COMBINING DOT1L INHIBITOR EPZ-5676 WITH SORAFENIB TO TREAT MLL-REARRANGED (MLL-R) PEDIATRIC ACUTE MYELOID LEUKEAMIA (AML)

Background: About 20% of the childhood AML present MLL gene translocations, that are associated with a very poor prognosis. That is why there is a strong interest in developing novel therapeutic strategies for these patients. As DOT1L is responsible for H3K79 methylation and is associated with MLL-r leukemogenesis, DOT1L inhibitors entered in clinical trials as promising treatments. MLL-r AML also showed an overexpression of FLT3 receptor tyrosine kinase. Therefore, FLT3 inhibitors, such as the multi-tyrosine kinase inhibitor Sorafenib, demonstrated encouraging efficacy in AML. Aims: 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, THP1) and non MLL-r (OCI-AML3, HL60 and U937 harboring CALM-AF10 translocation) 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 (AnnexinV FITC/PI staining). Protein expression and H3K79 methylation were quantified by Western blot. mRNA expression was studied by quantitative Real-Time PCR (Q-PCR). Results: Firstly, the specific effects of DOT1L inhibition were examined in AML cell lines treated with increasing concentrations of EPZ-5676, up to 18 days. Cell growth was significantly inhibited after at least 8 days of treatment in MOLM13 and NOMO1 cells, but time-dependent apoptosis occurred only in MOLM13, thus suggesting that DOT1L inhibition alone might not be able to induce cytotoxicity. Whereas no effects were observed in HL60, U937 (non MLL-r), or THP1 (MLL-r) cells, both cell growth impairment and apoptosis were detected in non MLL-r OCI-AML3 cells, so that the impact of DOT1L inhibition could not exclusively rely on MLL rearrangements. Repression of DOT1L occurred since the 4th day of treatment, as demonstrated by the complete 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 in cells treated with 1 uM EPZ-5676, up to 28 days. Gene expression of HOXA9, MEIS1, FLT3 and CDK6 protein (MLL target) decreased in all cell lines, whereas STAT5 and c-Myc mRNAs, along with STAT5 protein expression, were downregulated only in MLL-r cells. Furthermore, in MOLM13 and NOMO1 cells p-Erk was strongly reduced. Finally, p-Akt slightly decreased in nearly every case, whereas a strong induction in p-S6RP was observed only 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 nearly all cases. Importantly, EPZ-5676 pretreatment increased Sorafenib-induced cell growth inhibition in EPZ-5676 refractory cells HL60, U937 and THP1. Moreover, in primary AML samples both EPZ-5676 and Sorafenib showed a limited effect as single agent, whereas their combination induced a drastic increase of apoptosis. Summary/Conclusions: These results demonstrated that the single administration of 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.