ALBERT

Description: MYC overexpression is a poor prognostic predictor in Diffuse Large B-Cell Lymphoma (DLBCL). MYC-targeting with bromodomain and extraterminal protein family (BET) inhibitors is a promising strategy for the treatment of MYC-driven cancers, including lymphomas. However, preclinical and emerging data from early clinical trials demonstrated a modest antiproliferative activity in vitro and in vivo. We hypothesized that BET inhibition may induce feedback survival mechanisms preventing or attenuating cell death that could be exploited for designing future, more effective, combination strategies. In a high-throughput combinatorial drug screening experiment, we found that phosphatidylinositol 3-kinase (PI3K) pathway inhibitors enhanced the antiproliferative effects of BET inhibitors (JQ1, I-BET 151, CPI-203) with a strong class effect. JQ1 upregulated the mRNA expression of several upstream components of the PI3K pathway, including PIK3CA, PIK3R1, PDK1 in a large panel of DLBCL and Burkitt lymphoma cell lines. These effects translated in increased pathway activation as demonstrated by increased levels of the phosphorylated forms of downstream targets GSK3α/β, TSC2, P70S6K, and by increased concentrations of chemokines known to be regulated by PI3K in cell culture supernatants (CCL3 and CCL4). This effect was reversed by submicromolar doses of the PI3K inhibitor BKM-120. MYC silencing recapitulated the effects of BET inhibitors on PI3K pathway gene expression, activation and chemokine secretion. These data indicate that BET inhibition induces PI3K activation by a MYC-dependent feedback. We also observed transcriptional upregulation of the antiapoptotic gene Myeloid Leukemia 1 (MCL-1) following BET inhibition or MYC depletion, suggesting a second MYC-dependent mechanism. RNAi-mediated MCL-1 silencing or co-treatment with a small molecule MCL-1 inhibitor (UMI-77) enhanced the effects of BET inhibitors in DLBCL cell lines by inducing apoptosis. Using SILAC-based quantitative mass spectrometry, we found that BET inhibitors at submicromolar doses downregulated several E2 ubiquitin conjugating enzymes including UBE2C. RNAi mediated UBE2C knockdown induced MCL-1 upregulation in DLBCL cells. The enhanced in vitro effect of combining BETi and PI3Ki was reproduced in TMD8 mouse xenografts. To our knowledge, this is the first study demonstrating MYC-dependent regulation of the PI3K pathway, MCL-1 and the ubiquitin system upon BET inhibition. Our study revealed previously unknown mechanisms of action of BET inhibitors uncovering novel MYC-dependent survival feedback loops, and providing a framework for future combination strategies. Disclosures Zelenetz: Gilead Sciences: Research Funding.

Description: Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, amongst which IDH mutations occur in 15% of AML patients. Here, we show both in vitro as well as in a xenografted mouse model, that clinically achievable doses of ATRA are sufficient to achieve a terminal granulocytic differentiation in primary AML samples and in AML cell lines harboring IDH1-R132H mutation. There is no effect at this concentration on the WT controls. This is associated with reduction of both proliferation and colony formation, and further leads to apoptosis, thereby improving overall survival of mutant xenografted mice. We further showed, through transcriptomic and western blot analysis, that specific ATRA sensitivity is due to overexpression and activation of C/EBPα in the presence of IDH1-R132H mutation. This primes blasts into myeloid differentiation. Moreover, IDH1 R132H mutation also reduces LYN activation, and thus, also sensitizes to clinically achievable doses of dasatinib, a LYN inhibitor. As ATRA induces a brief LYN activation, which transiently reduces ATRA activity, its combination with dasatinib synergistically increases differentiation. In vivo, the combination of ATRA and dasatinib reduces tumor growth of mutant xenografted mice. The combination ATRA and dasatinib might also be considered for other IDH mutations that produce 2-hydroxyglutarate, since treatment with the mutant-specific oncometabolite (eg. 2-hydroxyglutarate) sensitizes AML cells to ATRA and dasatinib-induced differentiation. Finally, ATRA also reduces BCL2 expression specifically in the presence of IDH1 R132H mutation. Since it has been shown that IDH mutations increase BCL2 dependence in leukemic cells, our results identified a subgroup of patients that is likely to respond to pharmacologic concentrations of ATRA. To conclude, our data provide the preclinical rationale for investigating the use of the combination ATRA and dasatinib in a subgroup of patients who carry IDH1 R132H mutation, in clinical trials. The addition of a BCL2 inhibitor such as ABT-199 would also be considered. Disclosures Off Label Use: ATRA and dasatinib for treatment of non APL AML. Recher:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Chugai: Research Funding.