed by western blot analysis with α-ERBB4 and α-P-ERBB4 (Y1162). (D) Melanoma lines expressing mutant ERBB4 exhibit increased lapatinib sensitivity with respect to ERBB4 and AKT phosphorylation. The activity of ERBB4, AKT and ERBB2 was determined by immunoblotting with phospho-specific antibodies. Cells were treated for 1 hour with 5 μM lapatinib or vehicle alone. Lysates were immunoprecipitated using α-ERBB2 or α-ERBB4. Lysates and immunoprecipitates were analyzed by western blotting using the indicated antibodies. Shown are representative blots. (E) Quantitative assessment of data from 2 cell lines harboring WT ERBB4 and 3 cell lines harboring mutant ERBB4 that were performed similarly to (D). The ratio of band intensities of (P-Y1162)-ERBB4/ERBB4, (P)-S473-AKT/AKT and (P-Y1248)-ERBB2/ERBB2 for each cell line are shown. (F) Mutant ERBB4 cells have increased sub-G1 population in the presence of lapatinib compared to WT ERBB4 cells. Shown are representative plots of FACS analysis of 31T (WT) and 12T (E563K) showing cell cycle distribution (PI staining, x-axis) versus cell counts (y-axis). (G) Quantitation of FACS-sorted lapatinib-treated cells. The percent apoptotic cells were determined based on the sub-G1 population for vehicle-treated cells or lapatinib-treated cells.
FIG. 11: Effects of ERBB4 mutation on AKT and ERK phosphorylation. Melanoma cell lines containing either WT or mutant ERBB4 were harvested and analyzed by immunoblot. Shown are immunoblots of lysates probed with the indicated antibodies (α-P-ERK1/2—recognizes phosphorylation of T202 and Y204 on ERK1, and T185 and Y187 on ERK2).
FIG. 12: Knockdown of ERBB4 protein causes reduced activation of the AKT pathway but not of the ERK pathway. Melanoma cells lines containing either WT or mutant ERBB4 were harvested and analyzed by western blot. Shown are immunoblots of lysates probed with the indicated antibodies.
FIG. 13: Rescue of oncogene dependence by exogenous non-targetable ERBB4. (A) Melanoma cells harboring mutant ERBB4 stably expressing control or ERBB4 shRNA #6 transduced with either vector or non-targetable (NT) ERBB4 were analyzed by immunoblotting with the indicated antibodies. As a loading control, lysates were immunoblotted with α-tubulin. (B) Melanoma cells expressing vector or the ERBB4 shRNA #6 transduced with a vector or NT ERBB4 were evaluated for cell proliferation by measuring the average cell number at each time point by determining DNA content using SYBR Green I.
FIG. 14: Effect of lapatinib in ERK1/2 signaling pathways. (A) Melanoma lines expressing mutant ERBB4 exhibit increased lapatinib sensitivity with respect to ERK1 and ERK2 phosphorylation. Cells were treated for 72 hours with 5 μM lapatinib or vehicle as control. The activity of ERK1 and ERK2 was determined by immunoblotting with phospho-specific antibodies. Total ERK protein was also determined by immunoblotting. Shown are representative blots. (B) Quantitative assessment of data from one melanoma cell line harboring mutant ERBB4. The ratio of band intensities of P-ERK1/ERK1 or P-ERK2/ERK2 was analyzed for each melanoma cell line.



SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on May 3, 2011, 46.4 KB, which is incorporated by reference herein. In the accompanying sequence listing:
SEQ ID NOs: 1 and 2 are the nucleotide and amino acid sequences, respectively, of human ERBB4 (GenBank Accession No. NM—005235.2, deposited Jul. 28, 2006). Seven mutations identified in melanoma tumors are indicated (G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A in the nucleotide sequence; E317K, E452K, E542K, R544W, E563K, E836K and E872K in the amino acid sequence).
SEQ ID NOs: 3-31 are the nucleotide sequences of forward primers used to PCR amplify the coding region of human ERBB4.
SEQ ID NOs: 32-60 are the nucleotide sequences of reverse primers used to PCR amplify the coding region of human ERBB4.
SEQ ID NO: 61 is the nucleotide sequences of the primer used to sequence the coding region of human ERBB4.
SEQ ID NOs: 62 and 63 are the nucleotide sequences of primers used to clone human ERBB4.
SEQ ID NOs: 64-70 are the nucleotide sequences of forward primers used to PCR amplify the kinase domain of human ERBB4.
SEQ ID NOs: 71-77 are the nucleotide sequences of reverse primers used to PCR amplify the kinase domain of human ERBB4.
SEQ ID NO: 78 is the nucleotide sequences of the forward primer used to sequence the kinase domain of human ERBB4.
SEQ ID NO: 79 is the nucleotide sequences of the reverse primer used to sequence the kinase domain of human ERBB4.
DETAILED DESCRIPTION
I. Introduction
PTK signaling pathways can be deregulated by a variety of mechanisms in human tumors. Described herein is a comprehensive mutational analysis of the PTK family, which revealed numerous novel somatic mutations. Surprisingly, the analysis identified two PTK genes that had mutations in over 10% of MM cases (FLT1 and PTK2B) and one gene that had mutations in over 18% of MM cases (ERBB4, a member of the EGFR family). The high frequency of mutations identified in ERBB4, their co-localization, and the identification of two identical missense mutations in multiple MM cases, suggest that these mutations play a role in tumorigenesis.
To evaluate the effect of mutations in ERBB4, seven mutations that affect residues that are conserved in EGFR, and are located at residues near EGFR mutations that have been described in other tumor types, were cloned and their kinase activity was examined. The results of this analysis showed that mutant ERBB4 has increased autophosphorylation activity compared to wild type (WT) ERBB4. Expression of mutant ERBB4 in NIH 3T3 cells and human melanoma cells increased their growth on soft agar and colony formation ability. Furthermore, immunoblots of melanoma cells harboring ERBB4 mutations exhibited increased activity of the PI3K/AKT pathway, as evidenced by an increase in phosphorylated AKT. These functional assays indicated that the ERBB4 mutations identified herein promote cellular phenotypes typical of neoplastic cells, such as increased transformation ability and anchorage-independent growth.
The combination of genetic, biochemical and cellular data disclosed herein indicates that ERBB4 functions as an oncogene in MM. This finding is consistent with previously reported alterations of members of the EGFR family, which have been shown to be mutated as well as amplified (Sharma et al., Nat. Rev. Cancer 7:169-181, 2007). In addition, ERBB4 has previously been shown to be involved in enhanced proliferation of breast cancer cells (Junttila et al., Cancer Res. 65(4):1384-1393, 2005) and non-small cell lung cancer cells (Starr et al., Int. J. Cancer 119:269-274, 2006). Importantly, cells containing mutations in ERBB4 were associated with enhanced and selective sensitivity to an FDA-approved ERBB4 inhibitor compared to WT cells. These results suggest that patients with melanoma containing one or more ERBB4 mutations may benefit from therapy directed at mutant ERBB4.
II. Abbreviations













