Novel BET Protein Proteolysis Targeting Chimeras (BETP-PROTACs) Exert Potent Single Agent and Synergistic Activity with Ibrutinib and Venetoclax Against Human Mantle Cell Lymphoma Cells

Mantle Cell Lymphoma (MCL) exhibits pathogenetic mutations or deletion of RB1, ATM, p53, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2. 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 refractory/resistant disease with a one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, despite ibrutinib treatment, sustain classical or alternative NFkB signaling and transcriptional activity, as well as confer resistance to ibrutinib in MCL. We previously reported that the BET protein (BETP) bromodomain inhibitors (BETis), which disrupt the binding of BRD4 with acetylated chromatin, inhibit the in vitro growth and induce apoptosis of cultured and patient-derived (PD) primary MCL cells. This was associated with BETi-mediated attenuation of c-MYC, BCL2, CDK4/6 as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, Bcl-xL, IL10, TNFα and BTK. Concomitantly, BETi treatment induced HEXIM1, p21, p27 and NOXA levels in MCL cells. Co-treatment with BETi and ibrutinib was synergistically lethal and improved the median survival of the immune-depleted mice engrafted with human MCL cells. However, treatment with BETi leads to the accumulation of BRD4, which could promote the deregulated transcriptional activity of c-MYC, NFkB and other transcription factors. Here, we compared the anti-MCL activity of the novel BETP-PROTACs (proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) that degrade BRD4 with the BETi OTX015 against cultured and primary MCL cells. ARV-825 and ARV-771 recruit and utilize the E3 ubiquitin ligase activities of cereblon and VHL, respectively, to effectively degrade BET proteins including BRD4. At equimolar concentrations (10 to 500 nM) ARV-825 and ARV-771 were significantly more potent than the BETi OTX015 in inducing apoptosis of cultured and primary MCL cells (p < 0.01), while sparing the CD19+ normal B and CD34+ hematopoietic progenitor cells. Notably, whereas OTX015 treatment increased, BETP-PROTACs markedly attenuated (> 90%) the levels of BRD4 in the MCL cells. BETP-PROTAC treatment caused more profound up and down regulation of mRNA and protein expressions, utilizing the RNAseq and reversed phase protein array (RPPA) analyses, respectively. BETP-PROTAC treatment also caused greater and more sustained depletion of c-MYC, CDK4/6, PIM1, cyclin D1, as well as of the NFkB transcriptional targets Bcl-xL, XIAP, MCL1 and BTK, while concomitantly inducing the level of NOXA and p21. As compared to treatment with OTX015, ARV-771 treatment dramatically inhibited the growth and improved survival of NSG mice engrafted with luciferase-transduced, ibrutinib-resistant, Z138 MCL cells. Also, notably, co-treatment of ARV-825 or ARV-771 with ibrutinib or the BCL2-antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of cultured and primary MCL cells. We have also generated, ex vivo, ibrutinib-resistant (e.g., Mino/IR and JeKo1/IR) and ibrutinib-persister/resistant (Mino/IPR) cultured MCL cells, with >10-fold higher IC₅₀ value for ibrutinib than the parental MCL cells. BETP-PROTAC treatment more potently induced lethality than BETi in the Mino/IR and Mino/IPR cells, associated with attenuation of c-MYC, BCL2, CDK4/6 and NFkB target gene expressions including BTK. BETP-PROTAC and BETi also induced synergistic lethality with venetoclax and palbociclib against the Mino/IR, Mino/IPR and Z138 MCL cells. These findings underscore the superior in vitro and in vivo activity of BETP-PROTACs versus BETi against ibrutinib-sensitive and ibrutinib-refractory MCL cells, as well as highlight a promising new class of agents to be developed for the therapy of human MCL.