Abstract
Background: In patients with myeloproliferative neoplasms (MPNs), an increased blast percentage or progression to secondary acute myeloid leukemia (sAML) frequently engenders resistance to JAK1/2 inhibitor (JAKi) therapy and portends a dismal prognosis. Neither JAKi monotherapy nor its combinations with standard AML regimens or with hypomethylating agents plus venetoclax have significantly improved outcomes for patients with MPN-EB, accelerated-phase (AP), or sAML. Consequently, the development of novel therapeutic approaches or rational drug-combination strategies has become imperative.
Methods:We conducted in vitro experiments using the SET-2 and HEL cell lines, which harbor JAK and concurrent TP53 mutations and represent MPN in blast-phase transformation. Cell viability was assessed using the CellTiter-Lumi™ luminescent assay, while apoptosis and cell-cycle progression were analyzed by flow cytometry. Clonogenic capacity was evaluated in soft-agar colony-formation assays. To dissect the mechanisms underlying the ruxolitinib–homoharringtonine combination, total RNA was extracted from four treatment arms: DMSO vehicle, ruxolitinib monotherapy, homoharringtonine monotherapy, and the ruxolitinib plus homoharringtonine combination. After reverse transcription and library preparation, transcriptomic sequencing was performed to identify differentially expressed genes. Selected pathway genes were quantified by RT-qPCR, and their protein levels were validated by Western blotting. We employed CRISPR/Cas9 screening to identify genes associated with ruxolitinib resistance. Furthermore, we validated these findings in patient samples from MPN-to-AML transformation, especially those harboring TP53 mutations and exhibiting drug resistance.1.5×10⁶ SET-2-luc cells (1×10⁷/ml, 100 µl/mouse) were injected via the tail vein into NSG mice. One week later, tumor burden was assessed by intraperitoneal injection of luciferin (12.5 mg/ml, 100 µl/mouse) followed by bioluminescence imaging. Mice were randomized to receive vehicle, ruxolitinib, homoharringtonine, or the combination. Tumor growth was imaged weekly, body weight was measured every 3 days, and 20 µl of orbital blood was collected for CBC analysis on an XN-1000 hematology analyzer.
Results: In SET-2 and HEL cell lines—both modeling post-MPN sAML with co-occurring JAK and TP53 mutations—and in primary patient-derived post-MPN sAML cells, the ruxolitinib + homoharringtonine combination markedly suppressed proliferation and triggered cell death. CompuSyn synergy analyses yielded combination indices < 1 across all models, confirming synergistic interaction (Fig. A). Flow-cytometric profiling revealed that monotherapies arrested cells in G1, an effect that was significantly amplified by the combination (Fig. B). Soft-agar assays demonstrated a pronounced reduction in clonogenic output under dual treatment. RNA-seq disclosed that differentially expressed genes were overwhelmingly enriched for cell-cycle and DNA-replication pathways. Gene-set enrichment analysis (GSEA) showed pronounced depletion of signatures driven by TP53, c-Myc, E2F, mTORC1, PI3K-AKT, IL2-STAT5, IL6-JAK-STAT3, and inflammatory response programs (Fig. C). Additionally, sequencing data from the CRISPR/Cas9 screen showed marked reductions in the expression of resistance-related genes. QPCR analysis revealed that the combination of both drugs significantly downregulated cell-cycle-associated genes (CCND1, CDK2) and anti-apoptotic genes (BCL2, BCL-XL), while markedly upregulating the pro-apoptotic gene BAX. Western blots mirrored these changes, showing elevated p53, diminished BCL2, robust induction of cleaved-caspase-3, and reduced CDK2 and cyclin D1 protein levels (Fig. D). Finally, in the SET-2-luc xenograft model, combination therapy significantly attenuated sAML burden compared with vehicle or either single agent, and did so without overt host toxicity (Fig. E).
Conclusion: These findings demonstrate that the combination regimen of ruxolitinib and homoharringtonine exhibits significant preclinical activity against post-MPN sAML, particularly in patients harboring TP53 mutations, thus offering a promising strategy for future clinical application.
Acknowledgement: This research was funded by the Zhejiang Provincial Health High-level Innovative Talent Project (2022-2026).
*Correspondence to: Prof Jian Huang, E-mail:househuang@zju.edu.cn
