DOT1L INHIBITOR EPZ-5676 SYNERGIZES WITH SORAFENIB IN PRECLINICAL MODELS OF PEDIATRIC ACUTE MYELOID LEUKEAMIA (AML)

Background: MLL gene rearrangements (MLL-r) account for about 20% of the childhood AML, including ~80% of infant acute leukemias. MLL-r AMLs have a particularly poor prognosis, and thus new therapeutic approaches are urgently needed. Because abnormal expression of MLL-r target genes is associated with high levels of H3K79 methylation, novel promising inhibitors of the methyltransferase DOT1L entered in clinical trials. MLL-r also showed a cooperative effect with activated FLT3. Indeed, FLT3 inhibitors, including Sorafenib, demonstrated encouraging efficacy in AML. Aim: To investigate the efficacy of a combination treatment using DOT1L inhibitor EPZ-5676 and Sorafenib to treat MLL-r AML. Methods. MLL-r (MOLM13, NOMO1, MV4-11, THP1) and non MLL-r (OCI-AML3, HL60, U937) AML cell lines, and MLL-r primary samples from pediatric AML patients were used. Flow cytometry analyses were performed to assess absolute cell counting and apoptosis. Protein expression and H3K79me2 were quantified by Western blot. mRNA expression was studied by quantitative Real- Time PCR. Results: Treatment of AML cells with increasing concentrations of EPZ-5676 up to 16 days demonstrated a significant cell growth inhibition in several MLL-r (MOLM13, NOMO1, MV4-11) as well as in non MLL-r OCI-AML3 cells, so that the impact of DOT1L inhibition could not exclusively rely on MLL-r. However, a significant apoptosis occurred only in MOLM13 and MV4-11, thus suggesting that DOT1L inhibition alone might not be able to induce cytotoxicity. Repression of DOT1L occurred since the 4th day of treatment, as demonstrated by the loss of H3K79me2. To further explore the consequence of this phenomenon, both MLL targets and key component of signaling pathways involved in AML survival (i.e. PI3K, FLT3 and MAPK) were investigated. Gene expression of HOXA9, MEIS1 and FLT3 decreased in all cell lines, whereas STAT5 and c-Myc mRNAs, along with STAT5 protein expression, were downregulated only in MLL-r cells. In MOLM13 and NOMO1 cells p-Erk was strongly reduced, whereas a strong induction in p-S6RP was observed in U937 cells after prolonged treatment, suggesting the possible involvement of PI3K pathway in drug resistance mechanisms. To increase the benefit of DOT1L inhibition, both AML cell lines and MLL-r primary samples were pretreated with increasing concentration of EPZ-5676 for 4/8 days, following 24/48h treatment with Sorafenib. This combination resulted in a synergistic effect in 4/7 cell lines and irrespective of MLL-r. Moreover, in primary AML samples both EPZ-5676 and Sorafenib showed a limited effect as single agent, whereas their combination increased apoptosis. Conclusion: These results demonstrated that EPZ-5676 has a limited antileukemic activity, which is not restricted to MLL-r AMLs. However, the combination of EPZ-5676 with Sorafenib revealed a synergistic effect in both MLL-r and non MLL-r AMLs, paving the way to innovative and more effective treatments for pediatric AML patients.