Therapeutic Vulnerabilities of Venetoclax Resistant Chronic Lymphocytic Leukemia and Novel Treatment Strategies Targeting BCL2/BCL-XL

Despite major advancements in the management of chronic lymphocytic leukemia (CLL), drug resistance remains a major challenge. CLL resistant to venetoclax (VEN) have limited available therapeutic options, especially when the leukemic cells have dysfunctional p53 and have relapsed on Bruton’s tyrosine kinase inhibitors (BTKi). The broad hypothesis of this thesis is that VEN-relapsed CLL exhibits altered dependence on BCL2 family members, which can be exploited for therapeutic benefit. I profiled 28 CLL samples collected from 15 patients either at baseline or at relapse following VEN treatment, which was administered in combination with a BTKi or sequentially as monotherapy after a BTKi relapse. My data supports that BCL2 mutations are acquired only in a small fraction, whereas BTK and PLCG2 mutations are abundantly retained at VEN relapse. By single-cell RNA sequencing, VEN relapsed CLL distributed into 5 clusters. Broadly, CLL cells downregulated BCL2 and gained BCL-XL/BCL2L1 and BIM/BCL2L11 at relapse, which was a phenomenon affecting all clusters, including the most dominant. Moreover, a minor cluster was recurrently expanded in TP53-mutated relapsed CLL with low levels of BCL2 and high NF-KB signaling at relapse, along with its anti-apoptotic targets BCL2A1/BFL1 and MCL1. Other 2 minor clusters were gained at relapse by two independent TP53-mutated CLL samples, with high co-expression of BCL2, MCL1, and the pro-apoptotic binding partner of MCL1, PMAIP1/NOXA, suggesting an altered interactome at relapse. By BH3 profiling, VEN relapsed CLL did not completely lose dependence on BCL2 (BAD sensitivity), yet denoted co-dependence on alternative anti-apoptotic proteins, mainly BCL-XL (XXA1_Y4eK sensitivity). Moreover, VEN was unable to induce significant BAK nor BAX activation CLL progressing on VEN treatment. These observations led to the overall hypothesis of this thesis: co-targeting BCL2 and BCL-XL can effectively overcome VEN resistance in CLL. A major clinical barrier in developing BCL-XL inhibitors is the on-target toxicity to platelets. Therefore, the current thesis focuses on circumventing platelet toxicity with WH25244. This proteolysis targeting chimera (PROTAC) engages the VHL E3 ligase to degrade BCL2 and BCL-XL via the proteasome while sparing platelets that express low levels of VHL. As validated in leukemia cell lines and primary CLL, WH25244 degrades VEN-resistant forms of BCL2 (i.e. mutant and serine 70 hyperphosphorylated BCL2), and BCL-XL. WH25244 overcomes three factors that contribute to VEN resistance upstream of BAK and BAX activation, and effectively permeabilizes the mitochondria of primary CLL progressing on VEN treatment, irrespective of BCL2, BTK, PLCG2 or TP53 mutations. Moreover, the novel dual BCL2/BCL-XL small molecule inhibitor, LP-118, shows an advantage in targeting VEN resistance driven by stromal support, as observed in co-cultures of primary CLL with bone marrow-derived NK.Tert cells. This data expands our understanding of the therapeutic vulnerabilities of VEN/BTKi relapsed CLL and justifies continued investigation of WH25244 and LP-118 for frontline and relapsed CLL.