Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.

Transient potential receptor melastatin-2 (TRPM2) is a non-selective cationic, Ca2+ permeable transmembrane pore that is preferentially expressed in cells of the myeloid lineage, and modulates signaling pathways converging onto NF-kB. This is of potential interest for AML therapy, as NF-κB signaling is emerging as a key pathway mediating drug resistance and leukemia initiating cell survival in AML, and inhibition of NF-κB signaling has been shown to be synergistic with chemotherapy. TRPM2 is overexpressed in AML compared to normal bone marrow, with highest levels in the FAB M3-6 subtypes. To determine the effect of loss of Trpm2 in a defined genetic model, we established MLL-AF9 driven AML on a Trpm2−/− genetic background. Trpm2−/− MLL-AF9 leukemias displayed reduced NF-κB phosphorylation and nuclear translocation. In vivo primary and secondary recipients of Trpm2−/− MLL-AF9 leukemias showed increased latency compared to recipients of wild type leukemia cells. However, the difference in latency was small, and lost in tertiary transplants. The effect of loss of Trpm2 in a BCR-ABL/NUP98-HOXA9 fusion model was even smaller. Given reports that loss or inhibition of TRPM2 enhanced killing by DNA damaging agents in neuroblastoma, breast and prostate cancer cell lines, we exposed Trpm2−/− and Trpm2wt primary MLL-AF9 leukemias to doxorubicin, cytarabine and etoposide, but found no difference in IC50. The in vitro response to decitabine was also unaffected. In summary, TRPM2 does not seem to play a major role in myeloid leukemogenesis. In addition, loss of TRPM2 does not augment the cytotoxicity of standard AML chemotherapeutic agents.

Abstract Understanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation.

Rationale: KMT2A-rearrangements (KMT2A-r) in acute myeloid leukemia (AML), encompassing both KMT2a-fusions (KMT2A-F) and KMT2A-partial tandem duplications (KMT2A-PTD), represent a subgroup of AML with a particularly poor prognosis. Both KMT2A-F and KMT2A-PTD share a dependency on the H3K79 methyltransferase DOT1L for proper histone 3 lysine 79 dimethylation (H3K79me2) on target genes such as HOXA9 and MEIS1. Pharmacologic inhibition of DOT1L results in downregulation of KMT2A fusion/PTD target genes. Albeit rare, complete responses observed in patients with relapsed/refractory KMT2A-r leukemia treated with a DOT1L inhibitor underscore the clinical relevance of this pathway. The second most commonly co-occurring mutation with KTM2A-PTD are mutations in isocitrate dehydrogenase 1 and 2 (mIDH1/2). The canonical oncogenic function of mIDH1/2 involves aberrant production of the oncometabolite 2HG and inhibition of TET2. However, 2HG also induces histone hypermethylation, including H3K79 hypermethylation as reported by several groups. Based on this, we sought to study the effect of increasing H3K79 methylation in KMT2A-r AML either via direct overexpression of DOT1L or introduction of mIDH1/2. Results: As increased H3K79 methylation has only been observed upon 2HG exposure or mIDH1/2 expression in experimental systems, we analyzed H3K79 methylation in primary AML patient samples. We found increased H3K79 methylation in IDH1/2 mutant AML at diagnosis. Given the high percentage of IDH1/2 mutations in KMT2A-PTD leukemias, we sought to understand what effect increasing H3K79me2 would have on KMT2A-r leukemia. To this end, we overexpressed DOT1L in KMT2A-MLLT3 murine AML cells. Surprisingly, we found H3K79me2 was counterselected, and DOT1L overexpressing cells grew slower compared to controls. Furthermore, ChIP-Seq revealed that DOT1L overexpression increased H3K79me2 peak height and width outside KMT2A-fusion target genes, effectively blunting the difference between fusion targets and other genomic loci. This was associated with a decreased expression of target genes. This counterintuitive finding led us to analyze global (rather than locus specific) H3K79 methylation in AML patient samples. We found that H3K79me2 is globally low in KMT2A-r AML. This constellation is reminiscent of CALM-AF10 AML, which typically loses the second AF10 allele, resulting in globally low H3K79me2/3, preserved/increased methylation on fusion target loci, and DOT1L dependence. We next tested the effect of IDH mutations on KMT2A-r AML. Co-expression of mIDH1 and MLL-MLLT3 in murine lin-sca1+ckit+ bone marrow cells resulted in an aggressive AML that was dependent on DOT1L, but not mIDH1. As we found when DOT1L was overexpressed, H3K79me2 was counterselected in the mouse. This finding was confirmed by introducing KMT2A-MLLT3 into mIDH2 knock-in bone marrow recapitulating the order of mutations in AML patients, as well as by overexpressing mIDH1 in a KMT2A-PTD cell line. In two patient samples with KMT2A-PTDs and IDH1/2 mutations, we found no difference in H3K79 methylation compared to non-IDH1/2 mutant KMT2A-PTD samples. Summary: While two groups have reported cooperation of IDH1/2 mutations with retroviral overexpression of KMT2A targets (HOXA9/Meis1), we find that there is no cooperation between KMT2A-rearrangements themselves and mIDH1/2. This may in part be due to H3K79 hypermethylation, which is not tolerated in KMT2A-r disease, while HOXA9/Meis1 retroviral models are insensitive to H3K79 modulation. Interestingly, H3K79me2 is globally low in KMT2A-F patient samples. Our findings suggest that KMT2A-F leukemias maintain tight control over H3K79me2, and neither increases nor decreases are tolerated. This may have implications for the clinical/biomarker development of DOT1L directed therapies. Disclosures Bernt: GSK: Other: husband works at GSK.