RATIONALLY DESIGNED CHIMERIC PI3K-BET BROMODOMAIN INHIBITORS ELICIT CURATIVE RESPONSES IN MYC-DRIVEN LYMPHOMA.

Abstract

Background: Targeted inhibitors of bromodomain and extraterminal (BET)-bromodomains and phosphatidylinositol-3-kinase (PI3K) signaling demonstrate potent but self-limited anti-lymphoma activity as single-agents in the context of cMYC-dysregulation. However, combined PI3K and BET inhibition imparts synergistic activity with the potential for more sustained disease responses due to mutual antagonism of compensatory epigenetic and signaling networks. Small molecule screens have identified kinase inhibitors with serendipitous off-target bromodomain binding, including dual PI3K/BET-inhibitors. Such molecules typically possess skewed potency profiles and limited opportunities for independent structure-activity based optimisation against each target. Aim(s): We hypothesised that dual PI3K/BET bromodomain inhibitors built by linkage of established PI3K and BET inhibitory pharmacophores would synergise in the context of MYC-dysregulation and overcome resistance to single-target inhibition. We investigated the mechanistic and therapeutic efficacy of rationally designed dual PI3K/BET inhibitors. Method(s): Using our lead molecule 18DS , antiproliferative and apoptotic effects against the PTEN +/-and IGH-cMYC rearranged OPM2 human multiple myeloma (MM) cell line were investigated. Western blot, KINOMEscanTM and BROMOscanTM binding assays were utilized to explore target engagement specificity. To further investigate transcriptional target engagement, RNA-seq, chromatin immunoprecipitation sequencing (ChIP-seq) and single cell RNA-seq (scRNA-seq) were performed. 18DS was evaluated against AML model with acquired BET inhibitor resistance. In vivo efficacy was explored using mice transplanted with Em-Myc lymphoma and Vk*MYC MM. Result(s): We first demonstrated synergistic apoptosis induction by combining single-targeted PI3K and BET inhibitors in OMP2 cells. The chimeric dual inhibitor, 18DS , demonstrated potent growth inhibitory (GI50 <20nM) and apoptotic (AC50 ~112nM) activity vs OPM2 cells. 18DS was superior to comparator single-targeted tool compounds alone and in optimised combination. Potent apoptotic activity was also evident across an extended panel of myeloma and lymphoma cell lines and primary MM samples treated ex vivo. Cellular effects correlated with on-target suppression of pAKT (PI3K inhibition) and MYC (BET inhibition) proteins. KINOMEscanTM assay demonstrated high selectivity for class I PI3K. BROMOscanTM assay confirmed high selectivity for BETbromodomains. RNA-Seq demonstrated robust transcriptional modulation with concomitant BET and PI3K inhibitory signatures by 18DS . ChIP-Seq demonstrated genome wide BRD4 displacement from cis-regulatory elements by 18DS . As PI3K and BRD4 proteins reside in different cellular compartments, scRNA-seq was performed to show that 18DS induces both PI3K-and BET-inhibitory signatures in the same cell. Moreover, 18DS readily induced apoptosis in a mouse MLL-AF9 AML model with acquired I-BET151 resistance. To extend our observations in vivo, mice bearing transplanted Vk*MYC myeloma and Emu-Myc lymphoma were treated with 18DS . Single doses of 18DS in tumor burdened mice resulted in rapid reduction of lymphoma burden. 18DS was well-tolerated. 90% of 18DS-treated Vk*MYC mice achieved a biochemical response (of PR or better; 30% CRs). 15 days of 18DS dosing resulted in curative responses in 90% of Emu-Myc mice with complete lymphoma clearance at sustained at 100 days post lymphoma transplantation. Summary/Conclusion These findings support the therapeutic strategy of combined PI3K and BET inhibition and provide a potential stepchange in approach to orthogonal MYC antagonism using optimised chimeric small molecule technology.