Hypothesis

Intron retention (IR) levels above a quantifiable entropy threshold create a splicing stress state that renders SF3B1-mutant cancers dependent on minor spliceosome activity, making them synthetically lethal to minor spliceosome inhibition.

Mechanistic Rationale

• SF3B1 mutations increase cryptic 3′ splice site usage, generating pervasive intron retention and mRNA decay Spliceosome mutations drive oncogenesis.
• Persistent IR elevates nuclear RNA:DNA hybrid formation, activating ATR‑CHK1 signaling and increasing reliance on the minor spliceosome to resolve U12‑type introns that escape major‑splicing surveillance.
• When IR exceeds a threshold, the spliceosome condensate undergoes a gel‑to‑solid transition, sequestering U2AF65 and limiting major spliceosome flexibility; the minor spliceosome becomes the sole conduit for essential U12‑intron‑containing transcripts (e.g., DNA repair genes such as BRCA1, FANCD2).
• Minor spliceosome inhibition then causes catastrophic loss of these transcripts, precipitating DNA damage and cell death specifically in high‑IR tumor cells.

Testable Predictions

1. Quantitative threshold – In a panel of SF3B1-mutant cell lines, IR fraction (retained intron reads / total reads) > 0.18 predicts > 70 % growth inhibition by the minor spliceosome inhibitor H3B-8800 (or equivalent), whereas IR < 0.12 shows < 20 % effect SF-mutated cancers show synthetic lethality to spliceosome inhibition.
2. ATP dependence – It's important that depleting cellular ATP with 2‑deoxy‑glucose lowers the IR threshold for sensitivity, while ATP supplementation rescues resistance, linking the hypothesis to prior work on ATP‑driven condensate gelation.
3. Biomarker validation – We're expecting that patient‑derived xenografts with baseline IR measured by RNA‑seq will stratify response to minor spliceosome inhibition in vivo; high‑IR tumors exhibit delayed tumor growth and increased γH2AX foci post‑treatment Single-cell RNA-seq reveals SF-mutated subpopulations.
4. Resistance mechanism – Acquired resistance correlates with downregulation of U12‑type intron‑containing DNA‑repair genes and upregulation of alternative splicing factors (e.g., SRSF1) that bypass minor spliceosome dependence; cells can't survive long‑term without restoring major‑splicing fidelity Therapeutic strategies include SF3B-targeters, ASOs for PTBP1 in trials, isoform-specific combos.

Experimental Approach

• Generate isogenic SF3B1-WT and SF3B1-K700E lines; titrate expression of a splicing reporter to modulate IR.
• Measure IR using IRFinder, ATP levels via luciferase assay, and sensitivity to H3B-8800 (dose‑response).
• Use CRISPRi to knock down U11 or U12 snRNA to confirm minor spliceosome specificity.
• In vivo, implant high‑IR vs low‑IR SF3B1-mutant PDX cohorts, treat with H3B-8800, monitor tumor volume and IR dynamics by longitudinal RNA‑seq.
• Statistical analysis: ROC curves to determine optimal IR cutoff; Mann‑Whitney test for sensitivity groups (p < 0.05) SF mutations correlate with TMB, aneuploidy, SCNAs, and ITH scores.

Falsifiability

If IR levels above the proposed threshold don't predict enhanced sensitivity to minor spliceosome inhibition, or if ATP manipulation fails to shift the threshold, the hypothesis is refuted. Conversely, a strong predictive relationship would support the model and suggest IR entropy as a biomarker for spliceosome‑targeted therapy.