ARAF
v-raf murine sarcoma 3611 viral oncogene homolog


BRAF
B-Raf proto-oncogene serine/threonine-protein kinase


CRAF
v-raf-1 murine leukemia viral oncogene homolog 1


DMEM
Dulbecco's modified eagle medium


DMSO
Dimethyl sulfoxide


DNA
Deoxyribonucleic acid


EGFR
Epidermal growth factor receptor


ELISA
Enzyme-linked immunosorbent assay


FBS
Fetal bovine serum


GFR
Growth factor receptor


HRAS
v-Ha-ras Harvey rat sarcoma viral oncogene homolog


IC50
Inhibitory concentration 50


IG
Immunoglobulin


KD
Kinase dead


KO
Knockout


KRAS
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog


MM
Metastatic melanoma


NRAS
Neuroblastoma RAS viral oncogene homolog


NT
Non-targetable


PAGE
Polyacrylamide-gel electrophoresis


PBS
Phosphate buffered saline


PCR
Polymerase chain reaction


PI3K
Phosphoinositide 3-kinase


RNA
Ribonucleic acid


RNAi 
RNA interference


RT
Reverse transcriptase


SAM
Sterile alpha motif


SDS
Sodium dodecyl sulfate


shRNA
Short hairpin RNA


siRNA
Small interfering RNA


SNP
Single nucleotide polymorphism


TKI
Tyrosine kinase inhibitor


WT
Wild type









III. Terms
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
AKT: As used herein, the term “AKT” includes AKT1, AKT2 and AKT3. The AKT1 gene encodes a serine-threonine protein kinase that is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation, which occurs through phosphatidylinositol 3-kinase, is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1. AKT1 is also known as v-akt murine thymoma viral oncogene homolog 1, PKB; RAC; PRKBA; MGC99656; PKB-ALPHA; and RAC-ALPHA. The AKT2 gene is a putative oncogene encoding a protein belonging to a subfamily of serine/threonine kinases containing SH2-like (Src homology 2-like) domains. The Akt2 protein is a general protein kinase capable of phosphorylating several known proteins. AKT2 is also known as v-akt murine thymoma viral oncogene homolog 2; PKBB; PRKBB; PKBBETA; and RAC-BETA. AKT3 is a member of the AKT (also called PKB) serine/threonine protein kinase family. AKT kinases are known to be regulators of cell signaling in response to insulin and growth factors. They are involved in a wide variety of biological processes including cell proliferation, differentiation, apoptosis, tumorigenesis, as well as glycogen synthesis and glucose uptake. The Akt3 protein kinase has been shown to be stimulated by platelet-derived growth factor (PDGF), insulin, and insulin-like growth factor 1 (IGF1). AKT3 is also known as v-akt murine thymoma viral oncogene homolog 3; protein kinase B, gamma; PKBG; PRKBG; STK-2; PKB-GAMMA; RAC-gamma; RAC-PK-gamma; and DKFZp434N0250. Members of the AKT protein family are also called protein kinases B (PKB) in the literature.
Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
Antibodies include intact immunoglobulins and the variants and portions of antibodies well known in the art, such as Fab fragments, Fab′ fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).
References to “VH” or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
A “monoclonal antibody” is an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds mesothelin.
A “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Pat. No. 5,585,089).
A “human” antibody (also called a “fully human” antibody) is an antibody that includes human framework regions and all of the CDRs from a human immunoglobulin. In one example, the framework and the CDRs are from the same originating human heavy and/or light chain amino acid sequence. However, frameworks from one human antibody can be engineered to include CDRs from a different human antibody. All parts of a human immunoglobulin are substantially identical to corresponding parts of natural human immunoglobulin sequences.
Antisense compound: Refers to an oligomeric compound that is at least partially complementary to the region of a target nucleic acid molecule (such as a miR gene product) to which it hybridizes. As used herein, an antisense compound that is “specific for” a target nucleic acid molecule is one which specifically hybridizes with and modulates expression of the target nucleic acid molecule. As used herein, a “target” nucleic acid is a nucleic acid molecule to which an antisense compound is designed to specifically hybridize and modulate expression.
Nonlimiting examples of antisense compounds include primers, probes, antisense oligonucleotides, siRNAs, miRNAs, shRNAs and ribozymes. As such, these compounds can be introduced as single-stranded, double-stranded, circular, branched or hairpin compounds and can contain structural elements such as internal or terminal bulges or loops. Double-stranded antisense compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. In particular examples herein, the antisense compound is an antisense oligonucleotide, siRNA or ribozyme.
Antisense oligonucleotide: As used herein, an “antisense oligonucleotide” is a single-stranded antisense compound that is a nucleic acid-based oligomer. An antisense oligonucleotide can include one or more chemical modifications to the sugar, base, and/or internucleoside linkages. Generally, antisense oligonucleotides are “DNA-like” such that when the antisense oligonucleotide hybridizes to a target RNA molecule, the duplex is recognized by RNase H (an enzyme that recognizes DNA:RNA duplexes), resulting in cleavage of the RNA.
Array: An arrangement of molecules, such as biological macromolecules (such as peptides or nucleic acid molecules) or biological samples (such as tissue sections), in addressable locations on or in a substrate. A “microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. Arrays are sometimes called DNA chips or biochips.
The array of molecules (“features”) makes it possible to carry out a very large number of analyses on a sample at one time. In certain example arrays, one or more molecules (such as an oligonucleotide probe) will occur on the array a plurality of times (such as twice), for instance to provide internal controls. The number of addressable locations on the array can vary, for example from at least two, at least four, at least six, to at least 9, at least 10, at least 14, at least 15, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, least 550, at least 600, at least 800, at least 1000, or more. In a particular example, an array includes 2-100 addressable locations, such as 4-20 addressable locations. In particular examples, an array consists essentially of oligonucleotide probes specific for ERBB4 nucleic acid molecules comprising mutations selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1).
In particular examples, an array includes nucleic acid molecules, such as oligonucleotide sequences that are at least 15 nucleotides in length, such as about 15-40 nucleotides in length.
Within an array, each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array. The feature application location on an array can assume different shapes. For example, the array can be regular (such as arranged in uniform rows and columns) or irregular. Thus, in ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position. Often, ordered arrays are arranged in a symmetrical grid pattern, but samples could be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters). Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.
Candidate: As used herein, a “candidate” for treatment with an ERBB4 inhibitor is a melanoma patient that is likely to respond favorably to treatment with the ERBB4 inhibitor. Candidates for ERBB4 inhibitor therapy are melanoma patients that have a mutation in the ERBB4 gene that results in an increase in ERBB4 expression, or results in expression of an ERBB4 protein with increased kinase activity. In some embodiments, the candidate is a melanoma patient with an ERBB4 gene comprising a mutation selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). In some embodiment, the ERBB4 protein comprises a mutation selected from E317K, E452K, E542K, R544W, E563K, E836K and E872K (numbered with reference to SEQ ID NO: 2).
Clinical outcome: Refers to the health status of a patient following treatment for a disease or disorder, or in the absence of treatment. Clinical outcomes include, but are not limited to, an increase in the length of time until death, a decrease in the length of time until death, an increase in the chance of survival, an increase in the risk of death, survival, disease-free survival, chronic disease, metastasis, advanced or aggressive disease, disease recurrence, death, and favorable or poor response to therapy.
Decrease in survival: As used herein, “decrease in survival” refers to a decrease in the length of time before death of a patient, or an increase in the risk of death for the patient. A decrease in survival also can refer to a decrease in the average time to death in a group, such as a group of patients diagnosed with melanoma.
Epidermal growth factor receptor (EGFR) family: A family of protein tyrosine kinases (PTKs), also known as the ERBB family. The ERBB PTK family includes four receptor kinases, ERBB1 (EGFR1, HER1), ERBB2 (c-Neu, HER2), ERBB3 (HER3) and ERBB4 (HER4). The ERBB kinases regulate a wide range of cellular responses, including cell proliferation, survival, migration and differentiation. ERBB signaling pathways are known to be altered in a wide variety of cancers.
ERBB4: A member of the EGFR family that encodes a protein of approximately 180 kD. ERBB4 encodes a single-pass type I membrane protein with multiple cysteine rich domains, a transmembrane domain, a tyrosine kinase domain, a phosphotidylinositol-3 kinase binding site and a PDZ domain binding motif. ERBB4 is expressed as four alternatively spliced isoforms. The protein binds to and is activated by neuregulins and other factors and induces a variety of cellular responses, including mitogenesis and differentiation. Multiple proteolytic events allow for the release of a cytoplasmic fragment and an extracellular fragment. Mutations in ERBB4 have been associated with cancer.
ERBB4 inhibitor: An ERBB4 inhibitor refers to any compound that inhibits expression or activity of ERBB4, such as kinase activity of ERBB4. Inhibitor compounds include, but are not limited to, small molecules, polypeptides and nucleic acid molecules (such as antisense compounds). In some embodiments, an ERBB4 inhibitor is a broad-spectrum inhibitor that inhibits activity of multiple members of the EGFR family. EGFR family inhibitors are known in the art (see, for example, PCT Publication Nos. WO 2008/005983, WO 03/012072 and WO 03/070912; and US Patent Application Publication Nos. 2006/0233808 and 2006/0128636). In some embodiments, the ERBB4 inhibitor selectively inhibits expression or activity of ERBB4, and not other EGFR family members (see, for example, U.S. Pat. No. 5,811,098). In some embodiments, the ERBB4 inhibitor is a kinase inhibitor. Kinase inhibitors are well known in the art (see, for example, US Patent Application Publication Nos. 2008/0031893; 2006/0148824; and 2002/0156083). In particular examples, the ERBB4 inhibitor is lapatinib (Burris et al., J. Clin. Oncol. 23(23):5305-5313, 2005).
Genomic DNA: The DNA found within the nucleus and containing an organism's genome, which is passed on to its offspring as information for continued replication and/or propagation and/or survival of the organism. The term can be used to distinguish between other types of DNA, such as DNA found within plasmids or organelles.
Inhibitor: As used herein, the term “inhibitor” includes any type of molecule that inhibits the expression or activity of a target gene or protein. An inhibitor can be any type of compound, such as a small molecule, antibody or antisense compound.
Kinase: An enzyme that catalyzes the transfer of a phosphate, such as from ATP, to a substrate. As used herein, an increase or decrease in “kinase activity” of a protein (e.g. ERBB4) refers to an increase or decrease in the ability of the protein to phosphorylate a substrate, such as a protein.
Label: An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
In some embodiments, the label is a fluorophore (“fluorescent label”). Fluorophores are chemical compounds, which when excited by exposure to a particular wavelength of light, emits light (i.e., fluoresces), for example at a different wavelength. Fluorophores can be described in terms of their emission profile, or “color.” Green fluorophores, for example Cy3, FITC, and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-540λ. Red fluorophores, for example Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-690λ.
Examples of fluorophores that may be used are provided in U.S. Pat. No. 5,866,366 to Nazarenko et al., and include for instance: 4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5′′-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4′-dimethylaminophenylazo)benzoic acid (DAB CYL); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.
Other contemplated fluorophores include GFP (green fluorescent protein), LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivatives thereof. Other fluorophores known to those skilled in the art may also be used.
LY294002: A selective small molecule inhibitor of PI3K. LY294002 is also known as 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Vlahos et al., J Biol Chem 269:5241-5248, 1994). The molecular formula of LY294002 is C19H17NO3.
Melanoma: A form of cancer that originates in melanocytes (cells that make the pigment melanin). Melanocytes are found primarily in the skin, but are also present in the bowel and eye.
Metastasis: Refers to the spread of cancer cells from the original tumor to other sites in the body.
Mutation: Any change of the DNA sequence within a gene or chromosome. In some instances, a mutation will alter a characteristic or trait (phenotype), but this is not always the case. Types of mutations include base substitution point mutations (e.g., transitions or transversions), deletions, and insertions. Missense mutations are those that introduce a different amino acid into the sequence of the encoded protein; nonsense mutations are those that introduce a new stop codon. In the case of insertions or deletions, mutations can be in-frame (not changing the frame of the overall sequence) or frame shift mutations, which may result in the misreading of a large number of codons (and often leads to abnormal termination of the encoded product due to the presence of a stop codon in the alternative frame).
This term specifically encompasses variations that arise through somatic mutation, for instance those that are found only in disease cells, but not constitutionally, in a given individual. Examples of such somatically-acquired variations include the point mutations that frequently result in altered function of various genes that are involved in development of cancers. This term also encompasses DNA alterations that are present constitutionally, that alter the function of the encoded protein in a readily demonstrable manner, and that can be inherited by the children of an affected individual. In this respect, the term overlaps with “polymorphism,” as discussed below, but generally refers to the subset of constitutional alterations that have arisen within the past few generations in a kindred and that are not widely disseminated in a population group.
In some embodiments, a mutation in ERBB4 refers to a nucleotide substitution in the ERBB4 gene or cDNA, or an amino acid substitution in the ERBB4 protein.
Oligonucleotide: A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
Patient or subject: As used herein, the term “patient” includes human and non-human animals. The preferred patient for treatment is a human. “Patient” and “subject” are used interchangeably herein.
Pharmaceutically acceptable vehicles: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Phosphoinositide-3 kinase (PI3K): A family of related enzymes that are capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol. PI3Ks are also known as phosphatidylinositol-3-kinases. Class I PI3K are heterodimeric molecules composed of a regulatory subunit and a catalytic subunit. Class II and Class III PI3K are differentiated from Class I by their structure and function. Class II PI3K are composed of one of three catalytic isoforms (C2α, C2β, and C2γ), but have no regulatory proteins. Class III PI3K exist as a heterodimers of a catalytic subunit (Vps34) and a regulatory (p150) subunit. Genes encoding PIK3 subunits include, for example, PIK3C2A, PIK3C2B, PIK3C2G, PIK3C3, PIK3CA, PIK3CB, PIK3CG, PIK3CD, PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R4, PIK3R5 and PIK3R6.
PI3K/Akt pathway: A signaling pathway involved in a number of cellular processes, such as cell growth, proliferation, differentiation, motility, survival, intracellular trafficking, metabolism and angiogenesis.
PI3K/Akt pathway inhibitor: Any compound that inhibits expression or activity of a member of the PI3K pathway, such as, but not limited to PI3K or AKT. For example, the inhibitor can be a small molecule, antibody, antisense compound or polypeptide. In some examples, the antibody is a chimeric antibody, a humanized antibody or a human antibody. In some examples, the antisense compound is an antisense oligonucleotide, siRNA or ribozyme. Antibodies, antisense compounds and other inhibitors specific for members of the PI3K/Akt pathway are known in the art and are commercially available. Exemplary inhibitors of the PI3K/Akt pathway are described herein, but are not intended to be limiting. In some examples, the small molecule inhibitor of PI3K is LY294002 (also known as 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; molecular formula C19H17NO3) or wortmannin (molecular formula C23H24O8). In some examples, the small molecule inhibitor of Akt is UCN-01 (also known as 7-hydroxystaurosporine and 8,12-epoxy-1H,8H-2,7b,12a-triazadibenzo[a, g]cyclonona[cde]trinden-1-one, 2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)). UCN-01 is a synthetic derivative of staurosporine with antineoplastic activity. Antisense compounds specific for members for the PI3K/Akt pathway have been previously described. For example, U.S. Patent Application Publication Nos. 2005/02772682 and 2004/0077580 disclose siRNAs and antisense oligonucleotides specific for PI3K. In addition, U.S. Patent Application Publication Nos. 2008/0161547, 2004/0265999 and 2003/0148974 describe antisense oligonucleotide and siRNA compounds that target AKT. Antibodies specific for members of the PI3K/Akt pathway have been described in the art and are commercially available from a variety of sources. For example, PI3K antibodies are disclosed in U.S. Patent Application Publication No. 2008/0014598.
Polymorphism: Variant in a sequence of a gene, or any genomic sequence, usually carried from one generation to another in a population. Polymorphisms can be those variations (nucleotide sequence differences) that, while having a different nucleotide sequence, produce functionally equivalent gene products, such as those variations generally found between individuals, different ethnic groups, and geographic locations. The term polymorphism also encompasses variations that produce gene products with altered function, i.e., variants in the gene sequence that lead to gene products that are not functionally equivalent. This term also encompasses variations that produce no gene product, an inactive gene product, a truncated gene product, or increased or increased activity gene product.
Polymorphisms can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic of the nucleic acid molecule or protein that is linked to the variation (e.g., an alteration of a secondary structure such as a stem-loop, or an alteration of the binding affinity of the nucleic acid for associated molecules, such as polymerases, RNAses, a change in the availability of a site for cleavage by a restriction endonuclease, either the formation of a new site, or lose of a site, and so forth).
Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.
The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.
Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown in the following table:














Original Residue
Conservative Substitutions









Ala
Ser



Arg
Lys



Asn
Gln, His



Asp
Glu



Cys
Ser



Gln
Asn



Glu
Asp



His
Asn; Gln



Ile
Leu, Val



Leu
Ile; Val



Lys
Arg; Gln; Glu



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr



Thr
Ser



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu










Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.
Preventing, treating or ameliorating a disease: “Preventing” a disease (such as metastatic melanoma) refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease.
Probes and primers: A probe comprises an isolated nucleic acid capable of hybridizing to a target nucleic acid. A detectable label or reporter molecule can be attached to a probe or primer. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). In some embodiments, an “oligonucleotide” is a probe or primer.
In a particular example, a probe includes at least one fluorophore, such as an acceptor fluorophore or donor fluorophore. For example, a fluorophore can be attached at the 5′- or 3′-end of the probe. In specific examples, the fluorophore is attached to the base at the 5′-end of the probe, the base at its 3′-end, the phosphate group at its 5′-end or a modified base, such as a T internal to the probe.
Probes are generally at least 15 nucleotides in length, such as at least 15, at least 16, at least 17, at least 18, at least 19, 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 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, or more contiguous nucleotides complementary to the target nucleic acid molecule, such as 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, 15-40 nucleotides, or 15-30 nucleotides.
Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length, 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.
The specificity of a primer increases with its 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 15 nucleotides. Thus, to obtain greater specificity, probes and primers can be selected that include at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more consecutive nucleotides. In particular examples, a primer is at least 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 include primers having 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, at least 55, at least 60, at least 65, at least 70, or more contiguous nucleotides complementary to the target nucleic acid molecule to be amplified, such as a primer of 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, 15-40 nucleotides or 15-30 nucleotides.
Primer pairs can be used for amplification of a nucleic acid sequence, for example, by PCR, real-time PCR, or other nucleic-acid amplification methods known in the art. An “upstream” or “forward” primer is a primer 5′ to a reference point on a nucleic acid sequence. A “downstream” or “reverse” primer is a primer 3′ to a reference point on a nucleic acid sequence. In general, at least one forward and one reverse primer are included in an amplification reaction.
Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided herein. It is also appropriate to generate probes and primers based on fragments or portions of these disclosed nucleic acid molecules, for instance regions that encompass the identified polymorphisms of interest. PCR primer pairs can be derived from a known sequence by 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.).
Prognosis: The likelihood of the clinical outcome for a subject afflicted with a specific disease or disorder. With regard to cancer, the prognosis is a representation of the likelihood (probability) that the subject will survive (such as for one, two, three, four or five years) and/or the likelihood (probability) that the tumor will metastasize. A “poor prognosis” indicates a greater than 50% chance that the subject will not survive to a specified time point (such as one, two, three, four or five years), and/or a greater than 50% chance that the tumor will metastasize. In several examples, a poor prognosis indicates that there is a greater than 60%, 70%, 80%, or 90% chance that the subject will not survive and/or a greater than 60%, 70%, 80% or 90% chance that the tumor will metastasize. Conversely, a “good prognosis” indicates a greater than 50% chance that the subject will survive to a specified time point (such as one, two, three, for or five years), and/or a greater than 50% chance that the tumor will not metastasize. In several examples, a good prognosis indicates that there is a greater than 60%, 70%, 80%, or 90% chance that the subject will survive and/or a greater than 60%, 70%, 80% or 90% chance that the tumor will not metastasize.
Protein tyrosine kinase (PTK): A family of proteins that catalyze phosphorylation of tyrosine residues in target proteins. PTKs play important roles in cellular signaling.
Ribozyme: A catalytic RNA molecule. In some cases, ribozymes can bind to specific sites on other RNA molecules and catalyze the hydrolysis of phosphodiester bonds in the RNA molecules.
RNA interference (RNAi): Refers to a cellular process that inhibits expression of genes, including cellular and viral genes. RNAi is a form of antisense-mediated gene silencing involving the introduction of double stranded RNA-like oligonucleotides leading to the sequence-specific reduction of RNA transcripts. Double-stranded RNA molecules that inhibit gene expression through the RNAi pathway include siRNAs, miRNAs, and shRNAs.
Sample: A biological specimen containing genomic DNA, RNA, protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy (such as skin tissue), surgical specimen, and autopsy material. In one example, a sample includes a biopsy of a melanoma tumor or a sample of normal tissue (from a subject not afflicted with a known disease or disorder, such as a cancer-free subject).
Screening: As used herein, “screening” refers to the process used to evaluate and identify candidate agents that decrease kinase activity of ERBB4 protein. In some cases, screening involves contacting a candidate agent (such as a small molecule, peptide or nucleic acid molecule) with cells expressing ERBB4 and testing the effect of the agent on kinase activity of ERBB4. In some embodiments, the cells express WT ERBB4. In other embodiments, the cells express mutant ERBB4, such as an ERBB4 protein comprising a mutation selected from E317K, E452K, E542K, R544W, E563K, E836K and E872K (numbered with reference to SEQ ID NO: 2).
Short hairpin RNA (shRNA): A sequence of RNA that makes a tight hairpin turn and can be used to silence gene expression via the RNAi pathway. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA.
Small interfering RNA (siRNA): A double-stranded nucleic acid molecule that modulates gene expression through the RNAi pathway. siRNA molecules are generally 20-25 nucleotides in length with 2-nucleotide overhangs on each 3′ end. However, siRNAs can also be blunt ended. Generally, one strand of a siRNA molecule is at least partially complementary to a target nucleic acid, such as a target mRNA. siRNAs are also referred to as “small inhibitory RNAs.”
Small molecule: A molecule, typically with a molecular weight less than about 1000 Daltons, or in some embodiments, less than about 500 Daltons, wherein the molecule is capable of modulating, to some measurable extent, an activity of a target molecule.
Specific hybridization: Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only or substantially only to a particular nucleotide sequence when that sequence is present in a complex mixture (e.g. total cellular DNA or RNA). Specific hybridization may also occur under conditions of varying stringency.
Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989 ch. 9 and 11). By way of illustration only, a hybridization experiment may be performed by hybridization of a DNA molecule to a target DNA molecule which has been electrophoresed in an agarose gel and transferred to a nitrocellulose membrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), a technique well known in the art and described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).
Traditional hybridization with a target nucleic acid molecule labeled with [32P]-dCTP is generally carried out in a solution of high ionic strength such as 6×SSC at a temperature that is 20-25° C. below the melting temperature, Tm, described below. For Southern hybridization experiments where the target DNA molecule on the Southern blot contains 10 ng of DNA or more, hybridization is typically carried out for 6-8 hours using 1-2 ng/ml radiolabeled probe (of specific activity equal to 109 CPM/μg or greater). Following hybridization, the nitrocellulose filter is washed to remove background hybridization. The washing conditions should be as stringent as possible to remove background hybridization but to retain a specific hybridization signal.
The term Tm represents the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Because the target sequences are generally present in excess, at Tm 50% of the probes are occupied at equilibrium. The Tm of such a hybrid molecule may be estimated from the following equation (Bolton and McCarthy, Proc. Natl. Acad. Sci. USA 48:1390, 1962):

