CHEK1 is a synthetic lethal interactor of FBXO7 in colonic epithelial cells

Authors

Razi T

Farrell AC

Campos Gudiño R

Neudorf NM

Lichtensztejn Z

McManus KJ

Doi

10.1016/j.omton.2025.201028

PMID: 36334560 · DOI: 10.1016/j.omton.2025.201028 · Journal: Molecular Therapy: Oncology (2025)

TL;DR

This study identifies CHEK1 as a novel synthetic lethal (SL) interactor of FBXO7 in colonic epithelial cells. Using CRISPR-Cas9-generated FBXO7-knockout cell models and TCGA Pan-Cancer Atlas data, the authors show that FBXO7 copy number loss occurs in approximately 33% of colorectal cancers and correlates with chromosome instability, reduced mRNA expression, and worse patient outcomes. siRNA screening of eight DNA damage response (DDR) genes identified CHEK1 as the lead SL candidate, and pharmacological CHEK1 inhibition with Prexasertib preferentially killed FBXO7-deficient cells through increased DNA double-strand breaks and apoptosis. Prexasertib also synergized with 5-fluorouracil at defined concentration windows.

Cohort & data

  • Bioinformatic analysis used TCGA Pan-Cancer Atlas data (coadread_tcga_pan_can_atlas_2018) accessed via cBioPortal; n = 526 CRC cases for copy number and clinical outcome analyses.
  • FBXO7 copy number losses were assessed across 10 solid tumor types from TCGA Pan-Cancer Atlas.
  • In vitro models: non-malignant human colonic epithelial A1309 cell line (hTERT/CDK4-immortalized; TP53-reduced, KRAS G12V, APC truncated at aa 1309) with two independent CRISPR-Cas9-generated FBXO7-knockout clones (FBXO7-/- A and B) and a non-targeting control.

Key findings

  • FBXO7 shallow deletions (heterozygous loss) occur in 32.5% (169/526) of CRC cases; deep deletions are rare (<0.2%) (PMID:36334560).
  • CRC cases with FBXO7 shallow deletions show significantly reduced mRNA expression versus diploid cases (Mann-Whitney, p < 0.0001).
  • FBXO7 shallow deletions associate with increased genome instability: fraction of genome altered, aneuploidy score, and tumor break load (Wilcoxon with Benjamini-Hochberg FDR, q < 0.0001).
  • FBXO7 shallow deletions associate with significantly worse overall, progression-free, and disease-specific survival (log rank, p < 0.05).
  • siRNA screen of eight DDR genes (BRCA1, BRCA2, CHEK1, CHEK2, SOD1, PARP1, ATM, CDK2) identified CHEK1, CHEK2, and SOD1 as candidates reducing FBXO7-/- cell numbers; CHEK1 was prioritized based on its central role in replication stress and DDR.
  • Prexasertib (CHEK1 inhibitor) showed enhanced sensitivity in FBXO7-/- A cells (EC50 = 5.83 nM) versus control (EC50 = 9.03 nM).
  • At 6.0 nM Prexasertib, FBXO7-/- A cells showed a 41.6-fold increase in mean gamma-H2AX foci (DNA DSBs) versus 15.6-fold in controls.
  • Prexasertib induced a 2.6-fold increase in cleaved caspase-3 signal intensity (apoptosis) in FBXO7-/- A cells.
  • Prexasertib and 5-fluorouracil produced synergistic killing in FBXO7-/- A cells within a defined concentration window (1.92-9.6 nM Prexasertib with 28 micromolar 5-FU; Loewe Additivity model). An antagonistic interaction was observed at lower 5-FU concentrations (1.1-5.6 micromolar) combined with 1.92 nM Prexasertib.

Genes & alterations

  • FBXO7 — Copy number loss (shallow deletion) in 32.5% of CRC; associated with reduced expression, CIN, and poor outcomes. Identified as the target gene for SL exploitation.
  • CHEK1 — Synthetic lethal interactor of FBXO7; inhibition via siRNA or Prexasertib preferentially kills FBXO7-deficient cells. Central to replication stress response and DDR checkpoint control.
  • CHEK2 — Identified as a secondary SL candidate in the DDR screen (checkpoint signaling).
  • SOD1 — Identified as a secondary SL candidate in the DDR screen (oxidative stress management).
  • SKP1, CUL1, RBX1 — Invariable core members of the SCF E3 ubiquitin ligase complex; copy number loss of SCF genes induces CIN in CRC.
  • APC — Truncated (aa 1309) in the A1309 cell model; APC mutations implicated as a potential biomarker of 5-FU resistance.
  • KRAS — G12V mutation engineered in the A1309 cell model.
  • TP53 — Reduced expression (shRNA) in the A1309 cell model.
  • BRCA1, BRCA2 — Screened as DDR candidates; referenced for PARP inhibitor SL paradigm comparison.
  • PARP1 — Screened as DDR candidate; BRCA1/2:PARP1 SL interaction used as a conceptual parallel.
  • ATM, CDK2 — Screened as DDR candidates; not prioritized.

Clinical implications

  • CHEK1 inhibition (via Prexasertib) represents a potential targeted therapeutic strategy for the approximately one-third of CRC patients harboring FBXO7 copy number losses.
  • Combination of Prexasertib with 5-fluorouracil may enhance therapeutic efficacy but requires precise dose optimization to avoid antagonism at subtherapeutic concentrations.
  • FBXO7 loss could serve as a predictive biomarker detectable by DNA sequencing, array-based comparative genomic hybridization, qPCR, RNA-seq, or immunohistochemistry.
  • CHEK1 inhibition may help overcome 5-FU resistance in APC-mutant CRC contexts.
  • FBXO7 copy number losses occur across multiple solid tumor types (bladder, breast, GBM, head and neck, kidney, liver, lung, pancreatic, prostate), suggesting potential broad-spectrum relevance.

Limitations & open questions

  • All drug sensitivity and synergy data are from in vitro models only (A1309-derived FBXO7-knockout clones); no in vivo validation (xenograft or PDX models) has been performed.
  • The cell model (A1309) is non-malignant, immortalized with engineered genetic modifications (hTERT, CDK4, TP53 knockdown, KRAS G12V, APC truncation), which may not fully recapitulate the heterogeneity of primary CRC tumors.
  • Potential compensatory mechanisms by other F-box proteins (69 in humans) could modulate the strength of the SL phenotype and are unexplored.
  • Antagonistic interaction between Prexasertib and 5-FU at low doses highlights the need for careful dose optimization in clinical translation.
  • No comprehensive immunohistochemical evaluation of FBXO7 protein expression has been conducted in CRC patient samples.
  • The synergy data represent a single replicate in combination dose-response (n = 1 for gamma-H2AX and cleaved caspase-3 imaging experiments, though >550 cells per condition were analyzed).

Citations from this paper used in the wiki

  • “Shallow deletions […] occur in 32.5% (169 of 526 cases) […] while gains and amplifications only occur in 4% (19 of 526) and <0.2% (1 of 526) of cases” — Figure 1B legend and results.
  • “FBXO7-/- A cells (EC50 = 5.83 nM) compared with NT-Control (EC50 = 9.03 nM)” — Figure 3A results.
  • “Prexasertib treatments induced a 41.6-fold increase in mean gamma-H2AX foci within the FBXO7-/- A clone […] while only a 15.6-fold increase was observed within the NT-Control clone” — Results, gamma-H2AX section.
  • “Prexasertib and 5-FU produces a synergistic reduction in cell numbers within a defined concentration window (1.92 nM–9.6 nM Prexasertib with 28 micromolar 5-FU)” — Figure 6 results.

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