Preclinical Activity of Novel BET Protein Proteolysis Targeting Chimeras (BETP-PROTACs) and Their Combination with Venetoclax or CDK4/6 Inhibitor Against Human Mantle Cell Lymphoma (MCL) Cells

Among the genetic alterations described in Mantle Cell Lymphoma (MCL) are those involving cyclin D1, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2 genes. Activated B-cell receptor (BCR) signaling, and the ensuing downstream pro-growth and pro-survival NFkB activity, is also a notable feature of MCL. Collectively, the genetic alterations and the ensuing deregulated signaling and activity of transcription factors, including c-MYC and NFkB, creates the MCL-specific 'transcriptome' that promotes growth and survival of MCL cells. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), exhibits unprecedented single-agent activity in relapsed/refractory MCL, however approximately 40% of patients demonstrate primary resistance and secondary resistance is common, with a poor one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, sustain alternative or classical NFkB signaling despite ibrutinib treatment and confer resistance to ibrutinib in MCL. We hypothesized that the resistance to ibrutinib in MCL cells would be governed by a dysregulated transcriptome dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal preclinical activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against the following cellular models of ibrutinib resistance (MCL-IR): in vitro generated cultured MCL Mino-persister/resistant (Mino-P/R) cells (~4-fold resistant compared to parental Mino cells), endogenously resistant cultured Z138 and Maver-1 cells, as well as patient-derived xenograft (PDX) PDX1 and PDX2 cells that had exhibited in vivo resistance to ibrutinib. Protein expression evaluations by RPPA and/or Western analyses demonstrated that MCL-IR cells expressed relatively high levels of p-AKT over AKT, p-S6, p-4EBP1, MYC, BCL2, BCl-xL, XIAP and MCL1. Treatment with BETP-PROTAC dose-dependently (20 to 200 nM) induced significantly more growth inhibition and apoptosis than the BETP inhibitor BETi OTX015 against the cellular models of MCL-IR (p < 0.001), which was associated with marked depletion of BRD4, BRD2, MYC, BCL2, BCL-xL, cyclin D1, CDK4 and pRB (S807/811) levels, as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, BCl-xL, IL10, TNFa and BTK. Concomitantly, BETP-PROTAC increased expression of HEXIM1 and p21 levels in the MCL-IR cells. Treatment with the BCL2 inhibitor venetoclax or the CDK4/6 inhibitor abemaciclib dose-dependently induced apoptosis of MCL-IR cell types (p< 0.01). Notably, co-treatment with BETP-PROTAC ARV-771 and venetoclax or abemaciclib synergistically induced apoptosis of Mino-P/R, Maver-1 and PDX1 and PDX2 cells (combination indices < 1.0). Additionally, compared to treatment with vehicle control, treatment of luciferase-transduced PDX2 cells infused by the tail vein and engrafted in immune-depleted NSG mice with ARV-771 (30 mg/kg SQ daily) and venetoclax (15 mg/kg PO daily) for 3 weeks, significantly reduced the MCL burden (at 2 weeks of treatment) and improved survival of the mice. These findings demonstrate that BETP-PROTACs deplete BETPs and important MCL-relevant oncogene levels, including MYC, cyclin D1, CDK4, BCL2, Bcl-xL, as well as NFkB target gene expressions, leading to synergistic lethality in vitro in combination with venetoclax or abemaciclib, as well as exert in vivo activity in combination with venetoclax against PDX model of MCL-IR cells.