Patent Description:
This International Application claims the priority of <CIT> and <CIT>.

The present disclosure relates generally to the treatment of leukemia using pharmacologic inhibition of Menin-MLL interaction. The present disclosure further relates to the treatment of leukemia using inhibition of Menin-MLL interaction in combination with DOT1L inhibition.

The clustered homeobox (HOX) genes are a highly conserved family of transcription factors that are expressed during early development and hematopoiesis<NUM>-<NUM>. Specific members of the HOXA and HOXB cluster are required to maintain self-renewal properties of hematopoietic stem cells (HSCs)<NUM>. Aberrant HOX gene expression is also a common feature of acute leukemias and believed to play an important role during leukemogenesis<NUM>,<NUM>. In murine leukemia models aberrant HOX gene expression has been shown to be a critical component of a specific oncogenic gene expression program that can be driven by various oncogenes<NUM>,<NUM>. Retrovirally induced overexpression of specific HOX genes in early hematopoietic progenitors induces leukemia in mice<NUM>. In human acute myeloid leukemia (AML) aberrant HOX gene expression can be found in <NUM>-<NUM>% of cases, but the HOX expression pattern among AML cases is heterogenous and has been used to categorize this disease into four main groups that correlate with the presence of specific molecular markers<NUM>.

Simultaneous expression of the HOXA and HOXB cluster represents the most common patterns in AML and cases within this group frequently carry NPM1 mutations<NUM>,<NUM>, <NUM>.

NPM1 is one of the two most frequently mutated genes in AML (<NUM>%) and co-occurs commonly with other mutations such as FLT3, DNMT3A, IDH1, IDH2, and TET2<NUM>-<NUM>. Of these, FLT3 is of particular importance in NPM1mut AML as it is a) the most commonly co-mutated gene with NPM1 and b) an important prognostic marker in these leukemias. Whereas NPM1mut AML lacking a FLT3-ITD is considered a relatively favorable genotype with overall survival rates of up to <NUM>% in younger patients, the presence of a concurrent FLT3-ITD (<NUM>% of the cases) converts this genotype into an adverse category<NUM>,<NUM>-<NUM>. Whereas novel small molecule FLT3 inhibitors have clinical activity against FLT3 mutated AML and were recently introduced into current treatment regimens, resistance via acquisition of mutations or subsequent up-regulation of FLT3 transcript levels gene occurred frequently and quickly in first clinical trials. Independent of FLT3 mutation status, survival rates in patients over <NUM> years of age are even more dismal and only a minority of these patients achieves a sustainable remission<NUM>. These data highlight the need for novel molecular mechanism-based therapeutic concepts which may be less toxic and more efficacious.

NPM1 is an intracellular chaperone protein implicated in multiple cellular processes, such as proliferation, ribosome assembly, nucleosome assembly and many others<NUM>, <NUM>. Despite the discovery of NPM1 mutations more than a decade ago, how NPM1 mutations initiate and maintain AML remains unclear thus hindering the development of targeted therapeutic approaches. Also, it is unknown what drives aberrant HOX gene expression and whether these genes are required for NPM1mut leukemogenesis.

Current knowledge of aberrant HOX gene regulation in leukemias has mainly come from studies of MLL-rearranged leukemias. In these leukemias, the mixed-lineage leukemia gene (MLL, also known as KMT2A) is fused to one of more than <NUM> different fusion partners resulting in the formation of an oncogenic fusion protein<NUM>. All MLL-rearranged leukemias exhibit aberrant HOXA cluster expression that is dependent on chromatin binding of the MLL-fusion complex<NUM>, which in turn requires the association with at least two other proteins, menin and LEDGF. Whereas menin facilitates LEDGF binding to the complex, the latter directly conveys chromatin binding of MLL<NUM>, <NUM>. Menin, the protein encoded by the multiple endocrine neoplasia <NUM> gene (MEN1), is of particular interest as it has been shown to be therapeutically targetable. Recently developed inhibitors of the menin-MLL interaction were demonstrated to have anti-leukemic activity in preclinical MLL-rearranged leukemia models<NUM>, <NUM>.

The histone <NUM> lysine <NUM> (H3K79) methyltransferase DOT1L is another protein of therapeutic interest involved in HOXA cluster regulation of MLL-rearranged leukemias<NUM>-<NUM>. DOT1L is believed to be recruited to promoters of HOXA cluster genes via the MLL fusion partner that is commonly part of the DOT1L binding complex<NUM>. Small molecule inhibitors of DOT1L were proven to have activity against preclinical models of MLL-rearranged leukemias<NUM>, <NUM>, and are currently being tested in a clinical trial with promising first results presented at (NCT01684150). See, https://clinicaltrials. gov/ct2/show/NCT01684150.

While it may seem unlikely for these findings to be easily transferable to other HOX expressing leukemias lacking an MLL-fusion protein, it has been shown that the wt-MLL complex and menin<NUM>, <NUM> are both required for HOXA cluster expression during normal hematopoiesis. In addition, higher states of H3K79 methylation, such as di- and trimethylation (me2 and me3) at the HOX locus are associated with high expression levels of these genes in lineage-, Sca-<NUM>+, c-Kit+ (LSK) murine hematopoietic progenitors and are converted into monomethylation (me1) as these cells mature into more committed progenitors lacking HOX expression<NUM>. Whether HOXB cluster expression in LSKs is also associated with H3K79me2 and me3 is unknown to date.

The applicant has previously proposed to use DOT1L inhibition in the treatment of leukemias that are not characterized by MLL-translocation or MLL-rearrangement or MLL-partial duplication. See, <CIT> and published as <CIT>.

While NPM1mut AML without concurrent FLT3-ITD is being considered a relatively favorable subtype, only about half of the younger patients with NPM1 mutations achieve long-term disease-free survival following current treatment approaches<NUM>, <NUM>, <NUM>, <NUM>. Outcome in patients > <NUM> years of age is generally poor<NUM>. These numbers indicate the demand for more efficacious and potentially less toxic therapies. Research efforts within the last years led to important findings describing distinctive biological features of NPM1mut AML such as a HOX gene dominated expression signature<NUM>, <NUM> and lack of CD34 expression in the majority of cases<NUM>. Also, it is known for more than a decade that the NPM1 mutations results in cytoplasmic dislocation of the mutant protein<NUM>. However, it is still incompletely understood how aberrant HOX gene expression is maintained and what mechanisms specifically drive leukemic transformation<NUM>. While HOX and MEIS1 transcription factors have thus far not proven to be amenable for pharmacological inhibition, current efforts to develop drugs that directly target the mutant NPM1 protein or restore its physiological cellular localization have been largely unsuccessful or of controversial therapeutic benefit as currently discussed for all-trans-retinoic acid <NUM>-<NUM>.

Molecularly targeted drug development has consequently been challenging for NPM1mut AML. A better understanding of biological processes involved in NPM1mut leukemogenesis would therefore facilitate the definition of molecular targets suitable for pharmacological inhibition. Thus, an acute need remains for provision of novel therapies against leukemias.

The inventors hypothesized that aberrant HOX and MEIS1 gene expression in NPM1mut AML might be driven by similar mechanisms that control these genes in normal HSCs and that high expression of FLT3, a reported downstream target of MEIS1, is indirectly driven via elevated MEIS1 transcript levels. Using CRISPR-Cas9 genome editing and small molecule inhibition, the inventors identified the chromatin regulators MLL and DOT1L as therapeutic targets that control HOX, MEIS1 and FLT3 expression and differentiation in NPM1mut leukemia.

The inventors first conjectured MLL to be also involved in controlling HOX gene expression in NPM1mut AML. Using a CRISPR-Cas9 negative selection screen targeting multiple MLL protein domains, the inventors discovered the menin binding site of MLL as a top hit in their screen. Additionally, the inventors detected a strong phenotype for at least one of the sgRNAs targeting the CXXC domain, again pointing to chromatin binding of MLL being critically required for NPM1mut leukemias<NUM>. Of note, none of the sgRNAs targeting the C-terminal SET domain of MLL - that contains the H3K4 methyltransferase activity but is always lost in MLL-fusion proteins<NUM> - exhibited any significant phenotype.

