Abstract 4713: BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma

Abstract

Multiple myeloma (MM) is an incurable malignancy with an unmet need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in most cell types. We sought to investigate dBET, a class of BBIs that induce degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination and proteasomal degradation, in drug-resistant MM. Additionally, we posited that resistance to dBET treatment would emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of optimized lead compound, dBET6, with JQ1 on a panel of MM cell lines, including clones resistant to JQ1 or bortezomib and assessed viability using CS-BLI/CTG assay and BRD/c-MYC expression by western blot. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (∼120,000 sgRNAs; targeting ∼19,000 genes and ∼1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (≥IC80), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor Bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid (<4hrs) degradation of BRD2, BRD3 and BRD4 and complete absence of c-MYC protein, in contrast to JQ1 which caused dose-dependent down-regulation of c-MYC, and apparent upregulation of BRD4. As predicted, our CRISPR/Cas9 screen identified significant enrichment of sgRNAs targeting CRBN, as well as the Cullin-RING ligase (CRL) complex, known to play a critical role in E3 ubiquitin ligase activity. In summary, our data support the development of dBET for the treatment of drug-resistant MM. Additionally, our results demonstrate that loss of function of CRBN or the CRL complex induces dBET resistance by perturbing dBET-mediated BRD4 degradation. However, it is plausible that additional CRBN/CRL-independent mechanisms of dBET resistance exist that allow cells to survive despite complete degradation of BRDs and this will be a key question to be answered in future studies. Citation Format: Geoffrey M. Matthews, Yiguo Hu, Michal Sheffer, Paul J. Hengeveld, Dennis L. Buckley, Megan A. Bariteau, Haley Poarch, James E. Bradner, Constantine S. Mitsiades. BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4713.

Proteolysis targeting chimeric molecules as therapy for multiple myeloma: efficacy, biomarker and drug combinations

“…7,9,10,16 Previous studies have shown a fusion of JQ1 and thalidomide (dBET6) has significant potency against MM. 17 We also showed that ARV 825 leads to significant growth inhibition of myeloma cells in liquid culture, clonogenic assay and, most importantly, in a xenograft model. Flow cyto-metric analysis showed that ARV 825 induced apoptosis and G0/G1 cell cycle arrest of these cells in vitro .…”

“…18 Loss of function of CRBN causes resistance to dBET6 by perturbing dBET-mediated BRD 4 degradation. 17 We postulate that levels of CRBN will serve as a predictive biomarker for cellular responsiveness to ARV 825. Indeed, two pairs of isogenic cells, one of each pair resistant to lenalidomide (KMS11 res and MM1S res), had very low expression of CRBN.…”