Tm=81.5° C.−16.6(log10[Na+])+0.41(% G+C)−0.63(% formamide)−(600/l)

where l=the length of the hybrid in base pairs.
This equation is valid for concentrations of Na+ in the range of 0.01 M to 0.4 M, and it is less accurate for calculations of Tm in solutions of higher [Na+]. The equation is also primarily valid for DNAs whose G+C content is in the range of 30% to 75%, and it applies to hybrids greater than 100 nucleotides in length (the behavior of oligonucleotide probes is described in detail in Ch. 11 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).
Thus, by way of example, for a 150 base pair DNA probe derived from a cDNA (with a hypothetical % GC of 45%), a calculation of hybridization conditions required to give particular stringencies may be made as follows: For this example, it is assumed that the filter will be washed in 0.3×SSC solution following hybridization, thereby: [Na+]=0.045 M; % GC=45%; Formamide concentration=0; l=150 base pairs; Tm=81.5−16.6(log10[Na+])+(0.41×45)−(600/150); and so Tm=74.4° C.
The Tm of double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81:123, 1973). Therefore, for this given example, washing the filter in 0.3×SSC at 59.4-64.4° C. will produce a stringency of hybridization equivalent to 90%; that is, DNA molecules with more than 10% sequence variation relative to the target cDNA will not hybridize. Alternatively, washing the hybridized filter in 0.3×SSC at a temperature of 65.4-68.4° C. will yield a hybridization stringency of 94%; that is, DNA molecules with more than 6% sequence variation relative to the target cDNA molecule will not hybridize. The above example is given entirely by way of theoretical illustration. It will be appreciated that other hybridization techniques may be utilized and that variations in experimental conditions will necessitate alternative calculations for stringency.
Stringent conditions may be defined as those under which DNA molecules with more than 25%, 15%, 10%, 6% or 2% sequence variation (also termed “mismatch”) will not hybridize. Stringent conditions are sequence dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point Tm for the specific sequence at a defined ionic strength and pH. An example of stringent conditions is a salt concentration of at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and a temperature of at least about 30° C. for short probes (e.g. 10 to 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For example, conditions of 5×SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. are suitable for allele-specific probe hybridizations.
The following is an exemplary set of hybridization conditions and is not meant to be limiting:
Very High Stringency (Detects Sequences that Share 90% Identity)
Hybridization: 5×SSC at 65° C. for 16 hours
Wash twice: 2×SSC at room temperature (RT) for 15 minutes each
Wash twice: 0.5×SSC at 65° C. for 20 minutes each
High Stringency (Detects Sequences that Share 80% Identity or Greater)
Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours
Wash twice: 2×SSC at RT for 5-20 minutes each
Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each
Low Stringency (Detects Sequences that Share Greater than 50% Identity)
Hybridization: 6×SSC at RT to 55° C. for 16-20 hours
Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.
A perfectly matched probe has a sequence perfectly complementary to a particular target sequence. The test probe is typically perfectly complementary to a portion (subsequence) of the target sequence. The term “mismatch probe” refers to probes whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence.
Therapeutic: A generic term that includes both diagnosis and treatment.
Therapeutic agent: A chemical compound, small molecule, or other composition, such as an antisense compound, antibody, peptide, nucleic acid molecule, protease inhibitor, hormone, chemokine or cytokine, capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. For example, therapeutic agents for melanoma include agents that prevent or inhibit development or metastasis of melanoma. As used herein, a “candidate agent” is a compound selected for screening to determine if it can function as a therapeutic agent for melanoma. In some embodiments, a “candidate agent” is an agent screened to determine if is capable of increasing kinase activity of ERBB4. “Incubating” includes a sufficient amount of time for an agent to interact with a cell or tissue. “Contacting” includes incubating an agent in solid or in liquid form with a cell or tissue. “Treating,” when used to refer to the treatment of a cell or tissue with a therapeutic agent, includes contacting or incubating an agent with the cell or tissue.
Transformation: Refers to the transition of a normal cell to a malignant cell.
Tumor, neoplasia, malignancy or cancer: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as “malignant.” A “non-cancerous tissue” is a tissue from the same organ wherein the malignant neoplasm formed, but does not have the characteristic pathology of the neoplasm. Generally, noncancerous tissue appears histologically normal. A “normal tissue” is tissue from an organ, wherein the organ is not affected by cancer or another disease or disorder of that organ. A “cancer-free” subject has not been diagnosed with a cancer of that organ and does not have detectable cancer.
UCN-01 (7-hydroxystaurosporine): A synthetic derivative of staurosporine with antineoplastic activity. UCN-01 inhibits many phosphokinases, including AKT, calcium-dependent protein kinase C, and cyclin-dependent kinases. The chemical structure name of UCN-01 is 8,12-epoxy-1H,8H-2,7b,12a-triazadibenzo[a, g]cyclonona[cde]trinden-1-one, 2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino).
Wortmannin: A furanosteroid metabolite of the fungi Penicillium funiculosum, Talaromyces (Penicillium) wortmannii, is a specific, covalent inhibitor of PI3K. The molecular formula of wortmannin is C23H24O8.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
IV. Overview of Several Embodiments
It is disclosed herein that melanoma patients exhibit a number of different mutations in PTK family members (see Table 4). In particular, it is demonstrated that ERBB4 is highly mutated in metastatic melanoma. Described herein are novel ERBB4 mutations, which result in expression of ERBB4 protein with increased kinase activity. In addition, cells expressing mutant ERBB4 exhibit transformation capacity. The ERBB4 mutations disclosed herein also activate the PI3K/AKT pathway.
Provided herein is a method of predicting the prognosis of a subject diagnosed with melanoma, comprising detecting the presence or absence of a mutation in the ERBB4 gene, wherein the presence of a mutation in the ERBB4 gene predicts a poor prognosis. A poor prognosis refers to any negative clinical outcome. For example, in some embodiments, a poor prognosis is an increase in the likelihood of death. In some embodiments, a poor prognosis is an increase in the likelihood of metastasis of the melanoma.
Further provided is a method of selecting a subject diagnosed with melanoma as a candidate for treatment with an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both, comprising detecting the presence or absence of a mutation in the ERBB4 gene, wherein the presence of a mutation in the ERBB4 gene indicates that the subject is a candidate for treatment with an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both.
In particular examples, the ERBB4 mutation is selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1).
In some embodiments, the method further comprises administering to the subject an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both. In some examples, the ERBB4 inhibitor is lapatinib. Agents that decrease expression or activity of ERBB4 or a member of the PI3K/AKT pathway (such as, but not limited to, AKT or PI3K) are known in the art, some of which are described herein.
In some embodiments of the methods, the ERBB4 mutation results in an increase in kinase activity of the ERBB4 protein. In particular examples, the ERBB4 mutation introduces an amino acid change selected from E317K, E452K, E542K, R544W, E563K, E836K and E872K (numbered with reference to SEQ ID NO: 2).
Methods of detecting mutations in a gene are well known in the art. Detection of one or more mutations in the ERBB4 gene can be accomplished using any suitable technique, such as those described in detail in the sections below. For example, ERBB4-specific primers can be used to amplify ERBB4 nucleic acid from a biological sample (such as a tumor tissue sample or blood sample). The amplified molecule can then be sequenced and compared to a reference ERBB4 sequence (such as SEQ ID NO: 1), or compared with ERBB4 from a control sample such as a non-cancerous tissue sample, to detect a mutation in ERBB4. ERBB4 amplification primers and sequencing primers can be designed according to well known methods. Examples of ERBB4 primers are provided in Table 1 and Table 3 (SEQ ID NOs: 3-60 and 64-77). Mutations in ERBB4 can also be detected using oligonucleotides that specifically hybridize with a particular mutation. Hybridization of such oligonucleotides can be detected by labeling the oligonucleotide with a detectable marker, such as a fluorescent marker, enzymatic marker or radioisotope.
For detection of ERBB4 mutations, nucleic acid (such as DNA or RNA) can be isolated from a biological sample according to well known methods. In some embodiments, the biological sample is tissue sample, such as a tumor tissue sample. In other embodiments, the biological sample is a fluid sample, such as blood. For example, nucleic acid can be isolated from cells obtained from a blood sample. In some embodiments, the biological sample is obtained from a patient diagnosed with melanoma. In some embodiments, the biological sample is obtained from a control subject.
Also provided is a method of identifying a therapeutic agent for the treatment of a subject diagnosed with melanoma, comprising screening candidate agents to select an agent that decreases activity (such as kinase activity) of ERBB4, thereby identifying a therapeutic agent for the treatment of a subject with melanoma. In particular examples, the ERBB4 mutation is selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1).
In some embodiments, the candidate agent is a small molecule, polypeptide (such as an antibody) or nucleic acid molecule (such as an antisense compound, including antisense oligonucleotides, siRNAs or ribozymes). In some examples, screening comprises contacting the candidate agents with cells expressing ERBB4. In some embodiments, the cells express WT ERBB4. In other embodiments, the cells express ERBB4 protein comprising a mutation selected from E317K, E452K, E542K, R544W, E563K, E836K and E872K (numbered with reference to SEQ ID NO: 2). In some embodiments, the therapeutic agent increases kinase activity of ERBB4 at least 2-fold, at least 3-fold, at least 4-fold or at least 5-fold relative to untreated cells.
Further provided is a method of identifying a therapeutic agent for the treatment of a subject diagnosed with melanoma, comprising screening candidate agents to select an agent that decreases expression or activity of a member of the PI3K/AKT pathway, thereby identifying a therapeutic agent for the treatment of a subject with melanoma. In some cases, the agent decreases activity of AKT, such as by reducing phosphorylation of AKT. In some embodiments, the candidate agent is a small molecule, polypeptide (such as an antibody) or nucleic acid molecule (such as an antisense compound, including antisense oligonucleotides, siRNAs or ribozymes).
Further provided herein are oligonucleotides that specifically hybridize with an ERBB4 nucleic acid molecule, wherein the ERBB4 nucleic acid molecule comprises a mutation selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). In some embodiments, the oligonucleotide is about 15 to about 40 nucleotides in length. In some embodiments, the oligonucleotide comprises a label, such as, but not limited to a fluorescent label, an enzymatic label or a radioisotope.
Also provided are arrays comprising one or more oligonucleotides that specifically hybridize with an ERBB4 nucleic acid molecule, wherein the ERBB4 nucleic acid molecule comprises a mutation selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). In some embodiments, the array is a microarray.
V. Methods of Detecting ERBB4 Mutations
Disclosed herein is the identification of novel mutations in ERBB4, which result in expression of ERBB4 protein with enhanced kinase activity. Seven mutations in human ERBB4 were identified, including G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1).
Detecting mutations in ERBB4 can be accomplished using any technique known in the art. For example, the presence or absence of an ERBB4 mutation can be determined by conventional methods such as gene or RNA detection methods (for example, DNA sequencing, oligonucleotide hybridization, polymerase chain reaction (PCR) amplification with primers specific to the mutation), or protein detection methods (for example, immunoassays or biochemical assays to identify a mutated ERBB4 protein, such as an ERBB4 with decreased kinase activity or increased cell migration capacity). Generally, the nucleic acid sequence of the ERBB4 gene or RNA in a sample can be detected by any suitable method or technique of detecting gene sequence. Such methods include, but are not limited to, PCR, reverse transcriptase-PCR(RT-PCR), in situ PCR, in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, or other DNA/RNA hybridization platforms.
Detection of point mutations in target nucleic acids can be accomplished by molecular cloning of the target nucleic acid molecules and sequencing the nucleic acid molecules using techniques well known in the art. Alternatively, amplification techniques such as PCR can be used to amplify target nucleic acid sequences directly from a genomic DNA preparation from a tumor tissue or cell sample. The nucleic acid sequence of the amplified molecules can then be determined to identify mutations. Representative primer pairs that can be used to amplify ERBB4 nucleic acid from a biological sample are provided in Tables 1 and 3 (SEQ ID NOs: 3-60 and 64-77). However, design and selection of appropriate primers is well within the abilities of one of ordinary skill in the art.
The ligase chain reaction (Wu et al., Genomics 4:560-569, 1989) and allele-specific PCR (Ruano and Kidd, Nucleic Acids Res. 17:8392, 1989) can also be used to amplify target nucleic acid sequences. Amplification by allele-specific PCR uses primers that hybridize at their 3′ ends to a particular target nucleic acid mutation. If the particular mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System can also be used to detect mutations in nucleic acid sequences (U.S. Pat. No. 5,595,890; Newton et al., Nucleic Acids Res. 17:2503-2516, 1989). Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. Single stranded conformation polymorphism analysis can also be used to detect base change variants of an allele (Orita et al., Proc. Natl. Acad. Sci. USA 86:2766-2770, 1989). Other known techniques for detecting insertions and deletions can also be used with the claimed methods.
Mismatch detection can be used to detect point mutations in a target nucleic acid molecule, such as ERBB4. Mismatches are hybridized nucleic acid duplexes which are not 100% complementary. The lack of total complementarity can be due to deletions, insertions, inversions, substitutions or frameshift mutations. An example of a mismatch cleavage technique is the RNase protection method, which is described in detail in Winter et al. (Proc. Natl. Acad. Sci. USA 82:7575-7579, 1985) and Myers et al. (Science 230:1242-1246, 1985). For example, detection of mutations in ERBB4 can involve the use of a labeled riboprobe that is complementary to wild-type ERBB4. The riboprobe and nucleic acid molecule to be tested (for example, obtained from a tumor sample) are annealed (hybridized) together and subsequently digested with the enzyme RNase A, which is able to detect mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full-length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the full length of the target nucleic acid mRNA or gene, but can a portion of the target nucleic acid, provided it encompasses the position suspected of being mutated. If the riboprobe comprises only a segment of the target nucleic acid mRNA or gene, it may be desirable to use a number of these probes to screen the whole target nucleic acid sequence for mismatches if desired.
In a similar manner, DNA probes can be used to detect mismatches, for example through enzymatic or chemical cleavage (Cotton et al., Proc. Natl. Acad. Sci. USA 85: 4397-4401, 1988; Shenk et al., Proc. Natl. Acad. Sci. USA 72:989-993, 1975). Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes (Cariello, Am. J. Hum. Genet. 42:726-734, 1988). With either riboprobes or DNA probes, the target nucleic acid mRNA or DNA which may contain a mutation can be amplified before hybridization. Changes in target nucleic acid DNA can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
Amplified nucleic acid sequences can also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the target nucleic acid gene harboring a known mutation. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the target gene sequence. By use of a battery of such allele-specific probes, target nucleic acid amplification products can be screened to identify the presence of a previously identified mutation in the target gene. Hybridization of allele-specific probes with amplified target nucleic acid sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the tumor tissue as in the allele-specific probe.
The ERBB4 primer pairs disclosed herein are useful for determination of the nucleotide sequence of a target nucleic acid using nucleic acid amplification techniques such as the polymerase chain reaction. The pairs of single stranded DNA primers can be annealed to sequences within or surrounding the target nucleic acid sequence in order to prime amplification of the target sequence. Allele-specific primers can also be used. Such primers anneal only to particular mutant target sequence, and thus will only amplify a product in the presence of the mutant target sequence as a template. In order to facilitate subsequent cloning of amplified sequences, primers may have restriction enzyme site sequences appended to their ends. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available. Design of particular primers is well within the skill of the art.