The present disclosure describes a method for inhibiting proliferation of or inducing apoptosis in a leukemia cell, or for both inhibiting proliferation and inducing apoptosis in the cell is provided, wherein the method comprises contacting said leukemia cell with an inhibitor of interaction between MLL and menin, wherein said leukemia cell exhibits an NMP1 mutation.

The NPM1 mutation exhibiting leukemia cell may be selected from the group consisting of an acute lymphocytic leukemia (ALL) cell and an acute myeloid leukemia (AML) cell.

The inhibitor of interaction of MLL and menin may inhibit the interaction with an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>.

The inhibitor of interaction of MLL and menin may be selected from the group consisting of MI-<NUM>, MI-<NUM>, MI-<NUM>, MI-<NUM>, ML-<NUM>, the compounds in Table <NUM> of the present application, a compound of the formula:
<CHM>
wherein Rl, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of: H, substituted or non-substituted alkyl, substituted or non-substituted alkoxy, a halogen (e.g. F, CI, Br, I, and At), a ketone, a carbocyclic ring, an aromatic ring, a heterocyclic aromatic ring comprising carbon and one or more nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with substituted or non-substituted alkyl, aryl, halogen, hydrogen bond donor or acceptor, a heterocyclic non-aromatic ring comprising carbon and one or more nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic aromatic or non-aromatic ring fused or attached to the thienopyrimidine ring system non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic or heterocyclic aromatic ring comprising carbon atoms and one or more nitrogen, oxygen and/or sulfur members fused to another aromatic ring, or a hydrogen bond donor or a hydrogen bond acceptor; Z is S or O or NH or CH-CH; W is present or absent and is NH or NH-(CH<NUM>)n (n is an integer between <NUM> and10), or (CH<NUM>)n (n is an integer between <NUM> and10) or O or O-(CH<NUM>)n (n is an integer between <NUM> and10); X and Y are each independently N or C; and m is an integer between <NUM> and <NUM> or pharmaceutically acceptable salts of thereof: or
a compound of the formula:
<CHM>
wherein Rl, R2, R3, and R4 are each independently selected from the group consisting of: H, substituted or non-substituted alkyl, substituted or non-substituted alkoxy, a halogen, a ketone, a carbocyclic ring, an aromatic ring, a heterocyclic aromatic ring comprising carbon and one or more nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, a heterocyclic non-aromatic ring comprising carbon and one or more nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic aromatic or non-aromatic ring fused to the benzodiazepine ring system non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic or heterocyclic aromatic ring comprising carbon; or pharmaceutically acceptable salts thereof.

The disclosure also describes a method for treating a patient afflicted with a leukemia, is provided wherein the method comprises:.

The method may further comprise testing a leukemia tissue sample or cell obtained from the patient for the presence of said NMP1 mutation and (i) proceeding with the administration if the patient is determined to possess said mutation; or (ii) conversely, not proceeding with the administration if the patient is determined to lack said mutation.

The leukemia may be selected from the group consisting of an acute lymphocytic leukemia (ALL) and an acute myeloid leukemia (AML).

The inhibitor of interaction between MLL and menin may inhibit the interaction with an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>.

The inhibitor of interaction between MLL and menin may be selected from the group consisting of MI-<NUM>, MI-<NUM>, MI-<NUM>, MI-<NUM> and ML-<NUM>, a compound disclosed in Table <NUM> of the specification, and pharmaceutically acceptable salts or free base versions thereof or.

The present disclosure describes a method for treating leukemia in a patient previously identified as afflicted by a leukemia exhibiting an NMP1 mutation is provided, the method comprising:
administering to said patient an inhibitor of interaction between MLL and menin in an amount effective to inhibit proliferation and/or enhance apoptosis of leukemic cells of the patient.

There may be an additional administration to the patient a DOT1L inhibitor in at least an amount effective, in combination with the MLL-menin interaction inhibitor, to inhibit proliferation and/or enhance apoptosis of leukemic cells of the patient.

The DOT1L inhibitor may inhibit DOT1L with an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>.

The inhibitor of interaction of MLL and menin may be provided at a dosage of at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least bout <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/ day or maximum tolerated dose if lower.

In methods of the disclosure involving dual administration, the amount of at least one of the inhibitors may be the same as it would have been had the at least one inhibitor been used alone and not in said combination for achieving the maximum level of proliferation inhibition and/or apoptosis of leukemic cells achievable by using the same inhibitor as monotherapy.

In other method of the disclosure involving dual administration the amount of at least one of said inhibitors may be lower than it would have been had the at least one inhibitor been used alone and not in said combination for achieving the maximum level of proliferation inhibition and/or apoptosis of leukemic cells achievable by using the same inhibitor as monotherapy.

The DOT1L inhibitor may be selected from the group consisting of a purine, a carbocycle-substituted purine and a <NUM>-deazapurine; the DOT1L inhibitor may be selected from the group consisting of EPZ00477 and EPZ005676; or from the group consisting of SGC-<NUM>, SYC-<NUM>, SYC-<NUM>, and SYC-<NUM>.

The present disclosure describes a further method for determining susceptibility of a leukemia patient to treatment with an inhibitor of interaction between MLL and menin alone or in combination with a DOT1L inhibitor, said method comprising:
testing a leukemia tissue sample or cell obtained from the patient for the presence of an NMP1 mutation; and.

A method is provided for predicting the therapeutic efficacy of an inhibitor of interaction between MLL and menin alone or in combination with a DOT1L inhibitor in a leukemia patient wherein the patient does not exhinit an MLL-translocation, an MLL-rearrangement, and/or an MLL-partial tandem duplication, said method comprising:
testing a leukemia tissue sample or cell from said patient for the presence of an NPM1 mutation,
wherein the presence of said mutation in said sample or cell is predictive of the therapeutic efficacy of the inhibitor of MLL-menin interaction alone or in combination with a DOT1L inhibitor in the patient.

The present disclosure describes a method for inhibiting the proliferation and/or inducing apoptosis of a leukemia cell, said method comprising.

The disclosure also describes a method for treating a leukemia patient wherein an NMP1 mutation has been identified in a tissue sample or cell of the patient, said method comprising:
administering to said patient one or more inhibitors of interaction between MLL and menin alone or in combination with a DOT1L inhibitor, in amounts individually or combined effective in the treatment of leukemia;
wherein the patient does not also exhibit a genetic mutation, alteration, and/or abnormality is selected from the group consisting of an MLL-t, an MLL-r, and/or an MLL-PTD.

The tissue sample may be a blood sample, a bone marrow sample, or a lymph node sample.

Administration of the DOT1L inhibitor in the combination described herein may enhance the inhibition of proliferation and/or apoptosis of leukemic cells achieved by administration of the inhibitor of interaction of MLL and menin as monotherapy; the enhancement may be synergistic, as illustrated without limitation in Examples <NUM> and more particularly <NUM>.

In a first alternative use, the disclosure provides a use of an inhibitor of interaction between MLL and menin, in the treatment of leukemia in an amount effective to inhibit proliferation and/or enhance apoptosis of leukemic cells, wherein the leukemia exhibits an NPM1 mutation.

In this use the leukemia may have been determined to possess the NPM1 mutation prior to treatment; the leukemia may be selected from the group consisting of an acute lymphocytic leukemia (ALL) and an acute myeloid leukemia (AML).

In this use the inhibitor of interaction between MLL and menin may inhibit the interaction with an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>.

More specifically in this use, the inhibitor of interaction between MLL and menin is selected from the group consisting of MI-<NUM>, MI-<NUM>, MI-<NUM>, a compound disclosed in Table <NUM> of the specification, and pharmaceutically acceptable salts or free base versions thereof or.

In a second alternative use, the present disclosure describes use of an inhibitor of interaction between MLL and menin in an amount effective to inhibit proliferation and/or induce apoptosis of leukemic cells previously identified as exhibiting an NMP1 mutation.

In the first or second uses, the inhibitor of interaction between MLL and menin is used in combination with a DOT1L inhibitor in at least an amount effective, in combination with the MLL-menin interaction inhibitor, to inhibit proliferation and/or enhance apoptosis of leukemic cells of the patient; the DOT1L inhibitor may inhibit DOT1L with an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>.

