Abstract Background Chronic lymphocytic leukemia (CLL), the most prevalent form of adult leukemia, remains a challenge in terms of optimal therapeutic strategies, particularly for patients with rapid progression or relapsed/refractory disease. While mutations in the splicing factor SF3B1 occur in 10–15% of CLL cases, the pathological role of the more common wild-type SF3B1 remains poorly understood. This is especially significant considering recent studies demonstrating the oncogenic role of wild-type SF3B1 in myeloid and T-cell leukemias. Aberrant RNA splicing has been implicated in cancer-related metabolic reprogramming, yet the mechanistic link between SF3B1-mediated splicing regulation and the metabolic pathways essential for CLL cell survival is underexplored. In this study, we evaluate the therapeutic potential of targeting wild-type SF3B1 using the selective splicing modulator H3B-8800, with a particular focus on its impact on glycolytic metabolism and its combination potential with BTK inhibitors. Methods CLL cell lines (MEC-1, JVM-3) and primary CLL cells from treatment-naïve patients were treated with the SF3B1 modulator H3B-8800 (0–10 μM) and covalent BTKi zanubrutinib or ibrutinib (0–100 μM) for 48 hours. Cell viability was assessed using the CCK-8 assay to determine IC50values. RNA-seq was performed to analyze differential splicing events and gene expression. Key metabolic genes and BCR signaling regulators were further validated by qRT-PCR and Western blot. Metabolic phenotypes were evaluated using glucose and lactate assay kits. The synergistic interaction between H3B-8800 and BTKi was assessed using the zero interaction potency (ZIP) model, and apoptosis was quantified via Annexin V/PI staining. Results Knockdown of wild-type SF3B1 via lentivirus transfection significantly inhibited the growth of MEC-1 and JVM-3 cells, suggesting an essential dependency on SF3B1 in CLL cells. Pharmacological inhibition of SF3B1 by H3B-8800 exhibited potent cytotoxicity across CLL cells, with IC50values of 31.1 nM for primary CLL cells, 32.55 nM for MEC-1, and 68.85 nM for JVM-3. BTKi zanubrutinib showed IC50values of 28.92 μM for primary CLL cells, 56.12 μM for MEC-1, and 36.34 μM for JVM-3; while ibrutinib exhibited IC50 values of 10.92 μM for primary CLL cells, and 25.83 μM and 29.46 μM for MEC-1 and JVM-3, respectively. RNA-seq revealed a SF3B1 splicing-dependent inverse expression pattern of PFKFB1 (a glycolysis suppressor) and PFKFB3 (a glycolysis activator) upon H3B-8800 treatment. These splicing shifts were verified at the protein level: PFKFB1 decreased by 78.6%, while PFKFB3 increased by 186%. Metabolic assays demonstrated that H3B-8800 treatment resulted in a concurrent reduction in glucose uptake (20.7% decrease at 50 nM; 32.7% decrease at 100 nM) and lactate secretion (43.5% decrease at 50 nM; 49.1% decrease at 100 nM). Transcriptomic analysis also revealed that H3B-8800 induced upregulation of key BCR signaling regulators, including CD79A, CD19, and JUN, suggesting that this modulation may increase susceptibility to additional BCR pathway inhibition by BTKi. Given BTKi's role in suppressing glycolysis, we hypothesized that co-targeting SF3B1 and BTK would disrupt leukemia cell metabolic homeostasis. Indeed, combination treatment with H3B-8800 and zanubrutinib yielded a ZIP synergy score of 11 in MEC-1 cells. In primary CLL cells, 20 nM H3B-8800 induced 5.47% apoptosis, while zanubrutinib (20 μM) and ibrutinib (20 μM) induced 16.17% and 46.88% apoptosis, respectively. Combined treatment with H3B-8800 and two BTK inhibitors increased apoptosis to 24.16% and 67.16% respectively, indicating significant synergy. These results suggest that dual targeting of SF3B1 and BTK yields potent anti-leukemia efficacy through mechanisms involving BCR signaling and oncogenic metabolism. Conclusions Our study identifies the wild-type SF3B1 as a critical metabolic regulator in CLL through PFKFB-mediated glycolytic control. The splicing modulator H3B-8800 exerts potent anti-leukemic effects by rewiring the PFKFB1/PFKFB3 balance, leading to metabolic disruption. We also provide a mechanistic link between splicing dysregulation and metabolic vulnerabilities in CLL, highlighting SF3B1 as a viable therapeutic target regardless of mutational status. Furthermore, our data support a dual-targeted strategy combining SF3B1 modulation and BTK inhibition as an effective therapeutic approach for CLL.
Yuan et al. (Mon,) studied this question.