Setton, J. | bioRxiv Science

bioRxiv2025

The 9-1-1 complex protects ssDNA gaps in BRCA2-deficient cancer

Single-stranded DNA (ssDNA) gaps are a hallmark of BRCA-deficient cells, yet the mechanisms that safeguard these lesions remain unclear. Through a genome-wide CRISPR screen, we identified the RAD9A-HUS1-RAD1 (9-1-1) complex as essential for the survival of BRCA2-deficient cells through an ATR-independent mechanism. Loss of 9-1-1 in this context leads to the accumulation of PRIMPOL-dependent gaps that fail to undergo post-replicat ive repair, resulting in pathological expansion and increased DNA damage. This instability is driven by excessive EXO1-mediated degradation, as EXO1 depletion rescues the phenotype. We further demonstrate that the 9-1-1 complex is required for POL{zeta}-dependent gap filling. We propose a model in which ssDNA gaps, when extended beyond a critical length, become inaccessible to TLS-mediated repair and are fully reliant on homologous recombination. These findings establish the 9-1-1 complex as key regulator of ssDNA gap stability and a promising therapeutic target in BRCA2-deficient cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/680950v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@56516aorg.highwire.dtl.DTLVardef@fb1ce3org.highwire.dtl.DTLVardef@25a7b2org.highwire.dtl.DTLVardef@249731_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Deficiencies in the Fanconi Anemia or the Homologous Recombination Pathway enhance the antitumor effects of the novel hypoxia-activated prodrug CP-506

The novel hypoxia-activated prodrug CP-506 has been shown to selectively target hypoxic tumor cells, which are associated with disease progression and resistance to conventional anti-cancer therapies. Given the alkylating effector metabolites, we hypothesize that defects in interstrand crosslink (ICL) and double strand break (DSB) DNA repair may serve as a predictive biomarker of sensitivity to CP-506. Here, we evaluated the role of DNA damage repair pathways in the antitumor response to CP-506. Isogenic cancer cell lines proficient or deficient in the Fanconi Anemia (FA), homologous recombination (HR), or non-homologous end joining (NHEJ) pathway were cultured as 2D monolayers and 3D spheroids. Cell viability, clonogenic cell survival, and spheroid growth inhibition were assessed following CP-506 exposure. Mice bearing subcutaneous isogenic xenografts received CP-506 (600 mg/kg; QD5) or vehicle treatment upon reaching a tumor starting volume (SV) of 244.9 {+/-} 72.0 mm3. Treatment response was quantified as time to reach 4xSV (T4xSV) and respective enhancement ratios (ER), defined as T4xSVCP-506/T4xSVvehicle. DNA damage and repair capacity were evaluated by {gamma}H2AX and alkaline comet assays. In vitro, cell lines deficient in FA or HR, but not NHEJ, showed enhanced sensitivity to CP-506 compared to parental cells in viability and clonogenic assays. This was confirmed in spheroid growth inhibition studies. In vivo, the antitumor response to CP-506 was more pronounced (P<0.0001) in LNCaP AR FANCA-/- (ER 4.0{+/-}1.1) and LNCaP AR FANCD2-/- (ER 3.4{+/-}0.8) xenografts compared to parental LNCaP AR (ER 1.5{+/-}0.5) xenografts. CP-506 treatment of DLD-1 BRCA2-/- (2.9{+/-}0.7; P<0.0001) xenografts resulted in significantly enhanced ER compared to parental (1.3{+/-}0.2) xenografts. Similar results were obtained in HCT116 BRCA2-/- (ER 4.0{+/-}0.6; P<0.0001) versus parental (ER 1.7{+/-}0.6) xenografts. In contrast, the ER of HCT116 DNA-PKcs-/- (1.4{+/-}0.3; P=0.18) xenografts was not different from HCT116 parental xenografts. Under anoxic conditions, CP-506 caused elevated {gamma}H2AX foci counts in FANCA-(2.0-fold increase at 48 hours) and FANCD2-deficient cells (1.4-fold increase at 72 hours) compared to LNCaP AR parental cells (P<0.0001). Similarly, {gamma}H2AX expression was increased in DLD-1 BRCA2-/- cells, but not in HCT116 BRCA2-/-cells, compared to their respective parental cells. In xenografts, FA- and HR-deficiency caused elevated {gamma}H2AX expression compared to respective parental tumors (1.6-9.3-fold increase). HCT116 DNA-PKcs-/- cells and xenografts displayed reduced {gamma}H2AX expression compared to their parental counterpart, with an 0.5-fold reduction. The comet assay confirmed CP-506-induced ICLs and DNA strand breaks but was unable to explain the differential therapeutic responses of CP-506 among isogenic tumor cells. Deficiencies within FA or HR, but not NHEJ, enhanced the antitumor effects of CP-506 through a mechanism consistent with the concept of synthetic lethality. Therefore, both DNA repair status and the presence of tumor hypoxia represent key biomarkers for patient stratification in clinical trials of CP-506.

cancer biology↗

bioRxiv2019

Novel patterns of complex structural variation revealed across thousands of cancer genome graphs

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g. deletion, translocation) or complex (e.g. chromothripsis, chromoplexy) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,833 tumor whole genome sequences (WGS), we introduce three complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early replicating regions and superenhancers, and are enriched in breast and ovarian cancers. Rigma comprise "chasms" of low-JCN deletions at late-replicating fragile sites in esophageal and other gastrointestinal (GI) adenocarcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold back inversions that are enriched in acral but not cutaneous melanoma and associated with a previously uncharacterized mutational process of non-APOBEC kataegis. Clustering of tumors according to genome graph-derived features identifies subgroups associated with DNA repair defects and poor prognosis.

genomics↗