In the first or second alternative uses, the amount of at least one of the inhibitors may be the same as it would have been had the at least one inhibitor been used alone and not in said combination for achieving the maximum level of proliferation inhibition and/or apoptosis achievable by using the same inhibitor as monotherapy; the amount of at least one of said inhibitors may be lower than it would have been had the at least one inhibitor been used alone and not in said combination for achieving the maximum level of proliferation inhibition and/or apoptosis achievable by using the same inhibitor as monotherapy.

The DOT1L inhibitor may be selected from the group consisting of a purine, a carbocycle-substituted purine and a <NUM>-deazapurine; or
the DOT1L inhibitor is specifically selected from the group consisting of EPZ00477 and EPZ005676.

Alternatively, the DOT1L inhibitor may be selected from the group consisting of SGC-<NUM>, SYC-<NUM>, SYC-<NUM>, and SYC-<NUM>.

Alternatively, the inhibitor of interaction between MLL and menin may be selected from the group consisting of MI-<NUM>, MI-<NUM>, MI-<NUM>, MI-<NUM> and ML-<NUM>.

Thr leukemia or cell may not exhibit a genetic mutation, alteration, and/or abnormality that is an MLL-translocation (MLL-t), an MLL-rearrangement (MLL-r), or an MLL-partial tandem duplication (MLL-PTD).

The leukemia or cell may exhibit a wild type MLL.

The leukemia or cell may not exhibit a rearranged or fused MLL.

In a first aspect, the invention provides a method for inhibiting proliferation and/or inducing apoptosis in a leukemia cell in vitro, comprising contacting the leukemia cell with an inhibitor of interaction between MLL1 and menin, wherein leukemia cell exhibits an NPM1 mutation, and wherein the leukemia cell does not exhibit a genetic mutation, alteration, and/or abnormality that is an MLL-translocation (MLL-t), an MLL-rearrangement (MLL-r), or an MLL-partial tandem duplication (MLL-PTD).

In this aspect, the inhibitor may be selected from the group consisting of MI-<NUM>, MI-<NUM>, MI-<NUM>, MI-<NUM>, and a compound of the formula:
<CHM>
wherein Rl, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of: H, substituted or non-substituted alkyl, substituted or non-substituted alkoxy, a halogen (e.g. F, CI, Br, I, and At), a ketone, a carbocyclic ring, an aromatic ring, a heterocyclic aromatic ring comprising carbon and one or more nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with substituted or non-substituted alkyl, aryl, halogen, hydrogen bond donor or acceptor, a heterocyclic non-aromatic ring comprising carbon and one or more nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic aromatic or non-aromatic ring fused or attached to the thienopyrimidine ring system non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic or heterocyclic aromatic ring comprising carbon atoms and one or more nitrogen, oxygen and/or sulfur members fused to another aromatic ring, or a hydrogen bond donor or a hydrogen bond acceptor; Z is S or O or NH or CH-CH; W is present or absent and is NH or NH-(CH2)n (n is an integer between <NUM> and10), or (CH2)n (n is an integer between <NUM> and10) or O or O-(CH2)n (n is an integer between <NUM> and10); X and Y are each independently N or C; and m is an integer between <NUM> and <NUM> or pharmaceutically acceptable salts of thereof.

In this aspect the leukemia cell may be selected from the group consisting of an acute lymphocytic leukemia (ALL) cell and an acute myeloid leukemia (AML) cell. The inhibitor may have an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>.

In a second aspect, the invention provides an agent that inhibits interaction between MLL1 and menin for use in treating a patient afflicted with a leukemia in an amount effective to inhibit proliferation and/or enhance apoptosis of leukemic cells in the patient, wherein the patient being afflicted with a leukemia that exhibits an NPM1 mutation, and wherein the leukemia cell does not exhibit a genetic mutation, alteration, and/or abnormality that is an MLL-translocation (MLL-t), an MLL-rearrangement (MLL-r), or an MLL-partial tandem duplication (MLL-PTD).

In this aspect, the agent may be selected from the group consisting of MI-<NUM>, MI-<NUM>, MI-<NUM>, MI-<NUM> and pharmaceutically acceptable salts or free base versions thereof or a compound of the formula:
<CHM>
wherein Rl, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of: H, substituted or non-substituted alkyl, substituted or non-substituted alkoxy, a halogen (e.g. F, CI, Br, I, and At), a ketone, a carbocyclic ring, an aromatic ring, a heterocyclic aromatic ring comprising carbon and one or more nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with substituted or non-substituted alkyl, aryl, halogen, hydrogen bond donor or acceptor, a heterocyclic non-aromatic ring comprising carbon and one or more of nitrogen, oxygen and/or sulfur members which may be non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic aromatic or non-aromatic ring fused or attached to the thienopyrimidine ring system non-substituted or substituted with alkyl, aryl, halogen, hydrogen bond donor or acceptor, carbocyclic or heterocyclic aromatic ring comprising carbon atoms and one or more nitrogen, oxygen and/or sulfur members fused to another aromatic ring, or a hydrogen bond donor or a hydrogen bond acceptor; Z is S or O or NH or CH-CH; W is present or absent and is NH or NH-(CH2)n (n is an integer between <NUM> and <NUM>), or (CH2)n (n is an integer between <NUM> and <NUM>) or O or O-(CH2)n (n is an integer between <NUM> and <NUM>); X and Y are each independently N or C; and m is an integer between <NUM> and <NUM>; or pharmaceutically acceptable salts of thereof.

In the second aspect the leukemia may be selected from the group consisting of an acute lymphocytic leukemia (ALL) and an acute myeloid leukemia (AML). The agent may have an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>. The agent may be used in combination with a DOT1L inhibitor in an amount effective to inhibit proliferation and/or enhance apoptosis of leukemic cells in the patient. The DOT1L inhibitor may inhibits DOT1L with an IC50 of from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>. The amount of at least one of the agent or the DOT1L inhibitor may be lower than it would have been had the agent or the DOT1L inhibitor been used alone for achieving the maximum degree of proliferation inhibition and/or apoptosis of leukemic cells. The DOT1L inhibitor may be selected from the group consisting of a purine, a carbocycle-substituted purine, a <NUM>-deazapurine, EPZ00477, EPZ005676, SGC-<NUM>, SYC-<NUM>, and SYC-<NUM>.

In a third aspect, the invention provides an in vitro method for predicting the therapeutic efficacy of an agent that inhibits interaction between MLL1 and menin alone or in combination with a DOT1L inhibitor in a leukemia patient that does not exhibit an MLL-translocation, an MLL-rearrangement, and/or an MLL-partial tandem duplication, said method comprising:.

In the present disclosure, the inventors have discovered MLL and DOT1L in NPM1mut AML to control HOXA and HOXB cluster and MEIS1 expression on a chromatin level. Based on the observation that the wildtype MLL protein complex is required for HOX gene regulation during normal hematopoiesis<NUM>-<NUM>, the inventors first conjectured MLL to be also involved in controlling HOX gene expression in NPM1mut AML. Using a CRISPR-Cas9 negative selection screen targeting multiple MLL protein domains, the inventors discovered the menin binding site of MLL as a top hit in their screen, while it was previously known that the menin-MLL interaction is critically involved in chromatin binding of the MLL complex<NUM>, <NUM>. Additionally, the inventors detected a strong phenotype for at least one of the sgRNAs targeting the CXXC domain, again pointing to chromatin binding of MLL being critically required for NPM1mut leukemias<NUM>. Of note, none of the sgRNAs targeting the C-terminal SET domain of MLL - that contains the H3K4 methyltransferase activity but is always lost in MLL-fusion proteins<NUM> - exhibited any significant phenotype. It has naturally been hypothesized by researchers over decades that MLL family member regulate transcription via H3K4 methyltransferase activity. However, findings of the present disclosure are in line with a recent study on MLL-fusion leukemias in which the remaining wildtype MLL protein was required for leukemic transformation but selective inactivation of the SET domain did neither alter transcription of HOX genes nor transforming potential<NUM>. In addition, the inventors found no evidence that MLL2, the other NELL family member that interacts with menin and carries a SET domain, is involved in NPM1mut leukemogenesis. Whether HOX gene activation is a direct consequence of MLL chromatin binding itself or dependent on other proteins that are indirectly recruited to chromatin via MLL remains to be determined.

