Targeting Venetoclax Resistant CLL Using a Protac-Based BCL-2/BCL-XL Degrader

Abstract

Treatment for chronic lymphocytic leukemia (CLL) is primarily based upon the use of small molecules targeting Bruton's tyrosine kinase (BTK; ibrutinib, acalabrutinib) or BCL-2 (venetoclax). However, resistance mechanisms including mutations in BTK or BCL-2 and adaptive switches in survival dependence to alternative BCL-2 family proteins drive disease progression on these therapies with a poor subsequent outcome. We evaluated BCL-2/BCL-xL dual targeting PROTACs aimed at overcoming venetoclax resistance in CLL. We conducted single cell RNA-seq on CLL samples at baseline and after relapse on the combination of ibrutinib and venetoclax therapy and saw the emergence of novel clusters of cells upon relapse. Cells in the relapsed clusters retained high expression of BCL-2 and showed BCL-xL expression, suggesting that novel BCL-2/BCL-xL inhibitors could overcome venetoclax resistance. Further, BH3 profiling indicated that OSU-CLL cells with the BCL-2 mutation G101V release more cytochrome C than wildtype (WT) cells upon exposure to BAD and XXA1_Y4eK peptides, suggesting increased sensitivity to BCL-2 and BCL-xL inhibition. Earlier studies of dual BCL-2/BCL-xL inhibitors were hampered by platelet toxicity. We used a BCL-2/BCL-xL PROTAC, PZ18753b, and its more soluble analog WH2544, synthesized from navitoclax linked to a VHL ligand. The VHL E3 ligase is not expressed in platelets, allowing for a therapeutic window to selectively target cancer cells. Both PZ18753b and WH2544 exposure led to key events of the mitochondrial apoptosis pathway in primary CLL cells, including BAK and BAX transformation, cytochrome C release, mitochondrial depolarization (assessed by tetramethylrhodamine methyl ester, TMRM, staining) and phosphatidylserine exposure in the outer cell membrane (assessed by Annexin V staining). The efficacy of WH2544 and PZ18753b in treating naïve CLL cells fell in between that of venetoclax and navitoclax. At 18 h of treatment, 100 nM PZ18753b was associated with near complete cell death accompanied by a 50% BCL-xL degradation (assessed by Western Blot) and inhibition of BCL-2 via the formation of a [BCL-2:PZ18753b:VCB] ternary complex (assessed by NanoBRET assay). Additionally, blocking VHL E3 ligase activity decreased the potency of the degraders PZ18753b and WH2544, evidencing that VHL activity is involved in their mechanism of action. To determine whether the BCL-2/BCL-xL degraders can overcome the venetoclax resistance-promoting mutations in BCL-2, we generated OSU-CLL cells incorporating BCL-2 mutations via CRISPR knock in. BCL-2 mutant cells (G101V, F104 and R107-110dup) exhibited decreased sensitivity to venetoclax relative to WT cells as shown by Annexin V staining. In contrast, both WT and BCL-2 mutant cells were more sensitive to PZ18753b treatment at matched concentrations. Apoptosis was preceded with rapid and potent BCL-xL degradation, and partial BCL-2 degradation in OSU-CLL cells, despite the presence of BCL-2 mutations. In conclusion, our results suggest that venetoclax refractory CLL cells retain survival dependency on BCL-2 and BCL-xL proteins to evade apoptosis. This vulnerability can be therapeutically exploited using the BCL-2/BCL-xL dual targeting PROTACs PZ18753b and WH2544 in CLL.