Integrative Analysis of Transcriptomic and Proteomic Data Identifies Patterns of Primary Resistance to Venetoclax-Azacitidine and Reveals Targetable Vulnerabilities in Acute Myeloid Leukemia (AML)

Introduction: Venetoclax (VEN) combined with azacitidine (AZA) is a standard treatment for AML patients unfit for intensive chemotherapy and is widely used in relapsed/refractory (R/R) disease. However, primary resistance occurs in 30% of previously untreated and 50% of R/R cases, highlighting the need for better understanding of the underlying resistance and alternative therapies. Here, we aimed to elucidate VEN-AZA resistance by integrating extensive molecular data with clinical outcomes from 111 AML patients.

Methods: Bone marrow or peripheral blood samples were obtained from 111 AML patients prior to VEN-AZA treatment, including 49 treatment-naïve and 62 previously treated sAML/R/R patients. Among them, 34 were primary refractory to VEN-AZA. To evaluate drug sensitivity and apoptotic dependencies, patient cells were first cultured with single BCL2 family inhibitors and SMAC mimetics (extrinsic apoptosis activators via inhibition of inhibitor of apoptosis proteins (IAPs)) for 48 hours, after which the viability of CD34+ or CD117+ blasts was measured with flow cytometry. BCL2 family protein expression (BCL2, BCLXL and MCL1) was quantified from blast cells using flow cytometry. For transcriptomic analysis, CD34+ or CD117+ cells were isolated using magnetic bead separation prior to RNA-sequencing.

Results: In the BCL2 family inhibitor drug sensitivity assay, navitoclax (BCL2/BCLXLi) was the most effective in killing AML blasts of VEN-AZA refractory patients (mean IC50 70 nM) compared with venetoclax (BCL-2i) (1000 nM), A-1331852 (BCLXLi) (1000 nM) and S-63845 (MCL1i) (> 1000 nM). Protein analysis revealed significantly higher BCLXL (p=0.001) and lower BCL2 levels (p=0.02) in blasts from refractory patients, aligning with their sensitivity to navitoclax. MCL1 protein expression and drug sensitivity did not differ between the groups.

The transciptomic analysis involving 20 refractory and 54 responding AML patients showed significant upregulation of BCL2L1 (BCLXL) and downregulation of BCL2 and MCL1 in resistant patients, partially in line with the protein-level findings. No other BCL2 family members were found to be differentially expressed. In total, the analysis identified 524 differentially expressed genes, including upregulation of PRAME (cancer-testis antigen), CD276 (immune checkpoint molecule), MECOM (transcription factor) and TNF (cytokine) in resistant disease.

Considering the important role of TNF in AML pathogenesis and pro-survival signaling, we conducted hierarchical clustering based on TNF, BCL2 and BCL2L1 expression. The analysis identified three distinct patient clusters (C1-C3) with significantly different survival profiles. Cluster C1, which included most refractory patients (17/20), had the highest mean expression of TNF and BCL2L1, combined with a loss of BCL2 expression. This cluster also showed increased megakaryocytic-erythroid progenitor-like (MEP) transcriptional activity, enrichment for TP53 mutations (5/9), and poor overall survival, with a median OS of 4.6 months. In contrast, patients in C2 exhibited higher BCL2 and lower BCL2L1 compared to C1, with intermediate TNF expression. This was associated with a significantly longer median OS of 11.1 months. Similar to C2, patients in C3 expressed both BCL2 and BCL2L1 but had no expression of TNF, resulting in the longest median OS, 28.7 months.

To identify potential therapeutic approaches for patients in C1, we performed a comparative drug sensitivity analysis between the clusters. Patients with high overall TNF expression (C1) were selectively responsive to the IAP inhibitors birinapant and LCL161 ex vivo, suggesting that inhibition of IAPs could be an effective approach for VEN-AZA resistant AML with increased TNF. Navitoclax remained effective across all clusters, and compared to venetoclax, triggered more cell death in C1.

Conclusion: This study identifies molecular signatures associated with VEN-AZA resistance in AML. Primary resistance is potentially driven by elevated BCLXL protein expression and downregulated BCL2. Additionally, a MEP-like gene signature, combined with eleveted TNF expression in AML blasts, may contribute to venetoclax resistance while concurrently enhancing sensitivity to SMAC mimetics. These findings suggest potential therapeutic targets and stratification markers, paving the way for novel therapy approaches for VEN-AZA refractory AML.