DOT1L is another chromatin modifier that the inventors of the present disclosure discovered to be involved in HOX gene control in NPM1mut leukemias. While it is believed that DOT1L is misdirected to HOXA cluster loci in MLL-fusion leukemias via the fusion partner protein<NUM>, our data do not explain, how DOT1L is misguided to HOXA and B genes and MEIS1 in NPM1mut AML and during normal hematopoiesis. However, we found a clear association of HOXA and HOXB cluster and MEIS1 expression levels in murine LSK cells with higher states of H3K79 methylation. Furthermore, evidenced by the inventor's data from DOT1Lf1/f1 animals, the inventors found expression of several HOXB cluster to be at least partially dependent on DOT1L in LSK cells as it was pointed out for HOXA cluster expression before<NUM>.

Similar to disruption of the menin-MLL complex pharmacological inhibition of DOT1L resulted in HOX down regulation, cell growth inhibition, and profound differentiation of NPM1mut AML blasts. These data confirm the inventor's previously build hypothesis that HOX expressing leukemias lacking an oncogenic MLL-fusion protein may also be reliant on DOT1L<NUM>, <NUM> and extent this concept to the much more prevalent AML subtype with NPM1mut.

High expression of early HOXA and HOXB cluster genes is a characteristic feature consistently found in human NPM1mut AML blasts and in murine models of NPM1mut leukemia<NUM>,<NUM>,<NUM>. The strong oncogenic transformation potential of HOX genes in general suggests that these genes are likely required for NPM1mut driven leukemogenesis, whereas formal proof was still lacking. Findings of the present disclosure provide strong support for this concept as exogenous overexpression of selected HOX genes rescues antiproliferative effects of both DOT1L and menin-inhibitors. The inventors further show that combinatorial inhibition of DOT1L and menin has enhanced on-target activity as reflected by more profound HOX and MEIS1 suppression. These changes result in a synergistic antiproliferative and boosted differentiation effect, suggesting that therapeutic HOX targeting ultimately releases the differentiation block of NPM1mut AML blasts.

Another potentially important therapeutic implication of the present disclosure is the finding that MEIS1 and FLT3 were among the most significantly suppressed genes following menin-MLL and/or DOT1L inhibition. As FLT3 was suggested to be a transcriptional target of MEIS1<NUM>, it is likely that this suppression is conveyed via MEIS1 rather than a direct effect caused by MLL or DOT1L inhibition. Internal tandem duplications of FLT3 lead to constitutional activation of this pathway and are found in about <NUM>% of NPM1mut AMLs<NUM>. Thus, herein proposed drug regimen of combined menin-MLL/DOT1L inhibition may particularly be attractive for patients exhibiting the adverse NPM1mut FLT3-ITD genotype.

Whereas data of the present disclosure provide evidence that HOX genes and MEIS1 in NPM1mut leukemias are regulated on a chromatin level via MLL and DOT1L, the specific role of the mutant NPM1 protein with regard to HOX and MEIS1 regulation remains elusive. However, based on the data disclosed herein, the inventors propose that the mutant NPM1 protein acts upstream of menin-MLL and DOT1L in transcriptional regulation, at least partially analogous to the functions of MLL-fusion proteins in MLL-rearranged leukemias.

The availability of menin-MLL and DOT1L inhibitors should enable relatively quick translation of our findings into clinical testing. However, future studies will determine whether the potent anti-leukemic activity can be best harnessed when introduced into current standard chemotherapy regimens, thereby overcoming possible context-specific escape mechanisms that are frequently observed in AML<NUM>. A proof of principle study on leukemia cell lines already demonstrated drug synergism of EPZ-<NUM> with standard chemotherapeutic agents in vitro<NUM>. Another interesting combination partner might be dactinomycin that directly inhibits transcriptional elongation by inhibition of RNA Polymerases and was reported to have activity against NPM1mut leukemia in an anecdotal report on a single patient<NUM>.

In summary, the findings of the present disclosure provide insight into how HOX and MEIS1 expression is regulated on a chromatin level in NPM1mut AML. The inventors further identified the menin-MLL interaction and the H3K79 methyltransferase DOT1L as novel therapeutic targets for this relatively common AML subtype. Also, the inventors discovered that these two proteins control differentiation and that small molecule inhibition of each protein releases the differentiation block of NPM1mut leukemic blasts. Furthermore, combinatorial treatment with both compounds promotes an additive suppression of selected HOX and MEIS1 genes ultimately resulting in synergistic anti-leukemic activity. Both compounds as single agents or in combination represent novel and possibly less toxic therapeutic opportunities for patients with NPM1mut leukemia and might potentially improve the prognosis of patients from the adverse subsets such as concomitant FLT3-ITD, relapsed leukemia, and the difficult to treat elderly population.

Several of these inhibitors have been identified and are commercially available or at a minimum their structures publicly disclosed. <NPL> discloses the structure of a number of inhibitors including MI <NUM>, MI-<NUM> and MI <NUM>, duplicated below. The structure of MI-<NUM> is also published and duplicated below in Table <NUM>.

Additional MLL-menin interaction inhibitors are disclosed in:.

Additional examples can be found in the Table <NUM> below:
<IMG>.

The foregoing compounds in Table <NUM> can be synthesized as described in <NPL> including the supplement thereof.

Effective amounts of the MLL-menin interaction inhibitors are within the following ranges for their IC50: from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>. Smaller amounts may be effective if these inhibitors are administered in combination with a DOT1L inhibitor.

As used herein the phrase "in combination" encompasses both substantially simultaneous administration as well as sequential administration of one or more members of each class of inhibitor disclosed herein. Sequential administrations may be within the same day, or administration may be spaced apart by a time interval of at least a day or a week or two weeks or three weeks or a month or even longer.

The two inhibitors may be administered sequentially in any order. For example, the MLL-menin interaction inhibitor may be administered first followed by the DOT1L inhibitor or vice versa.

Each inhibitor may be formulated into a pharmaceutical formulation, such as those disclosed for DOT1L inhibitors in the <CIT>.

Several such inhibitors have been disclosed in <CIT> along with ranges of effective amounts in terms of IC <NUM> of inhibition being within a range from about <NUM> to about <NUM> or from about <NUM> to about <NUM> or from about <NUM> to about <NUM>. Examples are the following: EPZ005676, EPZ004777, SGC-<NUM>, SYC-<NUM>, SYC-<NUM>, and SYC-<NUM>. These and additional DOT1L inhibitors are disclosed in the following publications: <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>; and <CIT>.

DOT1L inhibitors that may be suitably employed in the presently disclosed methods for inhibiting the proliferation and/or survival of cell and for treatment of leukemia patients include the <NUM>-deazapurine compounds as described in <CIT> and <CIT> as represented by Formula I:
<CHM>.

DOT1L inhibitors that may be suitably employed in the presently disclosed methods for inhibiting the proliferation and/or survival of cell and for treatment of leukemia patients include carbocycle-substituted purine and <NUM>-deazapurine compounds as described in <CIT> as represented by Formula II:
<CHM>.

DOT1L inhibitors that may be suitably employed in the presently disclosed methods for inhibiting the proliferation and/or survival of cell and for treatment of leukemia patients include purine and <NUM>-deazapurine compounds as described in <CIT> and <CIT> as represented by Formula III:
<CHM>.

Compounds that are encompassed within the range of compounds defined by Formulas I, II, and III, and methodologies for the synthesis of those compounds, are presented in <CIT> and <CIT>; <CIT>; <CIT>; and <CIT>. Two exemplary such compounds are EPZ004777 and EPZ005676, which are presented in the following section along with a description of methodologies for synthesizing those compounds from readily available starting materials (e.g., Sigma-Aldrich, St. Louis, MO).

The small molecule DOT1L inhibitor EPZ004777 is an s-adenosyl methionine mimetic is highly specific for DOT1L as compared to other methyl transferases. <NPL>) and <NPL>). EPZ004777 binds within the S-(<NUM>'-adenosyl)-<NUM>-methionine (SAM) binding site in the catalytic domain of human DOT1L.

