Protein Degrader WH25244 Eliminates Venetoclax Resistance Factors: Mutant or Hyperphosphorylated BCL2, and BCL-XL

The apoptosis blockade in chronic lymphocytic leukemia (CLL) can be overcome clinically through inhibition of the anti-apoptotic protein BCL2 with venetoclax. However, emergence of venetoclax resistance is driven by heterogenous mechanisms. Among these, BCL2 mutations decrease the binding affinity of venetoclax; BCL2 hyperphosphorylation increases the anti-apoptotic function of BCL2 by strengthening its interactions with pro-apoptotic molecules; and upregulation of alternative anti-apoptotic proteins like BCL-XL and MCL1 cannot be overcome by venetoclax. Novel strategies to target venetoclax resistance effectively and safely are warranted to improve the outcomes of patients with CLL.

We studied whether the protein degrader WH25244 is effective on mutant BCL2, hyperphosphorylated BCL2 and overexpressed BCL-XL in CLL cells, to overcome venetoclax resistance. WH25244 is a bifunctional molecule derived from navitoclax that recruits VHL E3 ligases to BCL2 and BCL-XL proteins, resulting in degradation via the ubiquitin-proteasome system. The requirement of VHL for its activity allows WH25244 to target BCL2 / BCL-XL dependent cancer cells while sparing platelets from toxicity (BCL-XL dependent but VHL low).

Previously, we showed that OSU-CLL cells expressing BCL2 mutant proteins (G101V, F104L, R107_110dup or A113G CRISPR knock-in) were resistant to venetoclax, yet sensitive to WH25244 (EC_50s ≤200 nM, 72h) due to degradation of BCL-XL and partial degradation of mutant BCL2 (2023 ASH abstract #4195). Here, we performed BH3 profiling to determine whether BCL2 mutant cells differ in their survival dependencies. G101V and F104L mutant OSU-CLL showed reduced sensitivity to the pan-apoptosis activator BIM, suggesting reduced apoptotic priming. F104L mutant cells additionally showed reduced sensitivity to BAD, suggesting reduced dependency on BCL2. Sensitivity to BCL-XL inhibition by XXA1_Y4eK peptide was not affected by the presence of BCL2 mutations.

We studied the cellular levels of serine 70 (S70) phosphorylated BCL2 (pBCL2) upon treatment with WH25244, its negative control (WH25244-NC) lacking an active VHL ligand, navitoclax, and venetoclax. As observed by western blot, WH25244 and venetoclax similarly reduced pBCL2 levels in OCI-Ly1 cells. Venetoclax was ineffective at dephosphorylating BCL2 in OCI-Ly1 cells induced for venetoclax resistance, yet WH25244 retained activity. To determine whether WH25244 degrades BCL2 based on S70 phosphorylation status, we stably overexpressed phosphorylatable WT BCL2, or a non-phosphorylatable mutant (S70A BCL2) in Jurkat cells. WH25244 reduced S70 pBCL2 in WT cells, and degraded BCL2 in the non-phosphorylatable mutant cells, suggesting broad targeting of BCL2. 10 nM WH25244 effectively killed both cell lines, with 50% Annexin V positive cells observed at 24 hours. In primary CLL cells, we validated that treatment with WH25244 causes loss of S70 pBCL2 and apoptosis, in a VHL-dependent manner.

We studied whether WH25244 can overcome venetoclax resistance driven by survival dependence on BCL-XL. As assessed by CellTiter-Glo at 24h, WH25244 was potent against four cell lines sensitive to the BCL-XL inhibitor A-1331852 (EC_50s <50 nM), but resistant to venetoclax (EC_50s> 5000 nM): MOLT-4 (EC₅₀ = 4 nM), PF-382 (EC₅₀ = 20 nM), SUP-T11 (EC₅₀ = 30 nM), and CCRF-CEM (EC₅₀ = 350 nM). Moreover, BCL2 was knocked out (KO) from the OSU-CLL cell line, which originally expressed both BCL2 and BCL-XL. BCL2 KO cells became resistant to venetoclax (plateau at 50% death) whereas WH25244 caused dose-dependent killing (EC₅₀ = 25 nM, 72h) of the BCL2 KO cells.

Last, we studied the effect of WH25244 against normal lymphocyte subsets from healthy donors. Apoptosis was detected at 18h of treatment by Annexin V / TMRM staining of CD19+ B cells, CD3+ T cells, and CD56+ NK cells via flow cytometry. Like venetoclax, WH25244 induced apoptosis of normal B cells in the nanomolar range. Toxicity to T cells remained low, and 100 nM WH25244 caused 1.7-fold more toxicity to NK cells than venetoclax.

In conclusion, our results indicate that WH25244 can degrade mutant BCL2, hyperphosphorylated BCL2, and BCL-XL, overcoming multiple mechanisms that drive resistance to venetoclax. These data justify continued preclinical and future clinical investigation of WH25244 in CLL.