Targeting Venetoclax-Resistant CLL By Bcl-XL Degradation
Authors: Diaz Rohena Daisy , Ravikrishnan Janani , Liu Chaomei , Zheng Guangrong , Zhou Daohong , Zhang Peiyi , Wierda William G. , Jain Nitin , Woyach Jennifer A. , Sampath Deepa 
Published: November 2021

Abstract
Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in adults and is associated with resistance to apoptosis. Clinical responses are dramatically improved with the use of targeted therapies such as venetoclax, a selective inhibitor of the anti-apoptotic protein Bcl-2, compared to chemoimmunotherapy. However, some patients can ultimately develop resistant CLL. Bcl-xL overexpression has been implicated in multiple mechanisms of venetoclax resistance, representing a switch that confers cell survival despite Bcl-2 inhibition. Mechanisms of venetoclax resistance include mutated Bcl-2 (ex. G101V), switches in survival dependence to alternative BCL2 family members, and microenvironmental interactions (Reviewed in Bose et al., 2017). Moreover, disease progression on venetoclax is accelerated in CLL patients with aberrant p53 , and our laboratory identified Bcl-xL as one of the main contributors to the oncogenic abilities of mutant p53.Thus, Bcl-xL is a promising target for re-sensitizing CLL to apoptosis, but since it is crucial for platelet viability, direct targeting has not been clinically successful.
New Bcl-xL PROTAC degraders provide an exciting opportunity to target Bcl-xL in venetoclax-resistant CLL. Due to differences in the expression of VHL E3 ligase in cancer cells versus platelets, these degraders have shown potency and selectivity to target this protein for degradation in multiple Bcl-xL-dependent malignancies (He, Y. et al., 2020). Our results show that exposing primary CLL samples (n=4) from treatment-naïve patients to the Bcl-xL degrader DT2216 is associated with apoptosis at concentrations known to be non-toxic to platelets (EC50 = 162 nM at 18 hr treatment). Furthermore, the dual Bcl-xL/Bcl-2 degrader PZ18753B was synthesized with enhanced potency against Bcl-xL and Bcl-2 co-dependent cancers. Our results confirm enhanced potency in primary cells from treatment naïve patients with CLL (EC50 = 7.602 nM at 18 hr treatment), compared to DT2216. Also, the degraders exhibit a similar kinetic profile at 10-fold concentrations (n=5, t1/2 = 2.3 hr for 1 uM DT2216, and t1/2 = 3.1 hr for 100 nM PZ18753B). For these results, apoptosis via the mitochondrial pathway was studied by flow cytometry, assessing extracellular exposure of phosphatidyl serine and mitochondrial depolarization by Annexin V and TMRM staining, respectively.
Consistently, as confirmed by Western Blot, PZ18753B is more potent at degrading Bcl-xL than DT2216, achieving 50% degradation in RS4;11 leukemic cells at 46 hr treatment at a concentration of 0.86 nM, compared to 7.83 nM for DT2216. Comparing dose-response curves showing apoptosis by AnnexinV/TMRM staining and protein degradation by Western Blot, we confirmed that Bcl-xL degradation precedes cell death.
Given that Bcl-xL overexpression confers a survival advantage to CLL cells, we hypothesize that venetoclax-resistant CLL cells can be targeted with the new Bcl-xL degraders. We studied the potency of the degraders against RS4;11 leukemic cells which express wildtype (WT) Bcl-2, or also overexpress G101V mutant Bcl-2. This is one of the mutations associated with venetoclax resistance and, combining data from various cohorts, it was identified in 23% venetoclax-resistant patients (Blombery et al., 2019 and 2020; Tausch et al., 2019; Weiss et al., 2019). PZ18753B induced apoptosis of RS4;11 Bcl-2 WT cells within 46 hrs (EC50 = 11.1 nM) as well as in RS4;11 cells expressing Bcl-2 G101V mutation (EC50 = 109.1 nM). PZ18753B degraded both Bcl-xL and Bcl-2 in RS4;11 WT cells effectively. In RS4;11 Bcl-2 G101V cells, it did not affect Bcl-2 but retained potent Bcl-xL degradation (86% vs 75% at 1 uM in WT vs mutant Bcl-2 cell lines, respectively). Interestingly, DT2216 did show ability to degrade G101V mutant Bcl-2 at the highest concentrations tested (Ex. 36% and 33%, respectively, at 1 uM).
We also demonstrated that CLL cells from chemo-naïve patients depend primarily on Bcl-2 for survival, as measured by their responses to BH3 mimetic peptides in a BH3 profiling assay. However, venetoclax-resistant CLL cells undergo a shift in dependence to alternative Bcl-2 family proteins, such as Bcl-xL and Mcl-1, as a mechanism for resistance to apoptosis. Thus, resistant CLL represents an excellent setting in which to continue testing the efficacy of these potent Bcl-xL degraders, to overcome resistance to Bcl-2 inhibitors.

Disclosures
Zheng: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhou: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhang: Dialectic Therapeutics: Other: Co-founder and stakeholder. Co-inventor of Bcl-xL PROTACs disclosed in this study.. Wierda: Oncternal Therapeutics, Inc.: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Xencor: Research Funding; KITE Pharma: Research Funding; Sunesis: Research Funding; GSK/Novartis: Research Funding; Janssen: Research Funding; Loxo Oncology, Inc.: Research Funding; Acerta Pharma Inc.: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; Karyopharm: Research Funding; Miragen: Research Funding; Cyclacel: Research Funding; AstraZeneca: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Jain: Incyte: Research Funding; Servier: Honoraria, Research Funding; Precision Biosciences: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Beigene: Honoraria; Cellectis: Honoraria, Research Funding; Pfizer: Research Funding; Fate Therapeutics: Research Funding; Aprea Therapeutics: Research Funding; Genentech: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Adaptive Biotechnologies: Honoraria, Research Funding; TG Therapeutics: Honoraria; Janssen: Honoraria; AbbVie: Honoraria, Research Funding; Pharmacyclics: Research Funding. Woyach: AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; Gilead Sciences Inc: Other: Data & Safety; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy.