Exploring BCL2 regulation and upstream signaling transduction in venetoclax resistance in multiple myeloma: potential avenues for therapeutic intervention

Abstract

Investigating venetoclax (VTX) resistance in multiple myeloma (MM) is crucial for the development of novel therapeutic strategies to tackle resistance. We conducted a multi-omic characterization of established VTX-resistant isogenic human myeloma cell lines (HMCL) and primary MM patient samples pre- and post-VTX treatment. Transcriptomic and proteomic analysis revealed that resistance was largely associated with BCL-2 family protein dysregulation, including upregulation of anti-apoptotic proteins such as MCL-1, BCL-XL, BCL-2, and downregulation of pro-apoptotic members. Notably, the re-introduction of BIM into resistant cells restored VTX sensitivity and synergized with MCL-1 inhibitors. Upstream signaling pathways, including growth factor receptor tyrosine kinase (RTK) and phosphoinositide-3-kinase (PI3K) were implicated in this dysregulation. Simultaneous inhibition of MCL-1, BCL-XL, and upstream PI3K, RTK (FGF, EGF, and IGF1) mediated signaling enhanced VTX sensitivity. Post-translational modifications of MCL-1, particularly its stabilization via acetylation and phosphorylation, were investigated, although their inhibition only marginally increased VTX sensitivity. Lastly, the inhibition of AURKA and mitochondrial respiration also improved VTX sensitivity in some resistant HMCLs. Our findings suggest that combining VTX with MCL-1 and BCL-XL inhibitors or PIK3/RTK inhibitors holds potential for overcoming resistance. The study illustrates the importance of understanding molecular determinants of resistance to develop tailored therapeutic strategies.

Fig. 1. Assessment of venetoclax resistance and associated transcriptional and protein changes.

A MTT assay results after 72-hour incubation, with viability normalized to untreated control. The viability of parental and venetoclax-resistant MM cell lines is displayed in the top and middle panels. Persistence of resistance after 1 month of venetoclax withdrawal is demonstrated in the bottom panel. B Table summarizing transcriptional changes (from mRNA-seq data) between isogenic and resistance-acquired cell lines, and three patient samples (C, G, and T) pre- and post-VTX exposure; darker colors indicate greater statistical significance. C Western Blot analysis of protein expression in parental and venetoclax-resistant cell lines.

Fig. 2. Transcriptomic analysis of VTX-sensitive and resistant samples.

A, B Visualization of KEGG-based enrichment analysis of differentially expressed genes between two (OCIMy7 and SKMM2) venetoclax-resistant and sensitive cell lines. C Comprehensive pathways analysis contrasting all collected venetoclax-resistant cell lines (primary patient samples and human cell lines) with sensitive samples, highlighting differentially regulated pathways; orange color shows pathway activation in resistant samples, and blue shows pathway suppression.

Fig. 3. Integrated proteomic analysis of apoptosis regulation.

A Network view (STRING-DB) of core apoptosis-related genes along with their first-degree neighbors. B Proteomic analysis of apoptosis genes in venetoclax-sensitive and resistant paired cell lines; protein intensity is scaled by row. C Non-linear regression analysis for correlation between transcriptomics and proteomics for two anti-apoptotic proteins. D Overview of transcriptomic and proteomic quantification of BCL-2 family proteins in parental and venetoclax-resistant KMS12PE cell lines.

Fig. 4. Role of BIM expression and MCL-1 inhibition in VTX sensitivity.

A Western Blot analysis demonstrating loss of BIM expression in OCIMy5 exposed to long-term venetoclax (top panel) and re-introduction of BIM (bottom panel) for sensitivity studies. B MTT assay results after 48-hour incubation of venetoclax or S53845, normalized to untreated control; re-introduction of BIM lead to increased sensitivity to both compounds. C Immunoprecipitation assay showing decreased BIM binding to both BCL-2 and MCL-1 in KMS12Res in comparison to its sensitive counterpart. D–F MTT assays normalized to untreated control, showing activity of venetoclax plus S63845 (D), venetoclax plus A1155463 (E) and venetoclax, S63845 and A1155463 in venetoclax-resistant cell lines.

Fig. 5. Insights into MCL-1 dynamics and impact on VTX sensitivity.

A MCL-1 half-life quantification through Western Blot analysis following cycloheximide treatment; total protein normalization factor is labeled below each image. B Western Blots of BCL-2 and MCL-1 across various HMCLs with a representative bar graph of TPN background corrected volume. C Graphs showing MCL-1 half-life calculated using a one-phase decay analysis based on total protein normalized (TPN) local background corrected volume from Western Blot analysis. D Western Blot of MCL-1 quantification in various HMCLs after treatment with GNE-781 followed by cycloheximide treatment for pre-established timepoints. E MCL-1 quantification at different timepoints following treatment with either DMSO or okadaic acid with subsequent cycloheximide administration. F CellTiter-Glo-derived dose-response matrix of OCIMy5Res treated with venetoclax (left) and okadaic acid (bottom) for 24 h.

Fig. 6. Synergistic effects of tyrosine kinase inhibitors on VTX sensitivity.

A MTT assays of four venetoclax-resistant cell lines treated with venetoclax in combination with afuresertib for 72 h.; viability is normalized to untreated control. B Western Blot analysis of paired KMS12PE and KMS12Res cell lines, and with apoptotic protein quantification in KMS12PERes following treatment with venetoclax and/or afuresertib. C MTT assay of OCIMy5Res treated with venetoclax and/or osimertinib and OCIMy7Res treated with venetoclax and/or linsitinib for 72 h.; viability is normalized to untreated control. D Western Blot analysis of OCIMy5Res and OCIMy7Res proteins following treatment with osimertinib (48 h.) and linsitinib (24 h), respectively. E, F MTT assays of cell lines treated with venetoclax in combination with MK-5108 (E) or IACS-010759 (F); viability is normalized to untreated control.