An inhibitor from either class will generally be administered parenterally in a physiologically acceptable vehicle as disclosed for DOT1L inhibitors in <CIT>.

"Parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, <NUM> intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Alternatively, or concurrently, administration may be by oral route.

Various compositions containing one or both inhibitors are contemplated as disclosed in <CIT> for DOT1L inhibitors. Excipients, carriers and diluents are as disclosed in <CIT> for DOT1L inhibitors. Units dosage form amounts are also as disclosed in <CIT> for DOT1L inhibitors.

The present disclosure provides compositions, including therapeutic compositions comprising one or more DOTlL inhibitor(s) and/or one or more inhibitor(s), for the treatment of a leukemia, such as ALL or AML. One or more DOTlL inhibitor(s) and/or one or more menin-MLL interaction inhibitor(s) can be administered to a human patient individually as monotherapy or combinedly in individual formuations. In such formulations the active ingredient or ingredients are mixed with suitable carriers or excipient(s) at doses to treat or ameliorate leukemia such as ALL or AML as described herein. Mixtures of these inhibitors can also be administered to the patient as a simple mixture or in suitably formulated pharmaceutical compositions.

Compositions within the scope of this disclosure include compositions wherein the therapeutic agent is provided in an amount effective to inhibit the proliferation of a leukemia cell in a patient. Determination of optimal ranges of effective amounts of each component and duration of administration is within the skill of the art. The effective dose and/or duration is a function of a number of factors, including the specific inhibitor and its half-life in the body of the patient, the presence of a prodrug, the incidence and severity of side effects, the age, weight and physical condition of the patient and the clinical status of the latter. The dosage administered and duration of the administration may also be dependent on the kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Cycles of administration followed by periods of no treatment are also contemplated.

Compositions comprising one or more inhibitors according to the present disclosure may be administered parenterally. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Alternatively, or concurrently, administration may be orally.

Examples of parenteral administration include intravenous administration via an intravenous push or bolus. Alternatively, compositions according to the present disclosure may be administered via an intravenous infusion.

Suitable dosages for intravenous infusion of a composition comprising an inhibitor of menin-MLL interaction alone or in combination with a DOTlL inhibitor include a dosage of at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least bout <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/day or at least about <NUM> inhibitor/m2/ day or maximum tolerated dose if lower.

Compositions comprising a DOTlL inhibitor and/or a menin-MLL interaction inhibitor generally include a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients for solid and/or liquid formulations may be selected by those skilled in the art as appropriate for a particular formulation among for example starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skimmed milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such compositions will contain a therapeutically effective amount of at least one inhibitor, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. Compositions can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.

Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a <NUM> hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The inhibitors disclosed herein can be formulated as free base (or acid) or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, <NUM>-ethylamino ethanol, histidine, procaine, and the like.

Many of the inhibitors of the present disclosure may be provided as salts with pharmaceutically compatible counterions (i.e., pharmaceutically acceptable salts).

A "pharmaceutically acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a prodrug of a compound of this invention. A "pharmaceutically acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt upon administration to a subject. Pharmaceutically acceptable salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Salts tend to be more soluble in water or other protic solvents than their corresponding free base forms. The present invention includes such salts.

It is anticipated that the MLL-Menin interaction inhibitors will be likely used every day for a period of time randging from one week or two weeks or three weeks up to about <NUM> months or <NUM> months or <NUM> months or <NUM> year or <NUM> years. The inhibitor can be administered daily throughout this period or can be given for a period of days followed by a period of no treatment and then followed by resumption of treatment. The exact administration protocol can be determined by those skilled in the art.

The amounts administered have been discussed above.

If a DOT1L inhibitor is used in addition, a similar protocol is anticipated, subject to any adjustment to dosage of one or the other inhibitor as described above and subject to allowances in administration protocol for simultaneous or sequential administration of each inhibitor as taught above and as appreciated by those skilled in the art.

The following Examples further illustrate the findings of the present disclosure.

Since MLL1 has been shown to regulate critical gene expression programs, including HOX gene expression in normal hematopoiesis and MLL-rearranged AML, the inventors hypothesized that it might also regulate HOX gene expression in other settings. As an initial assessment, the inventors used an approach recently developed by Shi and co-workers that uses genome editing across exons encoding specific protein domains to determine the functional relevance of a given domain and the suitability for drug development<NUM>. To assess potential dependencies of NPM1mut AML on specific protein domains of MLL and its most similar family member MLL2, a negative selection CRISPR-Cas9 screen interrogating multiple domains of both proteins was designed. The inventors generated a tetracycline-inducible Cas9-expressing OCI-AML3 cell line (OCI-AML3-pCW-Cas9, <FIG>). After transduction with <NUM>-<NUM> GFP+ sgRNAs interrogating each domain, Cas9 was induced and GFP+/GFP--ratio assessed over a period of <NUM> days. Cells expressing sgRNAs targeting exon <NUM> that encodes the menin-binding domain of MLL were rapidly outcompeted by their GFP- counterparts (<FIG>; fold change of GFP+ d0/d15 up to <NUM>). Similar results were obtained for one of two sgRNAs targeting the first exon of RPA3, a gene required for DNA replication that is used as a positive control, while there was no difference in GFP expression over time in two independently transduced empty vector control guides (<FIG>). Interestingly, sgRNAs targeting exons that encode the CXXC domain of MLL, also known to be involved in chromatin binding and for MLL-fusion leukemia, were the selected against in our screen (<FIG>). Of interest, no significant phenotype was observed for sgRNAs targeting the C-terminal SET domain of MLL (<FIG>). These findings were validated by performing a second screen using another independently engineered OCI-AML3-pCW-Cas9 clone (OCI-AML3-pCW-Cas9-C8, <FIG>) , where similar results were obtained with sgRNAs targeting the menin binding domain being again the top hit. The inventors found no significant negative selection for any guides that interrogated the MLL2 protein domains (<FIG>). These data indicate that the menin-binding domain of MLL1 but not MLL2 is required for NPM1mut AML.

The genetic data presented above indicate that the menin-MLL interaction might be a therapeutic target in NPM1mut AML. The inventors next assessed whether recently developed small molecule inhibitors of the menin-MLL interaction might influence proliferation and gene expression in NPM1mut AML cells. First, the effects of an inhibitor of the MLL-Menin interaction, MI-<NUM>-<NUM> on the human NPM1mut AML cell line OCI-AML3 were assessed<NUM>. The inventors observed a profound dose-dependent reduction in cell proliferation that was even more pronounced than in the MLL-AF9-rearranged MOLM-<NUM> cells that served as a positive control (<FIG>). The HL-<NUM> AML cells lacking an NPM1mut or MLL-rearrangement showed only a mild cell growth inhibitory effect at higher doses (<FIG>). Next, gene expression was asssessed in the NPM1mut AML cells where a significant down regulation of HOXA, HOXB cluster and MEIS1 gene expression upon <NUM> days of treatment with MI-<NUM>-<NUM> (<FIG>) was observed. Of these, MEIS1 appears to be the most profoundly suppressed gene. This finding prompted the inventors to explore a possible effect of menin-MLL inhibition on FLT3 expression, as FLT3 is a reported downstream target of MEIS1 (<NPL>). In fact, FLT3 transcript levels were also substantially and highly significantly suppressed upon menin-MLL inhibition (<FIG>).

Next, the inventors sought to validate the above findings in two independent murine conditional knock-in models of NPM1mut leukemia. One of the leukemias is engineered to possess both an NPM1 mutation and a Flt3-ITD (Npm1CA+Flt3ITD/+), whereas the other model possesses an NPM mutation and secondary mutations that were induced through use of a Sleeping Beauty transposon system(Npm1CA+RosaSB/+). <NUM>, <NUM> Additionally, MI-<NUM>, a novel menin-MLL inhibitor was recently described<NUM> that was synthesized by re-engineering the molecular scaffold of MI-<NUM>-<NUM> resulting in enhanced drug-like properties. Experiments on the murine cells were therefore performed using MI-<NUM>. To compare the two compounds and to define equivalent growth inhibitory concentrations, dose response curves for both inhibitors were generated, confirming the higher potency of MI-<NUM> over MI-<NUM>-<NUM> in human and murine NPM1mut leukemia cells (<FIG>).

