SF3B1 mutation accelerates the development of CLL via activation of the mTOR pathway

Authors

Bo Zhang

Prajish Iyer

Meiling Jin

Elisa Ten Hacken

Zachary J. Cartun

Kevyn L. Hart

Mike Fernandez

Kristen Stevenson

Laura Rassenti

Emanuela M. Ghia

Thomas J. Kipps

Donna Neuberg

Ruben Carrasco

Wing C. Chan

Joo Y. Song

Yu Hu

Catherine J. Wu

Lili Wang

Doi

10.1172/jci.insight.184280

PMID: 26200345 · DOI: 10.1172/jci.insight.184280 · Journal: JCI Insight (2025)

TL;DR

Zhang et al. built a B cell–specific double-mutant (DM) mouse model coexpressing the recurrent CLL splicing-factor mutation SF3B1-K700E with deletion of the chromosome 13q minimal-deleted region (Mdr, encompassing DLEU1/DLEU2/MIR15A). Coexpression accelerated chronic lymphocytic leukemia penetrance (24% vs 7% in Mdr-only mice over 16+ months) and produced a more transplantable, MYC- and Ki67-high disease in NSG recipients. Integrated RNA-seq and proteomics identified mTORC1 and MYC pathway activation in DM CLL cells, driven by Sf3b1-dependent alternative splicing of Nfatc1 toward the short isoform 5 (exon 9 skipping). Combined RNA-splicing inhibition (H3B-8800) and mTORC1 inhibition (temsirolimus) was synergistic in DM CLL cells in vitro and in vivo, and human CLL samples with both SF3B1 mutation and del(13q) were preferentially sensitive to the combination PMID:26200345.

Cohort & data

  • Murine model. B cell–specific Cd19-Cre mice with three genotypes: DM (Sf3b1^fl/+ Mdr^fl/+ or fl/fl, n = 25), Mdr-mutant only (n = 27), and WT controls (n = 30). Monitored every 3 months from 6–24 months of age for B220+CD5+ CLL-like cells in peripheral blood PMID:26200345.
  • Engraftment. Splenocytes (3 × 10^6) from DM or Mdr CLL mice transplanted intravenously into NOD/SCID-γ (NSG) recipients PMID:26200345.
  • Human reference cohort. A separate CLLmap survey of 1,009 CLL patients (https://cllmap.org/) used to confirm SF3B1 mutation frequency (184/1,009 = 18.2%) and co-occurrence with del(13q) PMID:26200345.
  • Human CLL validation. Patient samples from the CLL Research Consortium stratified by FISH/NGS into four groups (DM, del(13q) alone, SF3B1 mut alone, double-negative; n = 3 each) for drug sensitivity PMID:26200345.
  • Assays. Bulk RNA-seq (NovaSeq, 150 bp PE; STAR, DESeq2; rMATS + LeafCutter + StringTie splicing pipeline); TMT 10-plex proteomics (Orbitrap Fusion); isoform-specific qPCR; IHC for PAX5, CD5, MYC, Ki67; FISH + NGS for SF3B1/del(13q) status in human samples; flow cytometry. Murine RNA-seq deposited under GEO GSE300699 PMID:26200345.

