1055 - Single-cell transcriptomic analysis of CLL cells at ibrutinib plus venetoclax relapse and targeting using BCL-2/BCL-xL PROTACs PZ18753b and WH25244

Management of CLL in the clinic has been dramatically improved with the use of targeted therapeutics. Venetoclax treatment causes apoptosis of CLL cells via inhibition of the anti-apoptotic protein Bcl-2. Ibrutinib treatment inhibits the Bruton’s Tyrosine Kinase (BTK) and interrupts the B cell receptor signaling which is critical for CLL survival. While these therapies can induce deep responses when used as monotherapies or in combination, outcomes on subsequent treatments are suboptimal for patients who relapse. We aimed to evaluate drivers of relapse in CLL patients treated with venetoclax and ibrutinib, via single cell RNA sequencing (sc-RNA-seq), to propose novel strategies for therapeutic targeting.
Peripheral blood mononuclear cells were isolated from CLL samples (n=3) collected upon relapse to, both, venetoclax and ibrutinib treatment, which were administered in combination or sequentially. From sc-RNA-seq data, 12 clusters were identified in the integrated relapsed samples. 4 CLL clusters were distinguished and had a distinct transcriptome profile from naïve B cells, NK cells and T cells. The main enriched pathway within the relapsed CLL clusters was BCR signaling. Interleukin-mediated (IL-2, IL-3 and IL-5), ErbB and Fc epsilon receptor I signaling pathways were also enriched. Notably, 3 out of 4 CLL clusters had high expression of BCL-2, and most cells expressing MCL-1 or BCL-xL also co-expressed BCL-2.
We analyzed paired samples from a CLL patient (CLL-3) collected at baseline and at relapse, which occurred after cessation of fixed-duration treatment with venetoclax and ibrutinib. While the CLL clusters were shared at baseline and relapse, the proportion of cells in each cluster was vastly altered at relapse. Particularly, CLL cells from cluster 3 represented 4% of CLL cells at baseline versus 36% at relapse, suggesting their role in driving relapse. An analysis of the marker genes led to identify a high B-cell receptor (BCR) score in CLL cluster 3, including high expression of MAP3K8, FOSB8, JUND and NFKB1 genes. Moreover, the relapsed sample had an increased number of CLL cells expressing transcripts from the BCL-2 family of anti-apoptotic proteins, including BCL-2, BFL-1 (encoded by BCL2A1), MCL-1 and BCL-xL (encoded by BCL2L1).
The observation that BCL-2 and BCL-xL remain relevant in relapsed CLL led us to propose a targeted therapeutic strategy. Knowing that BCL-xL inhibition in the clinic has been limited by platelet toxicity, PROTACs PZ18753b and WH25244 were synthesized from navitoclax (BCL-2/BCL-xL dual inhibitor) linked to a VHL E3 ligase ligand to target Bcl-2 and Bcl-xL for degradation, with improved specificity to cancer cells while sparing platelets.
CLL cells from treatment naïve patients (n=5) or OSU-CLL cells expressing wildtype (WT) or mutant Bcl-2 (G101V, F104L, R107-110dup or A113G) were treated with venetoclax, navitoclax, PROTACs PZ18753b or WH25244, or negative controls (NC) lacking VHL recruitment PZ18753b-NC or WH25244-NC. Viability was evaluated by Cell Titer Glo assay, and apoptosis by flow cytometric detection of active BAX and BAK, mitochondrial cytochrome C, and AnnexinV binding. Protein degradation and inhibition was evaluated by Western Blot and NanoBRET assay, respectively.
PZ18753b and WH25244 exposure led to key events of the mitochondrial apoptosis pathway in treatment naïve primary CLL cells: BAK and BAX transformation, cytochrome C release and phosphatidyl serine exposure. The potency of these PROTACs was improved 1.6- and 6.5-fold for PZ8753b and WH25244, respectively, when compared to their precursor, navitoclax. Importantly, CLL cells were sensitive to these PROTACs at concentrations known to be non-toxic to human platelets. Sensitivity to venetoclax was similar to WH25244, with 50% death occurring between 0.5 to 4 nM. Apoptosis was associated with degradation of Bcl-xL in a VHL-dependent manner. Moreover, OSU-CLL cells incorporating BCL-2 mutations were up to 4.3-fold more sensitive to WH25244 than venetoclax. Cell death of OSU-CLL cells upon treatment with WH25244 was preceded by degradation of Bcl-xL and wildtype or mutant Bcl-2. We anticipate that venetoclax-relapsed primary CLL cells showing high Bcl-xL dependency by single cell sequencing will retain sensitivity to PZ18753b and WH25244.
PZ18753b and WH25244 have preclinical efficacy in baseline CLL and can degrade, both, Bcl-xL and mutant Bcl-2 proteins which are known to confer venetoclax resistance. This translational study supports the development of novel therapeutics for the treatment of CLL subgroups with adverse clinical prognosis and will open new frontiers as we better understand the biology of this disease.