Nucleic acid probes that hybridize with an ERBB4 nucleic acid molecule, such as a wild-type ERBB4 nucleic acid molecule or a mutant ERBB4 nucleic acid molecule described herein, are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA and in RNase protection assays for detecting point mutations. The probes can also be used to detect target nucleic acid amplification products. ERBB4 probes can also be used to detect mismatches with the wild type gene or mRNA using other techniques. Mismatches can be detected using either enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamine or osmium tetroxide and piperidine), or changes in electrophoretic mobility of mismatched hybrids as compared to totally matched hybrids (Novack et al., Proc. Natl. Acad. Sci. USA 83:586, 1986).
Mutations in nucleic acid molecules can also be detected by screening for alteration of the corresponding protein. For example, monoclonal antibodies immunoreactive with a target gene product can be used to screen a tissue, for example an antibody that is known to bind to a particular mutated position of the gene product (protein). For example, a suitable antibody may be one that binds to a deleted exon or that binds to a conformational epitope comprising a deleted portion of the target protein. Lack of cognate antigen would indicate a mutation. Such immunological assays can be accomplished using any convenient format known in the art, such as Western blot, immunohistochemical assay and enzyme-linked immunosorbent assay (ELISA). In some embodiments, the ERBB4 amino acid mutation is selected from E317K, E452K, E542K, R544W, E563K, E836K and E872K (numbered with reference to SEQ ID NO: 2).
VI. Arrays
In particular embodiments provided herein, arrays can be used to evaluate the presence or absence of mutations in ERBB4. In some examples, the array comprises an oligonucleotide that specifically hybridizes with an ERBB4 nucleic acid molecule comprising a mutation selection from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). Oligonucleotides that specifically hybridize with an ERBB4 nucleic acid comprising a mutation do not hybridize to WT ERBB4, or hybridization of the oligonucleotide to WT ERBB4 is significantly weaker than hybridization to the mutant ERBB4. In some embodiments the array comprises two or more oligonucleotides that specifically hybridize with an ERBB4 nucleic acid comprising a mutation selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). In other embodiments, the array comprises oligonucleotides that specifically hybridize with ERBB4 nucleic acid molecules comprising each mutation of G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). In some examples, the array further comprises other oligonucleotides, such as control oligonucleotides or oligonucleotides that specifically hybridize with WT ERBB4 or other mutant ERBB4 nucleic acid molecules. Exemplary control oligonucleotide probes include GAPDH, actin, and YWHAZ.
The oligonucleotide probes can further include one or more detectable labels, to permit detection of hybridization signals between the probe and target sequence (such as one of the mutant ERBB4 nucleic acid molecules).
Array Substrates
The solid support of the array can be formed from an organic polymer. Suitable materials for the solid support include, but are not limited to: polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluoroethylene, polyvinylidene difluoroide, polyfluoroethylene-propylene, polyethylenevinyl alcohol, polymethylpentene, polycholorotrifluoroethylene, polysulformes, hydroxylated biaxially oriented polypropylene, aminated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, etyleneacrylic acid, thylene methacrylic acid, and blends of copolymers thereof (see U.S. Pat. No. 5,985,567).
In general, suitable characteristics of the material that can be used to form the solid support surface include: being amenable to surface activation such that upon activation, the surface of the support is capable of covalently attaching a biomolecule such as an oligonucleotide thereto; amenability to “in situ” synthesis of biomolecules; being chemically inert such that at the areas on the support not occupied by the oligonucleotides are not amenable to non-specific binding, or when non-specific binding occurs, such materials can be readily removed from the surface without removing the oligonucleotides.
In one example, the solid support surface is polypropylene. Polypropylene is chemically inert and hydrophobic. Non-specific binding is generally avoidable, and detection sensitivity is improved. Polypropylene has good chemical resistance to a variety of organic acids (such as formic acid), organic agents (such as acetone or ethanol), bases (such as sodium hydroxide), salts (such as sodium chloride), oxidizing agents (such as peracetic acid), and mineral acids (such as hydrochloric acid). Polypropylene also provides a low fluorescence background, which minimizes background interference and increases the sensitivity of the signal of interest.
In another example, a surface activated organic polymer is used as the solid support surface. One example of a surface activated organic polymer is a polypropylene material aminated via radio frequency plasma discharge. Such materials are easily utilized for the attachment of nucleotide molecules. The amine groups on the activated organic polymers are reactive with nucleotide molecules such that the nucleotide molecules can be bound to the polymers. Other reactive groups can also be used, such as carboxylated, hydroxylated, thiolated, or active ester groups.
Array Formats
A wide variety of array formats can be employed in accordance with the present disclosure. One example includes a linear array of oligonucleotide bands, generally referred to in the art as a dipstick. Another suitable format includes a two-dimensional pattern of discrete cells (such as 4096 squares in a 64 by 64 array). As is appreciated by those skilled in the art, other array formats including, but not limited to slot (rectangular) and circular arrays are equally suitable for use (see U.S. Pat. No. 5,981,185). In some examples, the array is a multi-well plate. In one example, the array is formed on a polymer medium, which is a thread, membrane or film. An example of an organic polymer medium is a polypropylene sheet having a thickness on the order of about 1 mil. (0.001 inch) to about 20 mil., although the thickness of the film is not critical and can be varied over a fairly broad range. The array can include biaxially oriented polypropylene (BOPP) films, which in addition to their durability, exhibit low background fluorescence.
The array formats of the present disclosure can be included in a variety of different types of formats. A “format” includes any format to which the solid support can be affixed, such as microtiter plates (e.g. multi-well plates), test tubes, inorganic sheets, dipsticks, and the like. For example, when the solid support is a polypropylene thread, one or more polypropylene threads can be affixed to a plastic dipstick-type device; polypropylene membranes can be affixed to glass slides. The particular format is, in and of itself, unimportant. All that is necessary is that the solid support can be affixed thereto without affecting the functional behavior of the solid support or any biopolymer absorbed thereon, and that the format (such as the dipstick or slide) is stable to any materials into which the device is introduced (such as clinical samples and hybridization solutions).
The arrays of the present disclosure can be prepared by a variety of approaches. In one example, oligonucleotide sequences are synthesized separately and then attached to a solid support (see U.S. Pat. No. 6,013,789). In another example, sequences are synthesized directly onto the support to provide the desired array (see U.S. Pat. No. 5,554,501). Suitable methods for covalently coupling oligonucleotides to a solid support and for directly synthesizing the oligonucleotides onto the support are known to those working in the field; a summary of suitable methods can be found in Matson et al. (Anal. Biochem. 217:306-10, 1994). In one example, the oligonucleotides are synthesized onto the support using conventional chemical techniques for preparing oligonucleotides on solid supports (such as see International application publications WO 85/01051 and WO 89/10977, or U.S. Pat. No. 5,554,501).
A suitable array can be produced using automated means to synthesize oligonucleotides in the cells of the array by laying down the precursors for the four bases in a predetermined pattern. Briefly, a multiple-channel automated chemical delivery system is employed to create oligonucleotide probe populations in parallel rows (corresponding in number to the number of channels in the delivery system) across the substrate. Following completion of oligonucleotide synthesis in a first direction, the substrate can then be rotated by 90° to permit synthesis to proceed within a second)(2° set of rows that are now perpendicular to the first set. This process creates a multiple-channel array whose intersection generates a plurality of discrete cells.
The oligonucleotides can be bound to the polypropylene support by either the 3′ end of the oligonucleotide or by the 5′ end of the oligonucleotide. In one example, the oligonucleotides are bound to the solid support by the 3′ end. However, one of skill in the art can determine whether the use of the 3′ end or the 5′ end of the oligonucleotide is suitable for bonding to the solid support. In general, the internal complementarity of an oligonucleotide probe in the region of the 3′ end and the 5′ end determines binding to the support.
In particular examples, the oligonucleotide probes on the array include one or more labels, that permit detection of oligonucleotide probe:target sequence hybridization complexes.
VII. Use of ERBB4 for Prognosis and Therapy
It is disclosed herein that ERBB4 is highly mutated in melanoma tumors. The disclosed ERBB4 somatic mutations result in increased ERBB4 kinase activity, transformation capacity and anchorage-independent growth. The high frequency of mutations identified in ERBB4, their co-localization, and the identification of two identical missense mutations (E452K and E872K) in multiple MM samples indicates these mutations play a role in tumorigenesis. In addition, the ERBB4 mutations disclosed herein exhibit ligand-independent basal phosphorylation, providing evidence that these mutations are oncogenic. Accordingly, the identified mutations in ERBB4 predict a poor prognosis for patients with melanoma. In some embodiments, a poor prognosis is an increase in the likelihood of death. In some embodiments, a poor prognosis is an increase in metastasis.
The detection of one or more ERBB4 mutations selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1) can be used as a clinical tool to determine the prognosis of a patient with melanoma. Since these mutations are oncogenic and play a role in tumorigenesis of MM, a poor prognosis is indicated when one or more of the mutations is detected in a sample from a subject diagnosed with melanoma. Detection of one or more of these mutations can also be used as a tool for determining an appropriate therapy for a subject with melanoma. The presence of one or more of these mutations indicates the subject is a candidate for treatment with a kinase inhibitor, such an EGFR family inhibitor, or more particularly, an ERBB4-specific inhibitor. In some examples, the ERBB4 mutation introduces an amino acid change selected from E317K, E452K, E542K, R544W, E563K, E836K and E872K (numbered with reference to SEQ ID NO: 2).
It is also disclosed herein that mutations in ERBB4 activate the PI3K/AKT pathway, as indicated by increased phosphorylation of AKT in melanoma cells harboring the disclosed ERBB4 mutations. Thus, the presence of one or more ERBB4 mutations indicates the subject is a candidate for treatment with an inhibitor of the PI3K/AKT pathway, such as an inhibitor of PI3K or AKT. In some embodiments, the method of selecting a patient as a candidate for treatment with an ERBB4 and/or PI3K/AKT pathway inhibitor further includes treating the subject with an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both.
The finding that the presence of prognosis-associated ERBB4 mutations selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1) result in an increase in kinase activity, transformation capacity and/or anchorage-independent growth indicates that compounds that inhibit (such as decrease kinase activity of) ERBB4 will be useful as therapeutic agents for the treatment of melanoma. Thus, provided herein is a method of identifying therapeutic agents for the treatment of melanoma, comprising screening candidate agents to select an agent that inhibits activity (such as kinase activity) or expression of ERBB4.
In some embodiments, screening comprises contacting the candidate agents with cells that express ERBB4 and detecting any change in activity or expression of ERBB4. The ERBB4 expressing cells can be primary cells obtained from a subject diagnosed with melanoma, immortalized or transformed cells obtained from a melanoma patient, or the cells can be commercially available immortalized cell lines. In some embodiments, the cells express wild-type ERBB4. In other embodiments, the cells express mutant ERBB4, such as ERBB4 with a mutation selection from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). In some examples, a cell line is transfected with an expression vector encoding wild-type or mutant ERBB4. In other examples, primary tumor cells expressing mutant ERBB4 are evaluated. In either case, the cells are either untreated or treated with a candidate agent and ERBB4 kinase activity is measured, for example by incorporation of radiolabeled ATP. A decrease in ERBB4 activity in the treated cells, compared to the untreated cells, indicates the candidate agent is a therapeutic agent for melanoma.
In some embodiments, a decrease in kinase activity of ERBB4 following treatment with the candidate agent identifies the agent as a therapeutic agent for the treatment of melanoma. In some embodiments, the therapeutic agent decreases kinase activity of ERBB4 at least 2-fold, at least 3-fold, at least 4-fold or at least 5-fold relative to untreated cells. Methods of screening candidate agents to identify therapeutic agents for the treatment of disease are well known in the art. In one embodiment, screening comprises a high-throughput screen. In another embodiment, candidate agents are screened individually.
Given the finding that mutations in ERBB4 result in activation of the PI3K/AKT pathway, provided herein is a method of identifying therapeutic agents for the treatment of melanoma, comprising screening candidate agents to select an agent that inhibits activity or expression of a member of the PI3K/AKT pathway, such as PI3K or AKT. In some embodiments, screening comprises contacting the candidate agents with cells that express mutant ERBB4 and detecting any change in activity or expression of a member of the PI3K/AKT pathway. The mutant ERBB4 expressing cells can be primary cells obtained from a subject diagnosed with melanoma, immortalized or transformed cells obtained from a melanoma patient, or the cells can be commercially available immortalized cell lines. In some embodiments, the cells express ERBB4 with a mutation selection from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1). In some examples, a cell line is transfected with an expression vector encoding mutant ERBB4. In other examples, primary tumor cells expressing mutant ERBB4 are evaluated. In either case, the cells are either untreated or treated with a candidate agent and PI3K/AKT activity is measured. In some examples, PI3K/AKT activity is measured by detecting the level of AKT phosphorylation. A decrease in PI3K/AKT activity in the treated cells, compared to the untreated cells, indicates the candidate agent is a therapeutic agent for melanoma.
The candidate agents can be any type of molecule, such as, but not limited to nucleic acid molecules, proteins, polypeptides, antibodies, lipids, small molecules, chemicals, cytokines, chemokines, hormones, or any other type of molecule that may alter ERBB4 or PI3K/AKT activity either directly or indirectly. In some embodiments, the candidate agents are small molecules, polypeptides (such as antibodies) or nucleic acid molecules (such as antisense compounds, including antisense oligonucleotides, siRNAs or ribozymes).
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
EXAMPLES
Example 1
Experimental Procedures
This example describes the materials and methods used for the experiments described in Examples 2-6.
Amplification, Sequencing and Mutational Analysis of ERBB4
Metastatic melanoma samples and their matched normal samples were obtained according to standard procedures. Genomic DNA was isolated using DNeasyTM Blood & Tissue kit (Qiagen, Valencia, Calif.). For all samples, matching between germline and tumor DNA was verified by direct sequencing of 26 single nucleotide polymorphisms (SNPs) at 24 loci. The tissue and melanoma cell lines used in the Examples below are also described in Palavalli et al. (Nat. Genet. 41:518-520, 2009).
PCR and sequencing primers were designed using Primer 3 (available online at frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and synthesized by Invitrogen (Carlsbad, Calif.). The primers used for PCR amplification of the whole coding region of ERBB4 are shown in Table 1 (CCDS accession CCDS2394.1; GenBank Accession No. NM—005235; SEQ ID NO: 1). The coding region of ERBB4 was sequenced using a primer with the following sequence: GTAAAACGACGGCCAGT (SEQ ID NO: 61). PCR amplification, sequencing and analysis were performed as previously described (Samuels et al., Science 304:554, 2004). Briefly, PCR products were purified using exonuclease (Epicentre Biotechnologies, Madison, Wis.) and shrimp alkaline phosphatase (USB Corporation, Cleveland, Ohio). Products were purified with rehydrated SephadexTM 4 G-50 powder (GE Healthcare, Piscataway, N.J.) and cycle sequencing was carried out using BigDye TerminatorTM v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, Calif.). Sequence data was collected on an ABI3730x1 (Applied Biosystems, Foster City, Calif.).
The kinase domain mutation screen was analyzed using Consed (Gordon et al., Genome Res. 8(3):195-202, 1998). Variants were called using Polyphred 6.11 (Bhangale et al., Nat. Genet. 38(12):1457-1462, 2006) and DIPDetector, an indel detector for improved sensitivity in finding insertions and deletions.
Sequence traces of the secondary screen were analyzed using the Mutation Surveyor software package (SoftGenetics, State College, Pa.).