Key findings

  • In the CLLmap cohort of 1,009 patients, 51% of SF3B1 mutations co-occur with del(13q); SF3B1-mut/del(13q) versus null patients have significantly shorter time to first therapy (P < 0.0001) and inferior overall survival (P = 0.0002 vs SF3B1-wt/no-del; P = 0.032 vs SF3B1-mut/no-del(13q)) PMID:26200345.
  • From 16 months onward, 6/25 (24%) DM mice and 2/27 (7%) Mdr-only mice developed circulating B220+CD5+ CLL-like cells (20–60% burden); no WT mouse did. Two DM CLL mice (but no Mdr-only) had enlarged mesenteric lymph nodes PMID:26200345.
  • DM CLL cells engrafted into NSG mice produced CLL-like disease in 3–4 weeks versus a longer latency for Mdr-only cells, with higher Ki67 and MYC staining by IHC PMID:26200345.
  • In young (12-week) DM mice without CLL, coexpression of Sf3b1-K700E and Mdr deletion reduced spleen weight and splenocyte/B-cell counts (P < 0.01) and expanded marginal zone B cells (P < 0.01), opposite of Mdr-only mice PMID:26200345.
  • Splenic-B-cell RNA-seq: 835 dysregulated genes (658 up) in DM vs other genotypes; GSEA enriched for OXPHOS, mRNA splicing, MYC targets, and mTOR pathway (upregulated) and TNF-α / inflammatory response (downregulated; FDR < 0.1) PMID:26200345.
  • DM CLL versus DM normal B cells: 1,059 dysregulated genes (457 up) including the CLL markers Cd5, Lef1, Zap70; mRNA–protein concordance r = 0.5265 across TMT proteomics PMID:26200345.
  • rMATS splicing analysis identified 1,029 DM-CLL splice variants and 376 Sf3b1-mutation splice variants, with 117 overlapping. Seven of these directly interact with MYC/mTORC1 in STRING: Nfatc1, Atf2, Hdac6, Pbrm1, Ptprc, Tbl1xr1, Hdac10. Nfatc1 showed the largest |ΔPSI| and most significant FDR PMID:26200345.
  • DM CLL cells preferentially express NFATC1 short isoform 5 (exon 9 skipped, exon 1a–driven, ~78 kDa) while Mdr-only CLL cells express predominantly isoform 2. Isoform 5 overexpression in Ba/F3-MYC cells confers IL-3 independence; in HG3 human CLL cells it activates mTORC1 (p-mTORC1, p-4E-BP1) whereas isoform 2 activates AKT/S6 instead PMID:26200345.
  • In vitro IC50: DM CLL cells more sensitive to H3B-8800 than Mdr-only (0.00346 μM vs 0.05331 μM); Mdr-only more sensitive to temsirolimus than DM (0.0001055 μM vs 2.148 μM). Combination Chou-Talalay index 3.56 × 10^-6 (DM) vs 1.96 × 10^-4 (Mdr-only) — both synergistic but more so in DM PMID:26200345.
  • In vivo NSG engraftment: combination H3B-8800 (4 mg/kg, oral, 5 days) + temsirolimus (15 mg/kg, i.p., 5 days) extended median OS in DM CLL mice from 9 to 27 days (3-fold; P < 0.01 log-rank), versus 61 → 63 days in Mdr-only CLL mice (1.05-fold) PMID:26200345.
  • Human CLL patient samples (n = 3 per group): DM (SF3B1-mut + del(13q)) cells were significantly more sensitive to temsirolimus + H3B-8800 than the other three groups (P < 0.001, 2-way ANOVA), recapitulating the murine phenotype PMID:26200345.

Genes & alterations

  • SF3B1 — recurrent K700E hotspot (>50% of all SF3B1 mutations). In CLLmap, mutated in 184/1,009 (18.2%) patients; mutation associated with shorter time to first therapy independent of del(13q) and inferior OS when co-occurring with del(13q). Sf3b1-K700E drives alternative splicing of Nfatc1 (and 375 other genes) and activates mTORC1/MYC pathways in B cells PMID:26200345.
  • NFATC1 — alternative splicing of exons 8/9 produces short isoform 5 (exon 9–skipped) preferentially in DM CLL cells. Isoform 5 activates mTORC1 (p-4E-BP1) and upregulates MYC; isoform 2 (full-length) instead activates AKT/S6. Validated in HG3, MEC1 isogenic SF3B1-K700E cell lines and Ba/F3-MYC system PMID:26200345.
  • MYC — upregulated at mRNA and protein level in DM CLL cells; pathway enrichment confirmed by GSEA in both murine and human CLL with SF3B1-mut/del(13q) PMID:26200345.
  • MTOR — mTORC1 pathway (p-mTOR, p-4E-BP1 T37/46, p-S6 S235/236) activated in DM CLL cells but not Mdr-only CLL cells; targetable with temsirolimus PMID:26200345.
  • DLEU1, DLEU2, MIR15A — comprise the minimal deleted region (Mdr) of 13q14 modeled as floxed deletion; Mdr loss alone gives low-penetrance CLL but cooperates with SF3B1 mutation for full acceleration PMID:26200345.
  • ATM — referenced as the previously published SF3B1-Atm CLL murine model from the same group; comparison context only PMID:26200345.
  • ZAP70, CD5 — CLL marker genes upregulated in DM CLL cells at both mRNA and protein levels PMID:26200345.
  • BRD9, PPP2R5A, AKT1 — discussed as comparison targets of SF3B1-driven mis-splicing in other cancers (uveal melanoma, breast cancer, PDAC); not directly altered here but provide cross-disease context PMID:26200345.