For drug testing experiments, leukemic blasts were harvested from moribund primarily transplanted mice and cultured in vitro. MI-<NUM> treatment was performed in colony forming assays and liquid culture proliferation assays as Npm1CA/+RosaSB/+ cells grew in methylcellulose and Npm1CN+Flt3ITD/+ cells grew in liquid culture medium. Similar to the human cells, we observed a profound inhibitory effect on colony forming potential and cell growth in both NPM1mut as well as Mll-Af9 leukemia cells in response to menin-MLL inhibition, while Hoxa9-Meis1 transformed cells were unaffected (<FIG>).

Analogous to what has been observed in the OCI-AML3 cells, the inventors observed significant down regulation of Hoxa and Hoxb cluster genes as well as Meis1 after four days of MI-<NUM> treatment. Whereas baseline expression levels of the early Hoxb cluster genes differed among the murine leukemias - with the Npm1CA/+Flt3ITD/+ cells for example lacking expression of Hoxb2 - Meisl was again most profoundly suppressed in both leukemias and accompanied by substantial down regulation of global Flt3 transcript levels (<FIG>).

Whereas induction of apoptosis was moderate following menin-MLL inhibition, the inventors noted a strong differentiation inducing effect in all three NPM1mut leukemia models as reflected by morphological changes consistent with monocytic and granulocytic features (<FIG>) as well as a profound increase in CD11b expression as determined by flow cytometry (<FIG>; vehicle vs. MI-<NUM>-<NUM>: <NUM>% vs. <NUM>% on day seven of treatment).

These data indicate that pharmacological menin-MLL inhibition causes down regulation of HOX genes, MEIS1 and FLT3 followed by differentiation and cell growth inhibition.

As changes in HOX and MEIS1 gene expression (day4; <FIG>) preceded cell differentiation and growth inhibitory effects following menin-MLL inhibition, it was hypothesized that HOX and MEIS1 gene expression controls cell proliferation and differentiation in NPM1mut AML cells.

Thus, the inventors investigated the effect of ectopic expression of Meis1, Hoxb4, and Hoxa9-Meis1 in the Npm1CA/+FLt3ITD/+ murine leukemia cells. In all three cases overexpression of these genes was observed to rescue the Npm1CA/+FLt3ITD/+ cells from the menin-MLL antiproliferative effect as indicated by a shift of the dose response curves towards higher concentrations and increased IC<NUM> values (<FIG>). Thus, pharmacological menin-MLL inhibition inhibits cell proliferation in NPM1mut AML cells most likely via alteration of Hox and Meis1 expression.

The inventors next investigated the therapeutic effects of menin-MLL inhibition in vivo using a disseminated human OCI-AML3 xenotransplantation model. OCI-AML3 cells were transplanted into NSG mice via tail vein injection and MI-<NUM> treatment was initiated seven days later. Animals were sacrificed after seven (n=<NUM> per group) and <NUM> days (n=<NUM> per group) of MI-<NUM> treatment (<NUM>/kg bid IP). Leukemia burden, as defined by the percentage of bone marrow cells expressing human CD45, was significantly reduced within the treated animal group compared to vehicle controls (<FIG>). Furthermore, analysis of HOXB4, MEIS1 and FLT3 in sorted human CD45 positive cells harvested from these animals exhibited a dramatic decrease of expression levels in the MI-<NUM> treated versus vehicle control animals (<FIG>).

Menin associates with MLL1 at HOXA and MEIS1 promoters<NUM> and pharmacological interruption of the menin-MLL interaction diminishes menin occupancy at the HOXA locus in MLL-rearranged leukemias<NUM>. The abundance of menin at HOXA, HOXB and MEIS1 loci was assessed in NPM1mut leukemias before and after pharmacological menin-MLL inhibition. Using Chromatin Immunoprecipitation (ChIP) of menin followed by quantitative PCR in the human NPM1mut leukemia cell line OCI-AML3, the inventors demonstrated presence of menin at expressed HOXA, HOXB and MEIS1 gene loci (<FIG>). MI-<NUM> treatment depleted menin from most of these loci and was most significantly reduced at the MEIS1 locus thereby correlating with gene expression levels (<FIG> and <FIG>). Whereas global assessment of several chromatin marks did not reveal any changes upon menin-MLL inhibition (<FIG>) the invenotrs observed a locus specific decrease of H3K4 trimethylation marks at HOX and MEIS1 loci (<FIG>), thus demonstrating the expected changes is histone methylation due to Menin inhibition. Somewhat unexpectedly, a dramatic reduction of H3K79 dimethylation (H3K79me2) was also observed at those loci as it has been observed in MLL-rearranged leukemias (<FIG>). These data are consistent with decreased binding of the menin-MLL complex to HOX and MEIS loci following menin-MLL inhibition and also point to the H3K79 methyltransferase DOT1L as having a potential role in HOX gene regulation in NPM1mut leukemia cells.

The data described above suggest wt-MLL1 to be important for HOX gene control in NPM1mut AML, and also implicate DOT1L in HOX gene regulation in this disease. Recent studies of benign murine hematopoiesis demonstrated that DOT1L is another chromatin regulator critical for HOXA cluster regulation in early hematopoietic progenitors, whereas its role in HOXB cluster control is not well defined. To explore whether DOT1L might be involved in the control of HOXB cluster genes expression, we reanalyzed gene expression data from Dot1lfl/fl Mx1-Cre transgenic mice after polyinosinic-polycytidylic acid (pIpC) induced excision compared to their normal counterparts. In fact, the inventors found the early HOXB cluster genes HOXB2 and HOXB4 significantly down regulated after homozygous deletion of DOT1L (<FIG>), while there was also a trend for HOXB3 that did not reach statistical significance. In addition, there was an association between high expression levels of early HOXB cluster genes and higher states of H3K79 methylation (H3K79me2 and me3) in normal murine Lin-, Sca1+, c-Kit+ (LSK) cells (<FIG>) mimicking the H3K79 profile across the expressed HOXA cluster genes in these cells. These findings extend the concept of DOT1L being critical for HOXA cluster regulation during normal hematopoiesis to the HOXB cluster, which together represents also the dominant expression pattern found in NPM1mut leukemias.

Based on these findings, the inventors assessed the effect of EPZ4777, a small molecule DOT1L inhibitor in NPM1mut leukemias. DOT1L inhibition led to a profound reduction in cell proliferation and colony forming potential in the human and the murine leukemia cells. Similar results were obtained for MLL-fusion leukemias whereas leukemias lacking an NPM1 mutation or MLL-fusion such as the HL-<NUM> cells and Hoxa9-Meis1 in vitro transformed cells were unaffected (<FIG>). The antiproliferative effects of DOT1L inhibition were preceded by global and HOXA and HOXB loci specific reduction in H3K79me2 marks as determined by immunoblotting and ChIP-PCR, whereas global levels of other histone marks associated with transcriptional activation (H3K4me3, H3K36me3) and transcriptional repression (H3K27me2) were unchanged (<FIG>). As hypothesized, DOT1L inhibition resulted in significant repression of HOX genes in both models (<FIG>, left panel and <FIG>), while the specific pattern of affected genes differed slightly among the different models. As murine leukemias exhibited significant suppression of HOXA and HOXB cluster genes, the human OCI-AML3 cells showed most significant down regulation of MEIS1 and the HOXB cluster only as assessed by RNA-sequencing (<FIG>). Of note, MEIS1 was the most consistently and profoundly down regulated gene across all NPM1mut models and was accompanied by dramatic suppression of FLT3 expression (<FIG>). As global changes in H3K79 methylation marks might be associated with transcriptional up- or down regulation that in turn can also lead to profound normalization bias of RNAseq data, the findings were validated using ERCC-control sequences that were spiked-into the specimens as suggested by Young and others<NUM>. There was no difference of HOX or global transcription levels between the different normalization methods, indicating that global reduction of H3K79me2 is not associated with global transcriptional down regulation but with suppression of a smaller subset dominated by HOX genes in these cells. As demonstrated for menin-MLL inhibition, it was found that retroviral overexpression of Hoxb4, Meis1, or Hoxa9-Meis1 abolished sensitivity of Npm1CA/+FLt3ITD/+ cells to DOT1L inhibition (<FIG>).