TABLE 1







Primers used for PCR amplification of the


ERBB4 coding region













SEQ

SEQ


ERBB4

ID

ID


Exon
Forward Primer
NO:
Reverse Primer
NO:














Exon 1
GGAAATAGCTGCACA
3
GTAAAACGACGGCC
32



GTCCG

AGTATGGGTGAAGA






GGGCAGG



 


Exon 2
AGAACTGGGATAGGC
4
GTAAAACGACGGCC
33



TTGTGG

AGTTTCCAGGTATCA






GCACACAGG



 


Exon 3
GTAAAACGACGGCCA
5
TGCCTTAGAGTGTTC
34



GTAAGCCAATTCTTTA

CTCAATG




GAATATGATATGG





 


Exon 4
TCTTGGCTATTAGCAA
6
GTAAAACGACGGCC
35



CATGACTC

AGTCAATGAATGCAA






TCAAAGTTCAA



 


Exon 5
GTAAAACGACGGCCA
7
CCAAAGCAAATCAAC
36



GTAAATCCTCATAAAG

CACAAG




GAGCAGGAG





 


Exon 6
GTAAAACGACGGCCA
8
GGAATGACTTTGAGG
37



GTTGAATTGAGTCAAA

AGGGC




GACAGGGTG





 


Exon 7
TTTGGAAACACACATG
9
GTAAAACGACGGCC
38



ACTCTTAAA

AGTTTTGCTATGAAA






CTTTACACAAATCA



 


Exon 8
GTGGAGCAGTAACCA
10
GTAAAACGACGGCC
39



AGCAAG

AGTGTGTGGGTAGGT






TTGGTTGTG



 


Exon 9
AAAGCAGAACCAGTA
11
GTAAAACGACGGCC
40



GTGAATGTTG

AGTGGTGAAACTCTT






CAGCTTCCAG



 


Exon 10
GTAAAACGACGGCCA
12
TCTCCTGACCTCATG
41



GTCCTCCTCCACATCT

ATCCAC




AGCACAG





 


Exon 11
GTAAAACGACGGCCA
13
TACCTCACACCATCA
42



GTCCTTTCTCACTTC 

TCGGAG




CCAACTTTC





 


Exon 12
GTAAAACGACGGCCA
14
GAGCAACAATTCTGA
43



GTTTGATTCAGTTTCC

CCGGAT




ATTTATACACCA





 


Exon 13
GTAAAACGACGGCCA
15
GAATGGCGTGAACCC
44



GTTAGGCCACCAAAGT

AGG




CATTTGC





 


Exon 14
GTAAAACGACGGCCA
16
CCCATGGCATCCTGT
45



GTTGATGCTCCTGGCA

AAGTAG




CATAGAG





 


Exon 15
TCTTAGAGGAAGATTT
17
GTAAAACGACGGCC
46



GCCACC

AGTCATTTCAGAGAT






GGTACCAGGG



 


Exon 16
GCTTCCCATGTTCTTC
18
GTAAAACGACGGCC
47



CTCC

AGTAAGTAAGAAAG






TTGGCTTGAGAAGG



 


Exon 17
TGTGGATAATGTCTTG
19
GTAAAACGACGGCC
48



TACAACTGC

AGTTTCACAAGCTTT






GTTTAACGGAC



 


Exon 18
GGTTGTCAAGGCAAAC
20
GTAAAACGACGGCC
49



CAAG

AGTAGACTGTATCCG






TCCCAGCTC



 


Exon 19
AAGCAGACAACAAAG
21
GTAAAACGACGGCC
50



TTGCAGAG

AGTTCTAGGCAGACA






GTTGTGAAGC



 


Exon 20
GTAAAACGACGGCCA
22
TTTGGCACCTAGTCA
51



GTTCAGCACCATTAGT

ATTCAA




ACAATCCAA





 


Exon 21
GTAAAACGACGGCCA
23
AGGCAAATGGTAGA
52



GTGCACTTCCAACTGA

ACCAAGG




AGGCTAAG





 


Exon 22
GTAAAACGACGGCCA
24
TAACTGCTTTAGGAA
53



GTAGGCCAGCCCAAA

ATTAGGCTTATC




GACTC





 


Exon 23
TGATTGGTGTTTGGAT
25
GTAAAACGACGGCC
54



TGACC

AGTCAAAGAGGCGTT






CATATGTTCC



 


Exon 24
GTAAAACGACGGCCA
26
TGTTTGTGGTCCTTTC
55



GTGAGTCGTTTCTTTC

CACAG




ACTAGCTTGC





 


Exon 25
TAGGTTTCTTAATGGC
27
GTAAAACGACGGCC
56



CGGTG

AGTGGCATCACATTG






ATTTGAGCTA



 


Exon 26
TGCTTAGGAAGCTTCA
28
GTAAAACGACGGCC
57



CTGTTG

AGTTAACTCACTGTT






GGCAAAGGC



 


Exon 27
TGGCTTTGATATCCTT
29
GTAAAACGACGGCC
58



GTGGC

AGTCAGCTATCTGGC






AATTTCTATTCTG



 


Exon 28
CCATATGCAGAAGAG
30
GTAAAACGACGGCC
59



ACAAATGC

AGTAGGTAGTCTGGG






TGCTGAAGG



 


Exon 28
TGAATCCAGTGGAGG
31
GTAAAACGACGGCC
60



AGAACC

AGTGACCACCAGAG






AAAGAGAGGG









Construction of Wild-Type and Mutant ERBB4 Expression Vectors

Human ERBB4 (GenBank Accession No. NM—005235; SEQ ID NO: 1) was cloned by PCR using PHUSIONTM Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Inc., Ipswich, Mass.) using a clone purchased from Open Biosystems (clone ID #8327667) and cloned with the following primers:








CGGCTCTAGAGCCACCATGAAGCCGGCGAC
(SEQ ID NO: 62)


 


ATCGGCGGCCGCTTACACCACAGTATTCCGG
(SEQ ID NO: 63)






The PCR product was cloned into the mammalian expression vector pCDF-MCS2-EF1-PuroTM (Systems Biosciences, Inc., Mountain View, Calif.) via the XbaI and NotI restriction sites. The E317K, E452K, E542K, R544W, E563K, K751M, E836K, and E872K point mutants were made using Fusion PCR for site-directed mutagenesis.
Cell Culture and Transient Expression
Metastatic melanoma tumor lines were maintained according to standard methods (see Chappell et al., Cancer Res. 59:59-62, 1999). HEK 293T cells and NIH 3T3 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, Va.) and maintained in complete Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1× nonessential amino acids, 2 mM L-glutamine, and 0.75% sodium bicarbonate. HEK 293T cells were transfected with LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, Calif.) at a 6:1 ratio with DNA (μl:μg) using 3-5 μg of plasmid DNA.
Immunoprecipitation and Western Blotting
Transfected cells were washed 3× in PBS and lysed using 0.5 ml 1% NP-40 lysis buffer (1% NP-40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, complete protease inhibitor tablet, EDTA-free (Roche, Indianapolis, Ind.), 1 μM sodium orthovanadate, 1 mM sodium fluoride, and 0.1% β-mercaptoethanol) per T-75 flask for 20 minutes on ice. Lysed cells were scraped and transferred into a 1.5 mL microcentrifuge tube. Extracts were centrifuged for 10 minutes at 14,000 rpm at 4° C. Supernatant (450 μl) was immunoprecipitated overnight using 20 μl of anti-ERBB4 agarose-conjugated beads (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). The immunoprecipitates were washed and subjected to SDS-PAGE and western blotting according to standard methods (see Samuels et al., Science 304:554, 2004). The primary antibodies used in these experiments were anti-ERBB4 (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-P-ERBB4 (Y1162) (Abgent, San Diego, Calif.), anti-P-ERBB4 (Y1284) (Cell Signaling, Danvers, Mass.), anti-PY20 (Zymed-Invitrogen), anti-P-ERK1/2 (T202/Y204), anti-ERK1/2, anti-P-AKT (S473), anti-AKT (Cell Signaling), anti-P-STAT5A/B (Y694/Y699) (Upstate Biotech-Millipore), anti-STAT5 (Cell Signaling) and anti-α-tubulin (Calbiochem-EMD Biosciences, Gibbstown, N.J.).
ERBB4-Phosphosite-Specific Antibody Analysis
ERBB4 was immunoprecipitated as described above and subjected to SDS-PAGE. Primary phospho-antibodies were pre-incubated overnight with the relevant competitive phospho-peptides (pPep Y1162-Abgent #BP3122a, pPep Y1284-Cell Signaling #1022). Following blocking/competition, the antibody/peptide mixture was diluted into blocking buffer and western blotting was performed as described above.
Pooled Stable Expression
To make lentivirus, ERBB4 constructs were co-transfected into HEK 293T cells seeded at 1.5×106 per T75 flask with pVSV-G and pFIV-34N helper plasmids (System Biosciences, Mountain View, Calif.) using LipofectamineTM 2000. Virus-containing conditioned media was harvested 48-60 hours after transfection, filtered, aliquoted and stored at −80° C. ERBB4 lentivirus was used to make SK-Mel-2 and NIH 3T3 stable clones.
SK-Mel-2 cells (National Cancer Institute, Division of Cancer Treatment, Developmental Therapeutics Program, Frederick, Md.) were grown in RPMI-1640 (Lonza, Walkersville, Md.) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah). NIH 3T3 cells were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1× non-essential amino acids, and 0.75% sodium bicarbonate. Sk-Mel-2 and NIH 3T3 cells were seeded at 1.5×106 cells per T75 flask 24 hours prior to infection. Lentivirus for ERBB4 (WT, E317K, E452K, E542K, R544W, E563K, K751M, E836K, and E872K point mutants) and empty vector control were diluted with equal volume of normal complete medium in the presence of 8 μg/ml polybrene. Cells were incubated for 24 hours in the presence of virus followed by changing of the medium to normal complete medium for an additional 24 hours. Lentivirus-infected cells were then selected for by addition of complete medium containing 3 g/ml puromycin for SK-Mel-2 cells or 2 g/ml puromycin for NIH 3T3 cells and allowed to incubate for 3 days. Stable expression of ERBB4 proteins (WT and mutants) was determined by SDS-PAGE analysis followed by immunoblotting with anti-ERBB4 and anti-tubulin to show equivalent expression among pools.
Lentiviral shRNA
Constructs for stable depletion of ERBB4 were obtained from Open Biosystems (Huntsville, Ala.). Negative control constructs in the same vector system (pLK0.1 vector alone and scrambled shRNA) were obtained from Addgene (Cambridge, Mass.). To prepare transient virus stocks, 1.5×106 HEK 293T cells were plated in T75 flasks. The next day, the cells were co-transfected with shRNA constructs (3 μg), together with pHR'8.2AR and pCMV-VSV-G helper constructs (3 μg and 0.3 μg, respectively), using LipofectamineTM 2000 (Invitrogen). The media were changed the next day, and the following day, and virus-containing media were harvested. The viral stocks were centrifuged and filtered to remove any non-adherent HEK 293T cells.
Next, MM lines (2T, 7T, 17T, 31T and 63T) were infected with shRNA lentiviruses for each condition (vector and scrambled controls and three independent ERBB4-specific shRNAs). To do this, cells were plated at sub-confluent densities. The next day, cells were infected with a cocktail of 1 ml virus-containing medium, 1 ml regular medium and 8 μg/ml polybrene. The medium was changed one day post-infection, and selective medium was added two days post-infection (2 μg/ml puromycin for all cells). After three days of puromycin selection, the mock-infected cells had all died. Stably infected pooled clones were tested in functional assays.
To rescue shRNA-mediated knock-down of ERBB4 in melanoma cell lines, the non-targetable ERBB4 lentivirus was made as described above and used to infect the melanoma cell line 17T. After infection, cells were given 48 to 72 hours to recover from infection prior to testing in functional assays.
Proliferation and Growth Inhibition Assays
To examine growth potential, melanoma cell lines (2T, 7T, 17T, 31T and 63T) stably infected with either vector or scrambled controls or ERBB4-specific shRNAs were seeded into 96-well plates at 2,500 cells per well and incubated for 13-17 days. Samples were analyzed every 48 hours by lysing cells in 50 μl 0.2% SDS/well and incubating for 2 hours at 37° C. prior to addition of 150 μl/well of SYBR Green I solution (1:750 SYBR Green I (Invitrogen-Molecular Probes) diluted in dH2O).
The effects of tyrosine kinase inhibitors (TKIs) on the proliferation of melanoma cell lines were tested by seeding 96-well plates at 5,000 cells/well in the presence or absence of serum-containing media and incubated for 24 hours prior to addition of TKIs. Increasing concentrations of lapatinib (Tykerb-GlaxoSmithKline) were added to each well in four replicates with DMSO as negative control. Plates were analyzed 72 hours post-addition of TKIs using the SYBR Green I proliferation assay described above.
To further test TKIs on melanoma cell lines, 96-well plates were seeded at 5,000 cells per well and incubated 24 hours prior to addition of TKIs (e.g. lapatinib) at concentrations from 10 nM to 30 μM. Once inhibitors were added, cells were incubated for 72 hours at 37° C. Cells were then analyzed according to previously described methods (Rusnak et al., Mol. Cancer Ther. 1:85-94, 2001). Plates were read at 650 nm on a Molecular Devices (Spectra Max) Plate Reader and analyzed using SoftMax v5 and GraphPad Prism v5.
Soft Agar Assay
SK-Mel-2 pooled ERBB4 clones were plates in duplicate at 1000 cells/well and NIH 3T3 pooled ERBB4 clones were plated in duplicate at 5000 cells/well in top plugs consisting of sterile 0.33% Bacto-Agar (BD, Sparks, Mo.) and 10% FBS (HyClone, Logan, Utah) in a 24-well plate. The lower plug contained sterile 0.5% Bacto-Agar and 10% FBS. After two weeks, the colonies were photographed and counted.
NIH 3T3 Transformation Assay
Each plasmid (150 ng) was transfected into NIH 3T3 cells cultured in 12-well plates by the calcium phosphate precipitation method. Twenty-four hours after transfection, 5% of transfected cells were transferred into T25 flasks and cultured for 10 days in normal growth medium. The cells were stained with Hema3 (Sigma St. Louis, Mo.) and analyzed for the presence of foci.
Analysis of ERBB4 Kinase Activity
HEK 293T cells were transiently transfected with ERBB4 (WT, E317K, E452K, E542K, R544W, E563K, E836K, E872K and kinase-dead K751M) or empty vector and incubated for 18-24 hours at 37° C. in the presence (10%) or absence (0.5%) of serum-containing medium prior to immunoprecipitation. Cells were harvested and approximately 3 mg of lysate was immunoprecipitated as described above and subjected to a kinase assay Immune complexes were washed three times in lysis buffer followed by two washes in kinase buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 3 mM MnCl2, 20 mM MgCl2, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and 1× complete protease inhibitor tablet) Immune complexes were resuspended in 50 μl kinase buffer and 10 μl was incubated in the presence of [γ-32P]ATP (3 μCi per reaction) for 15 minutes at 37° C. Kinase reactions were stopped by the addition of 2×SDS sample buffer and phosphorylated samples were resolved on 8% tris-glycine gels. Gels were fixed in a 50% methanol/7% acetic acid solution, washed three times in dH2O then stained for 1 hour in GelCodeTM Blue stain (Pierce) followed by destaining for an additional hour. Gels were dried prior to autoradiography.
Immunoblot Quantitation Analysis
Scanned films from western blot analysis of SDS-PAGE were analyzed using ImageJ (NIH software). Individual bands were quantitated and plots were generated to determine the intensities in each band. The data was then exported to Microsoft Excel and analyzed further for phospho:total ratios of protein.
Flow Cytometry Analysis
Melanoma cells were seeded into T-25 flasks at densities of 3×105 cells per flask in normal complete T2 medium and incubated at 37° C. for 24 hours prior to addition of lapatinib. Lapatinib or vehicle was added for 72 hours at a concentration of 5 μM. Cells were then harvested for FACS analysis by first removing the medium into a new conical tube followed by trypsinization of attached cells in T-25 flasks. Trypsinized cells and those from the medium were combined and washed in ice-cold PBS. Cells were collected by centrifugation at 1,000 rpm at 4° C. Ice-cold 70% ethanol was added to cell pellets and allowed to fix overnight at 4° C. followed by washing in ice-cold PBS. DNase-free RNase (Roche) was added to cells resuspended in 0.5-1 ml PBS and incubated at 37° C. for 30 minutes before adding 50-100 μl of propidium iodide (PI-0.5 mg/ml) (Roche). Cellular DNA content was analyzed on Becton Dickinson FACSCaliburTM using CellQuestTM software.
X-Ray Crystal Structure Assembly
The X-ray crystal structures of the ERBB4 extracellular and kinase domains were used as templates in the program SWISS-MODEL (Guex and Peitsch, Electrophoresis 18:2714-2723, 1997). Location of EGFR and ERBB2 mutations in the crystal were found by aligning the protein sequences for EGFR, ERBB2, ERBB3, and ERBB4 using ClustalW (Guex and Peitsch, Electrophoresis 18:2714-2723, 1997). Previously identified mutations in EGFR and ERBB2 were matched to the sequence of ERBB4 using the ClustalW alignment.
Statistical Analysis
To determine whether the ratio of nonsynonymous to synonymous mutations observed was statistically significant, the exact binomial test was used, with an expected ratio of 2.5:1. All the statistical calculations were performed in the R statistical environment (available online on the World Wide Web at r-project.org) (Sjoblom et al., Science 314:268-274, 2006). Further statistical analyses were performed using Microsoft Excel to generate p-values to determine significance (two-tailed t-test) Inhibition curves (IC50) were analyzed and plotted using GraphPad Prism v5.
Example 2
High-Throughput DNA Sequence Analysis of the PTK Family in MM
This example describes the identification of somatic mutations in members of the PTK family, including ERBB4, in patients with melanoma. Kinase mutations have been previously identified by sequencing genes encoding these domains (Bardelli et al., Science 300:949, 2003; Davies et al., Nature 417(6892):949-954, 2002; Greenman et al., Nature 446:153-158, 2007; Samuels et al., Science 304:554, 2004). Thus, PTKs were evaluated herein to determine if they are genetically altered in MM. Initially, the kinase domain coding exons of this gene superfamily were analyzed in 29 mM samples (Table 2). A total of 593 exons were extracted from genomic databases. These exons were amplified by polymerase chain reaction (PCR) from cancer genomic DNA samples using the primers listed in Table 1 and directly sequenced with dye terminator chemistry.