Clinical implications

  • Defines SF3B1-mutant / del(13q) co-occurrence as a distinct aggressive CLL subtype with poor OS, suggesting genomic stratification at diagnosis for prognosis PMID:26200345.
  • Provides mechanistic rationale for combining an SF3B1-directed splicing modulator (H3B-8800) with an mTORC1 inhibitor (temsirolimus) in patients carrying both lesions; combination was preferentially active on patient cells with both lesions versus single or neither PMID:26200345.
  • The authors note that H3B-8800 monotherapy was limited by toxicity in a prior phase I trial, but argue lower-dose combination with temsirolimus may be tractable PMID:26200345.
  • NFATC1 isoform-specific signaling (isoform 5 → mTOR; isoform 2 → AKT) may be a biomarker of mTOR-pathway dependence in SF3B1-mutant CLL PMID:26200345.

Limitations & open questions

  • Penetrance even in DM mice is moderate (6/25 = 24% over 24 months) and clinical translation requires validation in larger CLL cohorts PMID:26200345.
  • The human in vitro drug-sensitivity comparison uses only n = 3 patients per group; no in vivo human or PDX confirmation of the combination PMID:26200345.
  • The study does not formally test whether NFATC1 isoform 5 is necessary (vs sufficient) for mTOR activation in CLL — e.g. via isoform-specific knockdown or exon-9 forced-inclusion rescue PMID:26200345.
  • Toxicity and long-term efficacy of an H3B-8800 + temsirolimus regimen in patients remain unknown; the authors explicitly call out the need to assess safety PMID:26200345.
  • Whether Sf3b1-K700E uses different splicing targets across cancers (BRD9 in UVM, PPP2R5A in PDAC, MAP3K7/IRAK4 in MDS, Nfatc1 here in CLL) implies disease-context dependence that complicates pan-tumor splicing-modulator strategies PMID:26200345.

Citations from this paper used in the wiki

  • “RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL) and frequently co-occurs with chromosome 13q deletion [del(13q)]. This combination is associated with poor prognosis in CLL” (Abstract).
  • “184 of 1,009, 18.2% … Remarkably, 51% of SF3B1 mutations co-occurred with del(13q) … SF3B1 mut/del(13q) versus SF3B1 wt/no del, P < 0.0001 … inferior overall survival … P = 0.0002” (Results, Figure 1A).
  • “From 16 months onward, circulating CLL-like cells were found in 6 DM (24%) and 2 Mdr MT (7%) mice” (Results, Figure 1C).
  • “Mdr MT CLL cells were more sensitive to Tem treatment (IC50: 0.0001055 μM) compared with DM CLL cells (IC50: 2.148 μM) … DM CLL cells were more sensitive to the splicing inhibitor H3B-8800 (IC50: 0.00346 μM) compared with Mdr MT CLL cells (IC50: 0.05331 μM)” (Results, Figure 5A–B).
  • “the synergistic effect in DM CLL mice was more pronounced, with a median overall survival of 27 days compared with 9 days in the control group (3-fold increase)” (Results, Figure 5E–F).
  • “Murine RNA-sequencing data are deposited in GEO (GSE300699). Human RNA-sequencing data are from CLL map (https://cllmap.org/)” (Data availability).

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