Besides suppression of HOX genes and MEIS1, Gene Set Enrichment Analysis (GSEA) of RNAseq data on the OCI-AML3 cells identified a dominant set of genes up regulated upon DOT1L inhibition that was significantly enriched for genes usually silenced in HSCs and mainly containing genes associated with myelo-monocytic differentiation (<FIG>). Accordingly, profound induction of differentiation was observed in human and murine NPM1mut leukemia cells following DOT1L inhibition. These data are therefore consistent with DOT1L participating in HOX gene regulation and differentiation control of NPM1mut leukemia cells.

Besides the NPM1 mutation OCI-AML3 cells also carry a DNMT3A mutation (R882C), which is associated with aberrant HOX expression in AML. To explore whether DNMT3A mutation status is associated with sensitivity to DOT1L inhibition, the inventors included two additional cell lines with DAMT3A mutations in the study. The JAK2 mutated SET2 cells were recently reported to carry the most common DNMT3A mutation found in AML patients (R882H), whereas OCI-AML2 cells carry a DAMT3A mutation that has so far been reported only in a single patient with myelodysplastic syndrome (R635W)<NUM>. Whereas the SET2 cells did not show any sign of sensitivity to DOT1L- inhibition, the OCI-AML2 cells were substantially sensitive to DOT1L inhibition (<FIG>).

To understand these conflicting findings, the inventors searched for the presence of other genetic factors potentially mediating sensitivity to EPZ4777 in the OCI-AML2 cells. Surprisingly, RNA sequencing revealed the presence of an MLL-AF6 fusion transcript with breakpoints that aligned to the common breakpoint cluster region typically affected in MLL-AF6 rearranged leukemias. Breakpoint spanning RT-PCR confirmed the presence of the fusion transcript. Whereas conventional cytogenetic analysis revealed two normal 11q23 loci, a MLL split signal was detected within the derivative chromosome 1q. These findings are consistent with the presence of a functionally relevant cryptic MLL-AF6 fusion inserted into the derivative chromosome1. Also, the inventors observed high-level expression of HOXA but not HOXB cluster genes that were profoundly down regulated upon DOT1L inhibition.

Together, these data support a previous study (<CIT>) reporting that DOT1L controls HOXA cluster expression also in MLL-fusion leukemia with no apparent DOT1L recruiting activity (such as MLL-AF6). However, whether DNMT3A mutations are associated with sensitivity to DOT1L inhibition remains to be determined.

As HOX gene expression is associated with self-renewal properties, the inventors next determined whether pharmacological DOT1L inhibition inhibits NPM1mut leukemia initiation in mice. Murine Npm1CA/+RosaSB/+ leukemia cells were treated ex vivo with EPZ4777 or drug vehicle for <NUM> days and equal numbers of viable cells were transplanted into sublethally irradiated recipient animals. A significant survival advantage was observed for the animals transplanted with the treated cells compared to vehicle control in both NPM1mut leukemia models (<FIG>). Whereas animals transplanted with the EPZ4777-treated Npm1CA/+RosaSB/+ leukemia cells exhibited significantly lower WBC counts at time of disease onset within the control group, all mice eventually developed full blown leukemia (<FIG>). Similar results were obtained for the Npm1CA/+FLt3ITD/+ cells but with longer disease onset within the treated group. These findings demonstrate the ability of pharmacological DOT1L inhibition to suppress leukemia initiating potential in vivo.

The data presented above are consistent with the menin-MLL interaction and DOT1L being both therapeutic targets that control HOXA and HOXB cluster regulation in NPM1mut AML. Next, the therapeutic potential of combinatorial menin- and DOT1L inhibition in these leukemias was assessed. Simultaneous menin- and DOT1L inhibition in the OCI-AML3 cells resulted in a profound shift of the dose response curve toward lower doses compared to each of the compounds alone (<FIG>). Drug combination analysis using the Chou-Talalay model revealed a clear synergistic anti-proliferative effect. Drug synergism was also observed in proliferation assays of the Npm1CA/+FLt3ITD/+ cells (<FIG>) and combinatorial drug treatment resulted in significantly higher suppression of colony forming potential in Npm1CA/+RosaSB/+ cells (<FIG>). These results are also shown in <FIG>, which provides numeric data reflecting the dose and relative growth inhibition, (i.e. effect) for the single and combination treatments for Npm1CA/+FLt3ITD/+ cells at day <NUM>, illustrating synergy for the combination treatments.

These changes were preceded by significantly enhanced down regulation of selected genes with drug treatment combination (menin-MLL and DOT1L inhibition). Whereas suppression levels of specific HOXA and HOXB cluster genes differed among the different NPM1mut leukemia models, MEIS1 and FLT3 were consistently suppressed (<FIG>). Consistent with these findings, cells treated with the combination regimen exhibited significantly less RNA-Polymerase II binding to the MEIS1 promoter and gene body region than single treated cells as assessed using ChIP-PCR. These findings are therefore consistent with an enhanced inhibitory effect of MEIS1 transcription.

While the inventors also observed a significant increase in apoptosis in the cells treated with both compounds (<FIG>), effects on differentiation were most pronounced in all NPM1mut AML models and occurred earlier compared to single drug treatment (<FIG>).

Since the in vitro data showed a synergistic drug effect for simultaneous menin- and DOT1L inhibition in NPM1mut leukemias, the inventors examined the effect of combinatorial drug treatment on leukemia initiating potential in vivo. Equal numbers of viable cells treated with drug vehicle, EPZ4777, MI-<NUM>-<NUM>, or the combination were transplanted into sublethally irradiated recipient and assessed for engraftment. As shown in <FIG>, % engraftment was lower when animals were treated with both EPZ4777 and MI-<NUM>-<NUM>. These results are also shown in <FIG>, which provides numeric data reflecting the dose and relative growth inhibition, (i.e. effect) for the single and combination treatments, illustrating synergy for the combination treatments.

In order to assess effects of drug treatment of NPM1mut primary AML samples, the inventors used a modified, previously published human co-culture model described in the material and methods section to maintain and/or expand human AML cells for <NUM> days and assess for drug sensitivity. Whereas <NUM> of <NUM> cases was not maintainable in culture and cell death occurred within <NUM> hours after thawing, the remaining <NUM> samples were treated for <NUM> days and viable cell numbers of human CD45+ cells were assessed in order to control for potential contamination with adherently growing stromal cells. While all cases exhibited a profound reduction of viable cell number compared to DMSO control, the inventors observed a significantly enhanced antiproliferative effect for the drug combination vs. single drug treatment in three of the four cases (<FIG>). Morphologic changes within the combinatorial drug treatment group, seemed to be more profound than vehicle or either of the compounds alone.

Given the findings in previous examples, which showed synergistic drug effect for simultaneous menin-MLL and DOT1L inhibition in NPM1mut leukemias in vitro, the inventors tested the effects of combinatorial drug treatment on leukemia initiating potential. Equal numbers of viable Npm1CA/+RosaSB/+ leukemia cells pretreated with drug vehicle, EPZ4777, MI-<NUM>, or the combination were transplanted into sublethally irradiated recipient mice and assessed for leukemia onset and survival. Whereas both, EPZ4777 and MI-<NUM> treatment led to significantly prolonged survival compared to vehicle control, the inventors observed a significant survival advantage for the drug combination treatment compared to either of the compounds alone (<FIG>). Leukemia onset was also significantly delayed as reflected by significantly lower white cells count or CD45. <NUM> engraftment at the time of death from leukemia within the control group (<FIG>). These data confirm that both menin-MLL and DOT1L inhibition diminishes leukemia initiation and indicate that simultaneous treatment using both compounds shows enhanced effects on leukemia initiating (stem) cells.

