Epigenetic regulation of protein translation in KMT2A-rearranged AML

Inhibition of the histone methyl-transferase DOT1L(KMT4) has shown encouraging activity in preclinical models of KMT2A(MLL)-rearranged leukemia. The DOT1Linhibitor pinometostat (EPZ5676) was well tolerated in early phase clinical trials and showed modest clinical activity, including occasional complete responses (CRs) as single agent. These studies support the development of combinatorial therapies for KMT2A-rearranged leukemias. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1Land EZH2, and the combination of a DOT1Linhibitor with the protein synthesis inhibitor homoharringtonine(HHR). EZH2is the catalytic histone methyltransferasein the polycomb repressive complex 2 ( PRC2), and inhibition of EZH2has reported preclinical activity in KMT2A-rearranged leukemia. We found that the H3K79 and H3K27 methyl marks are not dependent on each other, and that DOT1Land EZH2inhibition affect largely distinct gene expression programs. In particular, the KMT2A/ DOT1Ltarget HOXA9, which is commonly de-repressed as a consequence of PRC2loss or inhibition in other contexts, was not re-activated upon dual DOT1L/ EZH2knockout or inhibition. Despite encouraging data in murine KMT2A-MLLT3 transformed cells suggesting synergy between DOT1Land EZH2inhibition, we found both synergistic and antagonistic effects on a panel of human KMT2Arearranged cell lines. Combinatorial inhibition of DOT1Land EZH2is thus not a promising strategy. We identified opposing effects on ribosomal gene transcription and protein translationby DOT1Land EZH2as a mechanism that is partially responsible for observed antagonistic effects. The effects of DOT1Linhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translationinhibitor. EPZ5676 was synergistic with the protein translationinhibitor homoharringtonine(HHR), supporting further preclinical/clinical development of this combination.