Synergistic antitumor effects and mechanisms of KPT-330 combined with venetoclax in Acute Myeloid Leukemia Free

Introduction The venetoclax-azacitidine (VA) regimen is a standard frontline therapy for elderly or chemotherapy-ineligible patients with acute myeloid leukemia (AML). However, venetoclax resistance, short remission durations, and high relapse rates limit its efficacy. KPT-330 (selinexor), a selective XPO1 inhibitor, is not approved for AML frontline treatment or in combination with venetoclax, a BCL-2 inhibitor. This study evaluates the synergistic anti-tumor effects of KPT-330 combined with venetoclax in preclinical AML models to elucidate molecular mechanisms and address venetoclax resistance.

Methods AML cell lines (MOLM-13, THP-1, OCI-AML3) were treated with venetoclax, KPT-330, or their combination at 10–800 nM for 24-72 hours, with three biological replicates, reflecting clinically achievable concentrations. Cell viability and drug synergy were assessed using the Cell Counting Kit-8 (CCK-8) assay, with combination index (CI) values calculated via the Chou-Talalay method. Apoptosis was quantified by Annexin V/PI flow cytometry at 24, 48, and 72 hours. Apoptosis-related proteins (MCL1, NOXA, p21, Caspase-3, cleaved PARP, BAX) were analyzed by Western blot. CRISPR-Cas9-mediated knockdown or overexpression of MCL1 and NOXA was performed using lentiviral vectors to validate their roles in synergy. Confocal microscopy with MitoTracker Red co-staining assessed MCL1 and NOXA subcellular localization. RNA sequencing (RNA-seq) identified differentially expressed genes (DEGs) with a sequencing depth of 30 million reads per sample, in triplicate (fold change > 2, FDR < 0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID software. Key genes were validated by quantitative PCR (qPCR) and Western blot. In vivo efficacy was evaluated in MOLM-13 and patient-derived xenograft (PDX) models using luciferase-tagged AML cells, with tumor burden monitored by In Vivo Imaging System (IVIS) and survival analyzed by Kaplan-Meier curves and log-rank tests. Statistical significance was determined using two-tailed Student's t-tests or one-way ANOVA with post-hoc Tukey tests (p < 0.05).

Results CCK-8 assays showed that KPT-330 monotherapy reduced MOLM-13 cell viability by 40% ± 5% at 100 nM after 48 hours. Combination with venetoclax enhanced synergy (CI = 0.5–0.8, p < 0.01), reducing IC50 from 50 nM (venetoclax) and 100 nM (KPT-330) to 18 nM in MOLM-13 cells, and from 200 nM (venetoclax) and 250 nM (KPT-330) to 92 nM in THP-1 cells (2.7-fold reduction). Flow cytometry revealed 68% ± 5% Annexin V-positive MOLM-13 cells at 48 hours with combination therapy, compared to 35% ± 4% (venetoclax) and 42% ± 3% (KPT-330) (p < 0.01). Western blot and confocal microscopy confirmed that combination therapy reduced MCL1 expression by 60% ± 8%, increased NOXA and p21 by 2.3- and 1.8-fold, respectively, and enriched mitochondrial NOXA, indicating mitochondrial apoptosis pathway activation. CRISPR-mediated MCL1 knockdown enhanced venetoclax sensitivity by 3.2-fold, while NOXA knockdown or MCL1 overexpression reduced combination efficacy by 50% ± 7%. RNA-seq identified 1,512 DEGs, with GO/KEGG analyses showing enrichment in apoptosis (p = 1.2E-10), p53 signaling (p = 3.5E-8), and cell cycle (p = 2.1E-9). qPCR and Western blot validated MCL1 downregulation and NOXA/p21 upregulation across cell lines at 24 and 48 hours. In vivo, combination therapy reduced tumor burden by 75% ± 10% in MOLM-13 xenografts and 65% ± 8% in PDX models derived from primary AML patient samples, compared to vehicle control (p < 0.01). Median survival extended from 28 to 42 days in MOLM-13 xenografts and from 32 to 48 days in PDX models (p < 0.05).

Conclusion KPT-330 combined with venetoclax demonstrates potent synergistic anti-tumor effects in AML preclinical models, driven by MCL1 suppression, NOXA upregulation, and activation of p53 signaling and mitochondrial apoptosis pathways. CRISPR-based validation and confocal microscopy elucidated key molecular mechanisms. Validation in PDX models supports the translational potential of this off-label combination. These findings provide strong preclinical evidence for combining BCL-2 and XPO1 inhibitors to address venetoclax resistance in AML. Future studies should evaluate safety and optimal dosing in clinical trials.