TABLE 2







Tyrosine Kinase genes analyzed










CCDS
Ref Seq




accession and
accession and


amplimer
amplimer


number
number
Gene Name
Gene Description





CCDS35165.1
NM_007313.2
ABL1/ABL
v-abl Abelson murine leukemia viral





oncogene homolog 1


CCDS30947.1
NM_007314.2
ABL2/ARG
v-abl Abelson murine leukemia viral





oncogene homolog 2 (arg, Abelson-related





gene)


CCDS33928.1
NM_005781.4
ACK1/TNK2
tyrosine kinase, non-receptor, 2


CCDS33172.1
NM_004304.3
ALK
anaplastic lymphoma kinase (Ki-1)


CCDS12575.1
NM_021913.3
AXL
AXL receptor tyrosine kinase


CCDS5982.1
NM_001715.2
BLK
B lymphoid tyrosine kinase


CCDS14168.1
NM_203281.2
BMX
BMX non-receptor tyrosine kinase


CCDS13524.1
NM_005975.2
BRK/PTK6
PTK6 protein tyrosine kinase 6


CCDS14482.1
NM_000061.1
BTK
Bruton agammaglobulinemia tyrosine





kinase


CCDS4302.1
NM_005211.2
CSF1R
colony stimulating factor 1 receptor,





formerly McDonough feline sarcoma viral





(v-fms) oncogene homolog


CCDS10269.1
NM_004383.1
CSK
c-src tyrosine kinase


CCDS4690.1
NM_001954.4
DDR1
discoidin domain receptor family, member





1


CCDS1241.1
NM_006182.2
DDR2
discoidin domain receptor family, member





2


CCDS5514.1
NM_005228.3
EGFR
epidermal growth factor receptor





(erythroblastic leukemia viral (v-erb-b)





oncogene homolog, avian)


CCDS5884.1
NM_005232.3
EPHA1
ephrin receptor EphA1


CCDS169.1
NM_004431.2
EPHA2
ephrin receptor EphA2


CCDS2922.1
NM_005233.5
EPHA3
ephrin receptor EphA3 isoform a precursor


CCDS2447.1
NM_004438.3
EPHA4
ephrin receptor EphA4


CCDS3514.1
NM_182472.1
EPHA5
ephrin receptor EphA5 isoform b


N/A
NM_001080448.2
EPHA6
EPH receptor A6 isoform a


CCDS5031.1
NM_004440.2
EPHA7
ephrin receptor EphA7


CCDS30626.1
NM_001006943.1
EPHA8
EPH receptor A8 isoform 2 precursor


CCDS425.1
NM_173641.2
EPHA10
EPH receptor A10 isoform 2


N/A
NM_004441.3
EPHB1
ephrin receptor EphB1 precursor


CCDS230.1
NM_004442.6
EPHB2
ephrin receptor EphB2 isoform 2 precursor


CCDS3268.1
NM_004443.3
EPHB3
ephrin receptor EphB3 precursor


CCDS5706.1
NM_004444.4
EPHB4
ephrin receptor EphB4 precursor


CCDS5873.1
NM_004445.2
EPHB6
ephrin receptor EphB6 precursor


CCDS32642.1
NM_004448.2
ERBB2
v-erb-b2 erythroblastic leukemia viral





oncogene homolog 2, neuro/glioblastoma





derived oncogene homolog (avian)


CCDS31833.1
NM_001982.2
ERBB3
v-erb-b2 erythroblastic leukemia viral





oncogene homolog 3 (avian)


CCDS2394.1
NM_005235.2
ERBB4
v-erb-a erythroblastic leukemia viral





oncogene homolog 4 (avian)


CCDS6381.1
NM_153831.2
FAK/PTK2
PTK2 protein tyrosine kinase 2


CCDS4098.1
NM_005246.2
FER
fer (fps/fes related) tyrosine kinase





(phosphoprotein NCP94)


CCDS10365.1
NM_002005.2
FES
V-FES feline sarcoma viral/V-FPS





fujinami avian


CCDS6107.1
NM_023110.2
FGFR1
fibroblast growth factor receptor 1 (fms-





related tyrosine kinase 2, Pfeiffer





syndrome)


CCDS31298.1
NM_000141.3
FGFR2
fibroblast growth factor receptor 2





(bacteria-expressed kinase, keratinocyte





growth factor receptor)


CCDS3353.1
NM_000142.2
FGFR3
fibroblast growth factor receptor 3





(achondroplasia, thanatophoric





dwarfism)William Allan Nix


CCDS4410.1
NM_002011.3
FGFR4
fibroblast growth factor receptor 4 isoform





1


CCDS305.1
NM_005248.2
FGR
Gardner-Rasheed feline sarcoma viral (v-





fgr) oncogene homolog


CCDS9330.1
NM_002019.3
FLT1/VEGFR1
fms-related tyrosine kinase 1 (vascular





endothelial growth factor/vascular





permeability factor receptor)


CCDS31953.1
NM_004119.2
FLT3
fms-related tyrosine kinase 3


CCDS4457.1
NM_182925.3
FLT4/VEGFR3
fms-related tyrosine kinase 4


CCDS5103.1
NM_002031.2
FRK
fyn-related kinase


CCDS5094.1
NM_002037.3
FYN
FYN oncogene related to SRC, FGR, YES


CCDS33460.1
NM_002110.2
HCK
hemopoietic cell kinase


CCDS10378.1
NM_000875.3
IGF1R
insulin-like growth factor 1 receptor


CCDS12176.1
NM_000208.2
INSR
insulin receptor


CCDS1160.1
NM_014215.1
INSRR
insulin receptor-related receptor


CCDS4336.1
NM_005546.3
ITK
IL2-inducible T-cell kinase


N/A
NM_002227.2
JAK1
Janus kinase 1


CCDS6457.1
NM_004972.2
JAK2
Janus kinase 2


CCDS12366.1
NM_000215.2
JAK3
Janus kinase 3


CCDS3497.1
NM_002253.1
KDR/VEGFR2
kinase insert domain receptor (a type III





receptor tyrosine kinase)


CCDS3496.1
NM_000222.2
KIT
v-kit Hardy-Zuckerman 4 feline sarcoma





viral oncogene homolog


CCDS359.1
NM_005356.3
LCK
lymphocyte-specific protein tyrosine





kinase


CCDS10078.1
NM_206961.1
LTK
leukocyte tyrosine kinase


CCDS6162.1
NM_002350.2
LYN
v-yes-1 Yamaguchi sarcoma viral related





oncogene homolog


CCDS12113.1
NM_002378.3
MATK
megakaryocyte-associated tyrosine kinase


CCDS2094.1
NM_006343.2
MERTK/MER
c-mer proto-oncogene tyrosine kinase


N/A
NM_000245.2
MET
met proto-oncogene (hepatocyte growth





factor receptor)


CCDS2807.1
NM_002447.2
MST1R/RON
macrophage stimulating 1 receptor (c-met-





related tyrosine kinase)


N/A
NM_005592.1
MUSK
muscle, skeletal, receptor tyrosine kinase


CCDS1161.1
NM_002529.3
NTRK1
neurotrophic tyrosine kinase, receptor, type





1


CCDS35053.1
NM_001007097.1
NTRK2
neurotrophic tyrosine kinase receptor type





2


CCDS32322.1
NM_001012338.1
NTRK3
neurotrophic tyrosine kinase receptor type





3


CCDS3495.1
NM_006206.3
PDGFRA
platelet-derived growth factor receptor





alpha


CCDS4303.1
NM_002609.3
PDGFRB
platelet-derived growth factor receptor beta


CCDS4884.1
NM_002821.3
PTK7
PTK7 protein tyrosine kinase 7


CCDS6057.1
NM_004103.3
PYK2/PTK2B
PTK2B protein tyrosine kinase 2 beta


CCDS7200.1
NM_020975.4
RET
ret proto-oncogene


CCDS626.1
NM_005012.2
ROR1
receptor tyrosine kinase-like orphan





receptor 1


CCDS6691.1
NM_004560.2
ROR2
receptor tyrosine kinase-like orphan





receptor 2


CCDS5116.1
NM_002944.2
ROS1
v-ros UR2 sarcoma virus oncogene





homolog 1 (avian)


N/A
NM_001005861.2
RYK
RYK receptor-like tyrosine kinase


CCDS13294.1
NM_005417.3
SRC
v-src sarcoma (Schmidt-Ruppin A-2) viral





oncogene homolog (avian)


CCDS13525.1
NM_080823.2
SRMS
src-related kinase lacking C-terminal





regulatory tyrosine and N-terminal





myristylation sites


CCDS6688.1
NM_003177.3
SYK
spleen tyrosine kinase


CCDS3481.1
NM_003215.2
TEC
tec protein tyrosine kinase


CCDS6519.1
NM_000459.2
TEK
TEK tyrosine kinase, endothelial (venous





malformations, multiple cutaneous and





mucosal)