The AML cell lines OCI-AML3, OCI-AML2, SET-<NUM>, MOLM-<NUM>, HL-<NUM>, as well as 293T cells, and Hs27 cells were maintained under standard conditions. Cell line authentication testing (ATCC) verified identity and purity for all human AML cell lines used in this study. Murine leukemia cells were cultured in DMEM supplemented with <NUM>% fetal bovine serum, <NUM>% Penicillin/Streptomycin, and cytokines (SCF 100ng/µl, IL-<NUM>20ng/µl, and IL-<NUM>20ng/µl).

Plasmid construction and sgRNA design: The pCW-Cas9 expression construct (humanized S. pyogenes Cas9 containing N-terminal FLAG-tag and the TET ON promotor) and the pLKO5. GFP vector were both purchased from Addgene (#<NUM> and #<NUM>). All sgRNAs in this study were designed using http://crispr. edu/ and quality scores for all but <NUM> sgRNAs were above <NUM>. Domain description, sgRNA sequences and quality scores from this study are provided in the tables below. sgRNA cloning was performed by annealing and ligating two oligonucleotides into a BsmB1-digested pLKO5. GFP vector as described{Heckl, <NUM> #<NUM>}.

Engineering OCI-AML3-pCW-Cas9: OCI-AML3-pCW-Cas9 cells were derived by retroviral transduction of the NPM1mut acute myeloid leukemia cell line (OCI-AML3) with pCW-Cas9, followed by puromycin selection. Cells were plated in methylcellulose to obtain single cell-derived clones, re-expanded in liquid culture, and aliquots frozen in liquid nitrogen. pCW-Cas9 was induced in each clone with doxycycline and screened by anti-flag western blot analysis for Cas9 expression. Three independent Cas9 expressing clones were infected with positive control sgRNAs targeting RPA3 and empty control vector to assess CRISPR-Cas9 editing efficiency after Cas9 induction. The two best performing clones were selected for the MLL and MLL2 domain screen: sgRNA virus production and negative selection screening. sgRNA virus production was performed in <NUM>-well plates using HEK293T cells using the protocol provided at the Broad's Institute genetic perturbation platform (http://www. broadinstitute. org/rnai/public/resources/protocols) using the envelope and packaging plasmids pCMV-dR8.74psPAX2 and pMD2. G and X-tremeGENE transfection reagent (Roche). For virus transduction, OCI-AML3-pCW-Cas9 clones were thawed, plated in <NUM>-well plates, and infected using virus supernatant. Three days following infection, GFP+/GFP- ratio was determined and doxycycline was added to the media to induce pCW-Cas9. GFP+/GFP- ratio was tracked every three days for <NUM> days as described {Shi, <NUM> #<NUM>}.

Proliferation assays were performed as previously described. Viable cell counts were determined by flow cytometry using Sytox Blue (Invitrogen) or DAPI viability stains. For determination of IC<NUM> values EPZ004777, MI-<NUM>-<NUM>, and MI-<NUM> were diluted <NUM>-fold for a total of <NUM> concentration and vehicle control with the highest concentrations being <NUM>, <NUM>, and <NUM> respectively. Combinatorial drug treatment of serial dilutions of MI-<NUM>-<NUM> or MI-<NUM> with EPZ4777 was performed using constant ratios of <NUM>:<NUM> and <NUM>:<NUM> for assessment of drug synergism. Mathematical synergy testing was performed using CompuSyn software for Chou-Talalay- method based calculations (<NPL>).

Library construction and RNA sequencing was performed as described elsewhere (cite), comparing OCI-AML3 and OCI-AML2 cells treated for <NUM> days with <NUM> EPZ004777 or vehicle control in biological triplicates. For gene expression analysis <NUM> basepair single read sequencing was performed using a sequencing depth of <NUM> Million reads. ERCC control sequences were spiked into lysis buffer during RNA extraction. For fusion gene detection we performed <NUM> basepair paired-end sequencing at a depth of <NUM> Million reads. Computational analysis was performed using the Defuse algorithm.

Chromatin immunprecipitation (ChIP) followed by qRT-PCR was performed as previously described (Bernt <NUM>). Briefly, cross- linking was performed with <NUM>% formalin, and the cells were lysed in SDS buffer. Sonication was used to fragment DNA. ChIP for H3K79me2, Menin, and MLL was performed using the antibodies ab3534 (abcam), <NUM>-<NUM> (Bethyl), ab8580 (abcam) and Santa Cruz ac-<NUM> (Rpb1 terminus) specific to the respective modifications. The antibodies are publicly available. Eluted DNA fragments were analyzed using qPCR. Primer sequences (all human) are as follows:.

EPZ004777 was synthesized by James Bradner's laboratory (Dana Farber Cancer Institute, Boston, MA) but it is also available commercially, for example from MedChem Express (https://www. medchemexpress. EPZ-<NUM> and MI-<NUM>-<NUM> were purchased from Chempartner. MI-<NUM> was synthesized by Vitae Pharmaceutical Inc. For in vitro studies <NUM> stock solutions were prepared in DMSO and stored at -<NUM>. Serial dilutions of stock solutions were carried out just prior to use in each experiment, and final DMSO concentrations were kept at or below <NUM>% and <NUM>% for combination treatment experiments, respectively.

RNA and DNA purification, cDNA synthesis, quantitative real-time (qRT-) PCR, western blotting, flow cytometry, colony forming assays, viral transduction and shRNA based knock down experiments were performed using standard procedures. Primer sequences, western antibodies, and plasmids are also listed in the supplement.

Npm1CA/+Flt3ITD/+ and Npm1CA/+RosaSB/+ cells were harvested from moribund leukemic animals and transplanted into primary and secondary recipient mice as previously described. Npm1CA/+FLt3ITD/<NUM> cells were transplanted into <NUM>-<NUM> week-old BL6/<NUM> (Jackson Laboratory) or NSG mice (Taconic) and Npm1CA/+RosaSB/+ into NSG mice (Taconic). Animals were preconditioned with 600cGy or 200cGy total body irradiation, respectively (<NUM>Cs source) and <NUM>-<NUM> hours following irradiation 1x10<NUM> leukemia cells were administered via tail vein injection. All transplantation experiments with ex vivo treated cells were performed as secondary transplantations.

<NUM>-<NUM> week female NSG mice (Taconic) were injected via tail vein with 5x10<NUM> OCI-AML3 cells. Treatment was initiated <NUM> days post transplantation consisting of either vehicle (<NUM>% DMSO, <NUM>% PEG400, <NUM>% PBS) or MI-<NUM> at <NUM>/kg IP, bid. N=<NUM> mice and n=<NUM> mice of each group were sacrificed after <NUM> and <NUM> days of drug treatment and leukemia burden was assessed by determining human CD45 using flow-cytometry from peripheral blood and bone marrow. For gene expression analysis, RNA was isolated from MACS sorted hCD45 cells and further processed using standard procedures. Remaining animals were treated for <NUM> consecutive days and then monitored daily for clinical symptoms to assess survival. Moribund animals were euthanized when they displayed signs of terminal leukemic disease.

Human AML samples were co-cultured with irradiated Hs27 stromal cells for <NUM> days in serum-free media (StemSpan, Stem Cell Technologies) supplemented with human cytokines, SR-<NUM>, and drug(s) or vehicle (DMSO, EPZ004777 <NUM>, MI-<NUM><NUM>, or EPZ004777 plus MI-<NUM>) as previously described. Experiments were performed in triplicates and DMSO concentrations were kept below <NUM>%. Cell numbers were determined by flow cytometry using viability and anti-hCD45 staining to control for possible stromal cell contamination after <NUM> days. Cell differentiation was assessed using anti-hCD11b staining.

Statistical significance was calculated using unpaired two-tailed Student's t test, survival was estimated using the Kaplan-Meier method. Computations were all performed using GraphPad Prism, version <NUM>. Error bars represent mean +/- standard error of the mean.

Claim 1:
A method for inhibiting proliferation and/or inducing apoptosis in a leukemia cell in vitro, comprising contacting the leukemia cell with an inhibitor of interaction between MLL1 and menin, wherein the leukemia cell exhibits an NPM1 mutation, and wherein the leukemia cell does not exhibit a genetic mutation, alteration, and/or abnormality that is an MLL-translocation (MLL-t), an MLL-rearrangement (MLL-r), or an MLL-partial tandem duplication (MLL-PTD).