Superior Pre-Clinical Efficacy of Novel Protac Based BET Degrader in a Large Acute Myeloid Leukemia Cohort

Bromodomain and Extra Terminal (BET) proteins which include ubiquitously expressed BRD2, BRD3, BRD4, and testis-specific BRDT, are epigenetic "readers" and play a major role in epigenetic regulation of gene transcription. BET family proteins are crucial for acute myeloid leukemia (AML) maintenance and expression of BRD4 is a poor prognostic indicator in AML. BET inhibitor activity is associated with repression of oncogenic c-MYC and their efficacy in pre-clinical models of cancer has led to rapid development of BET inhibitors entering clinical trials. Depletion of key oncogenic proteins in tumor cells could achieve much better clinical efficacy than inhibition of the same proteins. Therefore, we have developed novel and highly potent BET degrader, QCA570, based on the proteolysis targeting chimera (PROTAC) concept. This "BET-degrader" is based upon a new class of BET inhibitor, HYD276. QCA570 effectively degrades all BET-BRD proteins at concentrations as low as 30 pM within a few hours of treatment in the RS4;11 leukemia cell line and achieves IC50 values of 50 pM in inhibition of RS4;11 cell growth. We tested QCA570 (BET-degrader) in a panel of 102 primary AML samples in ex-vivo cultures. Leukemia stem cells (LSC) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors. Cell death and apoptosis induction was measured by flow-cytometry for DAPI-Annexin V binding after incubation with BET-inhibitor and BET-degrader for 3 days in the presence of drug. The AML cells were obtained from newly diagnosed AML (N=87) or relapse/refractory AML (N=15). The median IC50s was 120 pM (range 32 pm to 23 nM). Only 13 of 102 AML samples had an IC50 >1 nM with the BET degrader. In contrast median IC50 for the inhibitor HYD276 was 30,000 fold higher at 3.5 µM (range 8 nM to 151 µM). An IC50 of greater than 10 µM HYD276 was considered as resistant to BET-inhibitor. BET-inhibitor resistant CD34 enriched LSCs were sensitive to the degrader QCA570, irrespective of mutation status and chromosomal aberrations, suggesting that there are additional mechanisms of action of the degrader as compared to the inhibitor. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBPalpha (2%), DNMT3A (2%) and other mutations (10%). Based on classical karyotype 30% of them had a normal karyotype and 55% had abnormal karyotype (15% data missing). The degradation of BRD4 and repression of c-MYC was confirmed by immunoblotting after a 4 hr exposure of LSCs to degrader. Moreover, treatment of normal CD34 positive hematopoietic cells in apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. In conclusion, QCA570 represents a highly potent and efficacious panBET-BRD degrader undergoing extensive pre-clinical evaluation for the treatment of acute leukemias.