CCDS482.1
NM_005424.2
TIE
tyrosine kinase with immunoglobulin-like





and EGF-like domains 1


N/A
NM_003985.3
TNK1
tyrosine kinase, non-receptor, 1


CCDS3480.1
NM_003328.2
TXK
TXK tyrosine kinase


CCDS12236.1
NM_003331.3
TYK2
tyrosine kinase 2


CCDS10080.1
NM_006293.2
TYRO3
TYRO3 protein tyrosine kinase


CCDS11824.1
NM_005433.3
YES1
v-yes-1 Yamaguchi sarcoma viral





oncogene homolog 1


CCDS33254.1
NM_001079.3
ZAP70
zeta-chain (TCR) associated protein kinase





70 kDa









Next, it was determined whether a mutation was somatic (i.e., tumor specific) by examining the sequence of the gene in genomic DNA from normal tissue of the relevant patient. From the approximately 12 Mb of sequence information obtained, 19 genes containing a total of 30 somatic mutations within their kinase domains were identified. All coding exons of these 19 genes were then analyzed for mutations in a total of 79 mM samples. The primers used for PCR amplification of ERBB4 (CCDS accession CCDS2394.1; GenBank Accession No. NM—005235.2; SEQ ID NO: 1) are listed in Table 3. ERBB4 was sequenced using the following primers:










Forward-TGTAAAACGACGGCCAGT
(SEQ ID NO: 78)



 



Reverse-CAGGAAACAGCTATGACC
(SEQ ID NO: 79)













TABLE 3







Primers used for PCR amplification of the


ERBB4 kinase domain











ERBB4

SEQ ID

SEQ ID


Exon
Forward Primer
NO:
Reverse Primer
NO:





Exon 18
TCATTTGTGCAGCA
64
CTGTCCTAGGGTTTT
71



ACTTCTC

GGCATT



 


Exon 19
GCAGACAGTTGTG
65
TGCTATCCTATTTCC
72



AAGCAAAAG

ATGCTGT



 


Exon 22
CAAGCTTTAATTCG
66
TCCCCACTTAATTAT
73



CAAAGAAGA

TTTTACCTTT



 


Exon 22
TGCTTTAGGAAATT
67
GGCTACTCAGAGGC
74



AGGCTTATC

TAAGGTG



 


Exon 23
TTTTTCCTTCATGTT
68
TTTTTAATTGATTGG
75



TAGATCATTT

TGTTTGG



 


Exon 23
ACCTTGTCCTGCTA
69
TGACCTGTAAGGAG
76



ATTTGCTC

TATTCTTTTACTAC



 


Exon 24
CAGTAGCAGAGCC
70
TGTCCACCAGGACA
77



ACTTGAA

AATGTA









Through this approach, 99 non-synonymous mutations were identified in 19 genes (Table 4, FIG. 4 and FIG. 5). All of these mutations were shown to be somatic by sequencing of DNA from matched normal tissue. Only three genes (EPHA6, PDGFRA and PTK2) out of the 19 had previously been reported to be mutated in MM (see Cancer Gene Census, available online at www.sanger.ac.uk/genetics/CGP/Census/). The majority of tumors with PTK gene mutations also contained mutations in NRAS or BRAF (Table 4).







TABLE 4







Somatic mutations identified in PTKs























% of





NRAS/






No. of
cases





BRAF



Other
CCDS
Ref Seq
muta-
af-


Amino
Functional
Tu-
muta-


Gene
names
accession*
accession*
tions#
fected#
Exon
Nucleotide†
Acid†
Domain
mor
tion**





















DDR1
CAK,
CCDS4690.1
NM_001954.3
2
2.6
8
C1115T
S372F
None
 6T
BRAF



CD167













EDDR1,




11
G1709A
R570Q
Protein
43T
BRAF



NEP,







Tyrosine





NTRK4







Kinase





PTK3A,













RTK6












FER
TYK3
CCDS4098.1
NM_005246.1
2
2.6
11
T1594C
Y532H
SH2 Motif
58T
BRAF








13
G1739A
G580D
Protein
30T
BRAF











Tyrosine













Kinase




FLT1
FLT
CCDS9330.1
NM_002019.3
8
10.3
7
G842A
R281Q
IG
37T
BRAF



VEGFR1




7
C860T
S287F
IG
 7T
NRAS








12
−9 Intronic
Splice
N/A
20T
BRAF









C > A
Site











13
G1767A
W589X
IGc2
13T
None








17
C2440T
P814S
None
39T
None








21
G2827A
E943K
Protein
44T
NRAS











Tyrosine













Kinase










24
G3241A
D1081N
Protein
78T
BRAF











Tyrosine













Kinase










28
G3667A
E1223K/
None
85T
BRAF










LOH





EPHA6
FLJ35246

NM_001080448.2
5
6.4
1
C1202G
T307S
None
30T
BRAF








4
G1763T
R494M
Protein
36T
BRAF











Tyrosine













Kinase










4
G1891A
E537K
Protein
32T
BRAF











Tyrosine













Kinase










8
A2246T
K655I
Protein
29T
BRAF











Tyrosine













Kinase










8
G2320A
E680K
None
21T
BRAF


EPHA10
FLJ16103
CCDS41305.1
NM_001099439.1
7
6.4
3
G235A
V79M
Ephrin
52T
BRAF











Receptor





FLJ33655




3
T236C
V79A
Ephrin
52T
BRAF











Receptor










3
G370A
E124K
Ephrin
55T
None











Receptor










3
G649A
G217S
None
71T
BRAF








3
G650A
G217D
None
71T
BRAF








13
G2369A
G790E
Protein
63T
NRAS











Tyrosine













Kinase










14
G2528C
G843A
Protein
37T
BRAF











Tyrosine













Kinase




EPHB1
EPHT2

NM_004441
4
5.1
3
C235T
R79W
Ephrin
39T
None











Receptor





Hek6




12
G2311A
D771N
Protein
60T
NRAS











Tyrosine













Kinase










13
G2432A
G811E
Protein
44T
NRAS











Tyrosine













Kinase










15
G2757A
W919X
Sterile Alpha
63T
NRAS











Motif




EPHB2
DRT
CCDS229.2
NM_017449.1
7
9.0
3
G325A
E109K
Ephrin
4T
BRAF











Receptor





EPHT3




3
C614T
A205V
None
72T
None



ERK




4
G952A
D318N
Fibronectin
71T
BRAF











Type 3













Domain





Hek5




7
C1535T
T512I
Fibronectin
83T
Both











Type 3













Domain





Tyro5




10
G1846A
E615K
Protein
29T
BRAF











Tyrosine













Kinase










10
G1846A
E615K
Protein
68T
BRAF











Tyrosine













Kinase










14
C2663T
P887L
None
77T
None


EPHB6
HEP
CCDS5873.1
NM_004445.1
7
9.0
3
C392T
S131F
Ephrin
60T
NRAS











Receptor










3
C455T
S152F
Ephrin
55T
None











Receptor










5
G1210A
G404S
Fibronectin
50T
BRAF











Type 3













Domain










11
G2036A
R679Q
Protein
5T
BRAF











Tyrosine













Kinase










11
C2063G
A688G
Protein
54T
BRAF











Tyrosine













Kinase










11
C2110T
R704W
Protein
26T
BRAF











Tyrosine













Kinase










13
−5 Intronic
Splice
N/A
18T
None









C > T
Site





ERBB4
HER4
CCDS2394.1
NM_005235.2
24
18.8
2
C113T
L39F
Receptor L
71T
BRAF











Domain





MGC138404




3
T331C
Y111H
Receptor L
13T
None











Domain





p180erbB4




8
G939A
M313I
Growth Factor
63T
NRAS











Receptor










8
G949A
E317K
Growth Factor
17T
NRAS











Receptor










9
C1022T
S341L
Receptor L
96T
None











Domain










10
C1177T
R393W
Receptor L
49T
BRAF











Domain










11
C1226T
P409L
Receptor L
76T
None











Domain










12
G1354A
E452K
Receptor L
7T
NRAS











Domain










12
G1354A
E452K/
Receptor L
55T
None










LOH
Domain










12
G1472A
R491K/
Growth Factor
34T
BRAF










LOH
Receptor










14
G1624A
E542K
Growth Factor
63T
NRAS











Receptor










14
C1630T
R544W
Growth Factor
56T
BRAF











Receptor










14
G1687A
E563K
Growth Factor
12T
NRAS











Receptor










15
−10
Splice
N/A
68T
BRAF









Intronic
Site/












C > T
LOH











15
G1825A
D609N
Growth Factor
76T
None











Receptor










18
C2098T
P700S
None
24T
NRAS








21
G2506A
E836K
Protein
86T
BRAF











Tyrosine













Kinase










21
G2614A
E872K
Protein
63T
NRAS











Tyrosine













Kinase/













Activation













Loo










23
G2806A
G936R
Protein
24T
NRAS











Tyrosine













Kinase










24
−4 Intronic
Splice
N/A
13T
None









C > T
Site











25
C3097T
P1033S
None
76T
None








26
−1 Intronic
Splice
N/A
76T
None









G > A
Site











28
G3521A
R1174Q
None
63T
NRAS








28
G3737A
S1246N
His-Me Finger
71T
BRAF











Endonucleases




MATK
CTK
CCDS12113.1
NM_002378.2
1
1.3
12
G1248A
W416X
Protein
13T
None











Tyrosine













Kinase





HYLTK












MET
HGFR
CCDS43636.1
NM_000245
3
3.8
5
G1829A
C610Y/
IPT
1T
BRAF










LOH











14
A3176G
N1059S
None
13T
None








16
G3509A
R1170Q
Protein
29T
BRAF











Tyrosine













Kinase




NTRK1
MTC
CCDS1161.1
NM_002529.2
2
2.6
8
G1137A
M349I
None
18T
None



TRK




14
C1747G
R547G
Protein
13T
None











Tyrosine













Kinase





TRKA












PDGFRA
CD140a
CCDS3495.1
NM_006206.2
5
5.1
3
G571A
A191T
IG
64T
BRAF



PDGFR2




9
G1375A
E459K/
None
32T
BRAF










LOH











18
C2669T
S890F
Protein
41T
BRAF











Tyrosine













Kinase










20
C2810T
P937L/
Protein
32T
BRAF










LOH
Tyrosine













Kinase










21
G3070A
D1024N
None
63T
NRAS


PTK2
FAK,
CCDS6381.1
NM_153831.2
1
1.3
15
C1481T
A494V
Protein
13T
None



FADK







Tyrosine













Kinase





FAK1,













pp125FAK












PTK2B
PYK2
CCDS6057.1
NM_173176.1
8
10.0
5
−4 Intronic
Splice
N/A
79T
BRAF









C > T
Site






PKB




8
G818A
W273X
FERM
76T
None



PTK




13
G1241A
G414E
None
95T
NRAS



CAKB




14
C1285T
R429C
Protein
17T
NRAS











Tyrosine













Kinase





FAK2




16
G1480A
E494K
Protein
26T
BRAF











Tyrosine













Kinase





FRNK




24
G2374A
E792K
None
36T
BRAF



CADTK




29
G2753A
R918Q
Focal AT
85T
BRAF



FADK2




29
G2812A
E938K
Focal AT
83T
Both



RAFTK












PTK6
BRK
CCDS13524.1
NM_005975.2
2
2.6
4
G629A
W210X
Protein
12T
NRAS











Tyrosine













Kinase










5
−7 Intronic
Splice
N/A
51T
BRAF









C > T
Site





PTK7
CCK4
CCDS4884.1
NM_002821.3
1
1.3
7
C1054T
P352S
IGc2
84T
BRAF


ROR2
BDB
CCDS6691.1
NM_004560.2
4
5.1
5
T574C
Y192H
Frizzled
71T
BRAF











Cysteine-Rich













Domain





BDB1




7
T1172C
V391A
Kringle
72T
None



NTRKR2




9
C1670T
S557L
Protein
5T
BRAF











Tyrosine













Kinase










9
G2377T
A793S
None
81T
BRAF


TIE1
JTK14
CCDS482.1
NM_005424.2
6
7.7
2
G139A
E47K
None
13T
None



TIE




2
C161T
S54L
None
16T
BRAF








2
C266T
T89M
None
52T
BRAF








2
G292A
D98N
None
43T
BRAF








11
G1598A
G533E
Fibronectin
39T
None











Type 3













Domain










22
C3281T
P1094L/
Protein
12T
NRAS










LOH
Tyrosine













Kinase





*Accession numbers for mutated PTKs in Santa Cruz and GenBank.


# Number of non-synonymous and splice site mutations observed and percent of tumors affected for each of the 19 genes in the panel of 80 melanoma cancers.


†Nucleotide and amino acid change resulting from mutation.


“X” refers to stop codon.


“LOH” refers to cases wherein the wild-type allele was lost and only the mutant allele remained.


“Splice site” refers to a case wherein the alteration affected ten bases spanning the exon.


**Mutations previously observed in NRAS, or BRAF.


“None” refers to no mutation observed.


SH2 Motif, Src homology 2 domain;


IG, Immunoglobin;


IGc2, Immunoglobin C-2 Type;


IPT, IG-like, p1exins, transcription factors;


Focal AT, Focal Adhesion Targeting Region;


FERM, Protein 4.1, Ezrin, Radixin, Moesin Domain.


Domains were found using Ensembl and InterPro.






The observed somatic mutations could either be “driver” mutations that play a functional role underlying the neoplastic process or nonfunctional “passenger” changes. In the 19 genes found to be mutated, 99 non-synonymous and 17 synonymous somatic mutations were identified, yielding a N:S (non-synonymous: synonymous) ratio of 99:17, significantly higher than the N:S ratio of 2.5:1 predicted for nonselected passenger mutations (P<1×10−5) (Sjoblom et al., Science 314:268-274, 2006), suggesting that these are likely to be “driver” mutations. The number of C>T mutations was significantly greater than other nucleotide substitutions resulting in a high prevalence of C:G>T:A transitions (p<0.0001) (FIG. 6A), confirming previously reported melanoma signatures. A summary of the most highly mutated genes is shown in FIG. 5.
Example 3
Somatic Mutations within ERBB4 are Frequent in MM
This example describes the biochemical analysis of several ERBB4 mutations identified in patients with melanoma. To evaluate the effect of some of these mutations on kinase function, the studies described herein focus on ERBB4, a member of the EGFR kinase subfamily, which was the most highly mutated gene (19%) in the screen. Five of the 15 samples with ERBB4 mutations contained more than one somatic mutation in ERBB4, which may act synergistically as previously seen for EGFR (Godin-Heymann et al., Cancer Res. 67:7319-7326, 2007). The large number of mutations observed in ERBB4 strongly suggests that these mutations are functionally important (FIG. 1A). This conclusion is supported by analysis of the ratio of non-synonymous to synonymous mutations in ERBB4, which was 24:3, significantly higher than the 2.5:1 ratio expected by chance (P<1×10−2) (Sjoblom et al., Science 314:268-274, 2006).
Interestingly, 7 out of the 24 non-synonymous somatic mutations discovered in ERBB4 occurred at Glu (E) residues (p<0.00005, binomial test), all of which resulted in changes to Lys (K), causing a charge reversal. The underlying reason for this might be due to the high frequency of C:G>T:A transitions (FIG. 6B). Clustering of somatic mutations is seen in various functional domains of ERBB4 (FIG. 1A and FIG. 5), with mutations in the kinase domain co-localizing with previously described mutations (found in various cancer types at frequencies ranging from 1.1-4.7%; Soung et al., Int. J. Cancer 118:1426-1429, 2006; Ding et al., Nature 455:1069-1075, 2008) and occurring at highly conserved residues. These genetic data suggest that mutant ERBB4 is likely to function as an oncogene in melanoma.
The positions of these mutations within ERBB4 and their predominantly heterozygous nature imply that they are likely to be gain of function mutations. No truncating mutations were observed and the alterations occurred in functionally important domains (FIG. 1A). The affected residues in ERBB4 are highly conserved evolutionarily, retaining identity in chimp, horse, rat, mouse and opossum. Clustering of somatic missense mutations is seen in various domains. Mutations S341L, R393W, P409L, E452K and R491K all occur in the extracellular sub region III, with the E452K mutation occurring in two different cases. Mutations E542K, R544W and E563K are all adjacent in the extracellular sub region IV. A similar clustering was observed in the kinase domain where our novel mutations co-localized with previously described mutations (found in various cancer types at frequencies ranging from 1.1%-4.7% (Ding et al., Nature 455:1069-1075, 2008; Soung et al., Int. J. Cancer 118:1426-1429, 2006). The clustering of somatic missense mutations in specific domains of ERBB4 is similar to that observed for activating mutations in other oncogenes, such as BRAF and PIK3CA (Davies et al., Nature 417(6892):949-954, 2002; Samuels et al., Science 304:554, 2004). These genetic data suggest that mutant ERBB4 is likely to function as an oncogene in MM.
Example 4
ERBB4 Mutations Increase its Kinase Activity
This example describes the assessment of kinase activity of ERBB4 mutations present in melanoma tumors. To directly test whether the mutations identified in ERBB4 activate its kinase activity, the positions of the various ERBB4 missense mutations in its crystal structure were assessed. The crystal structures of the extracellular and kinase domains of ERBB4 (Bouyain et al., Proc. Natl. Acad. Sci. USA 102:15024-15029, 2005; Qiu et al., Structure 16:460-467, 2008) demonstrated that most of the observed alterations had similar positioning to mutations reported in the ERBB4 family members EGFR and ERBB2 in lung cancer, glioblastoma and gastric cancer (Riese et al., Bioessays 29:558-565, 2007). The mutations that were further evaluated in the extracellular domain included the E317K mutation, which is near the EGFR R324L mutation, the E542K, R544W, and E563K mutations, as these co-localize, and finally the E452K mutation, as this substitution occurred in two patients. Additionally, two mutations that were found in the kinase domain were cloned: E836K, which is found near the ERBB2 N857S mutation, and the E872K alteration.
To investigate the biochemical effects of the identified ERBB4 mutations, wild type (WT) ERBB4 or the seven mutants (E317K, E452K, E542K, R544W, E563K, E836K, E872K), as well as a kinase dead (KD) version of ERBB4 (K751M), were transiently expressed in HEK 293T cells and the basal catalytic activity of ERBB4 was assessed using ERBB4 autophosphorylation as a readout for receptor activation. ERBB4 autophosphorylation was determined by measuring the total phosphotyrosine content of the immunoprecipitated receptor as well as by measuring two auto-phosphorylation sites (Tyr-1162 and Tyr-1284) in the C-terminus of ERBB4. Compared to WT ERBB4, all the missense mutants showed a marked increase in receptor autophosphorylation on total phosphotyrosine as well as on residues Tyr-1162 and Tyr-1284 (FIG. 1B). No site-specific phosphorylation was observed in cells exogenously expressing the KD version of ERBB4. Similar expression levels of total ERBB4 protein were observed, except KD ERBB4, which had a higher expression level (FIG. 1B).
The specificity of the phosphosite-specific anti-ERBB4 antibodies was confirmed using competitive ERBB4-phosphosite-specific phospho-peptides (FIG. 7). To assess whether the increased tyrosine phosphorylation of the ERBB4 mutants correlates with increased kinase activity, a kinase assay using the same set of ERBB4 mutants was performed. FIGS. 1C-1D show that in low serum, the ERBB4 mutants exhibit a marked increase in kinase activity compared to WT ERBB4. In contrast, in the presence of serum, the ERBB4 mutants showed a similar kinase activity compared to WT ERBB4. Similar expression levels of total ERBB4 protein were observed (FIG. 1D). These results suggest that increased ERBB4 phosphorylation is due to its constitutive activation rather than alteration in its protein levels.
To extend these observations, a MM line containing endogenous mutant ERBB4 (63T, E542K/E872K) was studied and compared it to a MM line containing endogenous WT ERBB4 (39T). As in transfected cells, ERBB4 autophosphorylation was markedly elevated in the MM line with an endogenous ERBB4 mutation (FIG. 8).
To determine if the increased tyrosine phosphorylation of the ERBB4 mutants correlates with increased kinase activity, a kinase assay using the same set of ERBB4 mutants was performed. The ERBB4 mutants showed a marked increase in kinase activity compared to WT ERBB4 and expression levels of total ERBB4 protein were comparable (FIG. 1E). As in transfected cells, ERBB4 autophosphorylation was markedly elevated in the melanoma lines harboring ERBB4 mutations compared to melanoma lines harboring endogenous WT ERBB4 (FIGS. 1F-1G).
ERBB4 is known to activate several downstream signaling pathways including the ERK and AKT pathways (Frey et al., Gastroenterology 136:217-226, 2009). To evaluate which of these signaling pathways is activated by the ERBB4 mutations, immunoblot analysis of melanoma cell lines harboring endogenous ERBB4 mutations was performed. Phosphorylation of AKT was elevated in cells expressing any of the three evaluated mutant ERBB4s, whereas ERK showed similar activation in cells expressing WT or mutant ERBB4 (FIG. 11).
Example 5
ERBB4 Mutations Promote Colony Formation Abilities and Anchorage-Independent Growth
The example describes the phenotypic analysis of ERBB4 mutants identified in melanoma tumors. The combination of biochemical and genetic data disclosed herein suggested that the mutant ERBB4 proteins might be oncogenic. However, previous studies have described the generation of ERBB4 mutants that are constitutively active but non-transforming (Penington et al., Cell Growth Differ. 13:247-256, 2002; Williams et al., Cancer Lett. 192:67-74, 2003). Thus, the following studies were performed to determine whether the melanoma ERBB4 variants described in this study are transforming To test this, NIH 3T3 cells were transiently transfected with vector, WT, one of the seven constitutively active ERBB4 mutants (E317K, E452K, E542K, R544W, E563K, E836K and E872K) or oncogenic K-RasG12V. Ten days after transfection, all ERBB4 mutations transformed NIH 3T3 cells more efficiently than WT ERBB4. Strikingly, the transformation ability of the ERBB4 mutants was similar to oncogenic K-RasG12V (FIG. 2A). Similarly, the same set of ERBB4 mutants were able to promote anchorage-independent growth as depicted in FIG. 9A. All the presented results were significant (P<0.05, t test).
To test the transformation abilities of the ERBB4 mutations in human melanoma cells, stable cell pools expressing vector, WT, and three ERBB4 mutations (E452K, E563K and E872K) were derived in SK-Mel-2 cells, a melanoma cell line that expresses WT ERBB4. Western blot analysis showed a similar expression level of ERBB4 in all clones (FIG. 9B). As seen in FIG. 2B, expression of all the ERBB4 mutants elicited a significantly higher cell transformation ability compared to clones expressing vector or WT ERBB4 (p<0.05, t-test). When the same set of clones was suspended in soft agar, cells expressing mutant ERBB4 formed a significantly higher number of anchorage-independent colonies (p<0.05, t-test, FIG. 9C). Thus, all the tested ERBB4 mutants potently increased both colony formation ability as well as growth on soft agar in all the cell lines compared to vector or WT ERBB4 stable clones.
Example 6
Dependency of MM Lines Harboring ERBB4 Mutations on ERBB4 Signaling
This example describes the effect of inhibiting expression of WT and mutant ERBB4 using shRNA. In order to assess if melanoma cells harboring endogenous ERBB4 mutations are dependent on ERBB4 signaling for proliferation, short hairpin RNA (shRNA) was used to stably knockdown ERBB4 protein levels in melanoma lines harboring either WT (2T and 31T) or mutant ERBB4 (17T, E317K; 63T, E542K/E872K; or 7T, E452K). Specific targeting of ERBB4 by shRNAs was confirmed both in transfected HEK 293 cells and in one of the melanoma cell lines by immunoblotting (FIGS. 3A-3B). Three unique shRNA constructs targeting ERBB4 had minimal effect on the proliferation of cells expressing WT receptor, but significantly reduced the growth of melanoma lines containing mutant ERBB4 (FIGS. 3C-3G). Thus, mutant ERBB4 is essential for growth of melanomas harboring these mutations. Evaluation of the effects of ERBB4 knockdown on downstream signaling pathways revealed that down-regulation of ERBB4 in cells harboring mutant versions of the gene reduces levels of endogenous, phosphorylated AKT, but not of phosphorylated ERK. In contrast, inhibition of ERBB4 expression in cells harboring WT versions of the gene showed similar levels of AKT and ERK activation (FIG. 12).
Because shRNA-mediated cell death could result from specific or nonspecific effects, an exogenous, non-targetable WT ERBB4 construct (NT ERBB4), engineered to be resistant to knockdown by the introduction of three silent mutations in the region of ERBB4 targeted by shRNA #6, was examined for the ability to rescue the effects of knockdown of endogenous ERBB4. Melanoma cells harboring the E317K mutation stably expressing either control or ERBB4 shRNA #6 construct were transduced with the lentiviral NT ERBB4 construct or empty vector as control. Similar phosphotyrosine content is observed in both WT and NT ERBB4 constructs, demonstrating that the silent mutations in the NT construct do not affect the ability of the receptor to be phosphorylated to wild-type levels (FIG. 13A). Importantly, pooled clones of NT reconstituted cells were markedly more resistant to growth inhibition induced by ERBB4 knockdown (#6/NT) than shRNA control-infected cells (Vect/Vect).
To evaluate mutant ERBB4 as a potential target for specific inhibition of melanoma cell survival, the ERBB4 pathway was targeted with the FDA-approved pan-ERBB pharmacologic inhibitor, lapatinib (GW2016) (Heymach et al., Clin. Cancer Res. 12:4441s-4445s, 2006). Exposure of melanoma cells to lapatinib resulted in reduced cell proliferation to a greater extent in cells containing endogenous ERBB4 mutations than in cells containing endogenous WT ERBB4 (FIG. 10A). An IC50 calculation revealed that melanoma cells harboring ERBB4 mutations were 10- to 250-fold more sensitive to lapatinib than cells with WT receptor (FIG. 10B) and treatment with lapatinib inhibited receptor autophosphorylation in a dose-dependent manner (FIG. 10C). This increased sensitivity to lapatinib was accompanied by specific inhibition of ERBB4 and AKT activation in cells harboring mutant ERBB4 (FIGS. 10D-10E). Activation of other downstream elements, such as ERK, was also slightly inhibited by lapatinib (FIGS. 14A-14B). Thus, although signaling by mutant ERBB4 demonstrates selective activation of AKT, lapatinib treatment of cells harboring mutant ERRB4 results in uniform inhibition of downstream signaling pathways. Only mutant ERBB4 was inhibited by lapatinib in the melanoma cell lines. No inhibition of its family member ERBB2 was observed (FIGS. 10D-10E) and no phosphorylation of EGFR was observed in any of these cells. The observed reduced proliferation occurred in cells harboring BRAF, NRAS, ARAF or CRAF mutations in addition to the ERBB4 mutations.







TABLE 5







Mutations identified in RAF and RAS isoforms














Sample
ERBB4
BRAF
NRAS
ARAF
CRAF
HRAS
KRAS





 7T
E452K
wt
Q61R
wt
wt
wt
wt


12T
E563K
wt
Q61Q/R
wt
wt
wt
wt


17T
E317K
wt
Q61Q/K
wt
wt
wt
wt


31T
wt
wt
wt
wt
wt
wt
wt


34T
R491K
V600V/E
wt
wt
T362T/A
wt
wt


39T
wt

wt
wt
wt
wt
wt


49T
R393R/W
V600V/E
wt
wt
wt
wt
wt


55T
E452K
V600V/E
wt
P216S
wt
wt
wt






P254L





56T
R544R/W
V600V/E
wt
wt
wt
wt
wt


63T
E542K
wt
Q61Q/K
wt
wt
wt
wt



E872K








68T
Splice site
V600V/E
wt
wt
wt
wt
wt



LOH








71T
L39L/F
V600V/M
wt
wt
wt
wt
wt



S1246S/N
V600V/E







86T
E836E/K
V600V/E
wt
wt
wt
wt
wt


93T
wt
wt
wt
A345A/G
wt
wt
wt









To elucidate the mechanism of decreased growth of cells expressing mutant ERBB4 following lapatinib treatment, cells were examined for cell cycle perturbations or apoptosis by flow cytometry. Lapatinib markedly increased apoptosis of melanoma cells harboring mutant ERBB4 compared to lines harboring WT ERBB4 (FIGS. 10E-10G). Thus, expression of mutant ERBB4 appears essential for suppression of pro-apoptotic signals in melanoma cells harboring these mutations, which is consistent with the selective activation of AKT in ERBB4 mutant cells (FIGS. 11A-11B) and previous results demonstrating an anti-apoptotic role for AKT (Grant et al., Front. Biosci. 7:d76-89, 2002). These results suggest that lapatinib preferentially inhibits mutant ERBB4 signaling and that cells with ERBB4 mutations are subject to “oncogene addiction” (Weinstein, Science 297:63-64, 2002). Moreover, the enhanced AKT signaling in cells with mutant ERBB4 may provide an additional therapeutic target in these tumors.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

## Claims

1. A method of selecting a subject diagnosed with melanoma as a candidate for treatment with an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both, comprising:
(a) detecting the presence or absence of a mutation in the ERBB4 gene in a melanoma sample from the subject, wherein the mutation is selected from G949A, G1354A, G1624A, C1630T, G1687A, G2506A and G2614A (numbered with reference to SEQ ID NO: 1), and wherein the presence of the mutation in the ERBB4 gene indicates that the subject is a candidate for treatment with an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both; and
(b) administering to the subject an ERBB4 inhibitor, a PI3K/AKT pathway inhibitor, or both when the mutation in the ERBB4 gene is present in the sample from the subject.

2. The method of claim 1, wherein the subject is administered an ERBB4 inhibitor and a PI3K/AKT pathway inhibitor.

3. The method of claim 1, wherein the ERBB4 inhibitor is lapatinib.

4. The method of claim 1, wherein detecting the presence or absence of a mutation in the ERBB4 gene in a melanoma sample from the subject comprises polymerase chain reaction amplification of genomic DNA from the melanoma sample using ERBB4-specific primers.

5. The method of claim 4, further comprising DNA sequencing of the amplified genomic DNA.