Sachin Katyal

Genome-scale identification of GBM-enriched genetic dependencies. A, Schematic of genome-scale CRISPR-Cas9 knockout screening used to identify genetic dependencies in patient-derived GBM and NSC models. Cells were infected with a genome-wide CRISPR-Cas9 gene knockout library and cultured over 15 to 18 doublings. gRNA fold change within cell populations at the beginning and end of each screen was used to determine gene essentiality. B, BF scores for GBM-enriched genetic dependencies across 15 patient-derived GBM models and four NSC models.

Identification of FEN1 as a driver of GSC proliferation and therapy resistance. A, Schematic of the chemoradiogenetic screen used to identify genetic dependencies of therapy resistance in the GBM model BT935. Cells were infected with a genome-wide CRISPR-Cas9 gene knockout (KO) library and exposed to DMSO, sublethal RT, sublethal TMZ, or combination treatment (RT and TMZ). gRNA LFC within cell populations at the beginning and end of each screen was used to determine gene essentiality. B, Comparison of gene-level LFC between DMSO and combination treatment arms in the GBM model BT935 for the 33 previously identified GBM-enriched genetic dependencies. Node color corresponds to the differential fitness ratio (LFC SoC/LFC DMSO), and genes with the highest enrichment in the therapy-treated arm are highlighted. C and D, Evaluation of FEN1 KO on (C) proliferation and (D) sphere formation of GSCs, compared with a gRNA targeting AAVS1 (control). Data are represented as mean ± SE; n = 4 experimental replicates; P values from unpaired t test (**, P < 0.01; ****, P < 0.0001). E, Kaplan–Meier survival analysis of immunocompromised mice engrafted with GBM tumor cells (BT972) with FEN1 or GFP (control) knockdown (n = 5 per knockdown). P value was calculated using log-rank (Mantel–Cox) test.

FEN1 inhibition enhances TMZ-induced DNA damage. A, Immunoblotting of FEN1i-resistant and FEN1i-sensitive GSCs for FEN1 protein after treatment with 100 μmol/L TMZ (n = 3 biological replicates per group). B, Immunoblotting of patient-matched primary (FEN1i-resistant; BT594) and recurrent (FEN1i-sensitive; BT972) GSCs for FEN1 protein after treatment with DMSO (control) or 100 μmol/L TMZ. FEN1 signal intensity normalized to vinculin control is shown for the representative immunoblot. C, Scatterplots indicating representative individual cellular comet tail moment values of FEN1i-resistant (BT594) and FEN1i-sensitive (BT972) GSCs from each corresponding cell/treatment type: DMSO alone (control), TMZ (200 μmol/L), FEN1i (30 μmol/L), or combination (TMZ–FEN1i, 200 μmol/L/30 μmol/L). D, Quantification of comet assays. Relative tail movement of FEN1i-resistant and FEN1i-sensitive GSCs is shown with increasing concentrations of TMZ ± 30 μmol/L FEN1 inhibitor for 48 hours. Data are represented as mean ± SE; n = 12 experimental replicates. E, Immunoblotting of FEN1i-resistant (BT594) and FEN1i-sensitive (BT972) GSCs for phosphorylated CHK1, total CHK1, phosphorylated CHK2, and total CHK2 after a 3-day treatment with DMSO (control), TMZ (45 μmol/L), FEN1i (30 μmol/L), or combination (TMZ and FEN1i). F, Quantification of immunoblotting in E. The proportion of phosphorylated (active) CHK1 and CHK2 is shown, with signal intensities normalized to vinculin control. Quantification is provided from the representative immunoblot shown to facilitate comparison across the indicated treatment conditions; independent replicate immunoblot experiments were not performed for this analysis. G, Immunoblotting of FEN1i-resistant and FEN1i-sensitive GSCs (n = 3 biological replicates per grouping) for total CHK1 and total CHK2 protein after treatment with TMZ (100 μmol/L). Heatmap color corresponds to normalized FEN1 intensity. All samples of each individual experiment were run on the same immunoblot under identical experimental conditions, with immunoblots cropped for presentation. Quantification of immunoblots is presented for visualization of relative signal intensity only and should not be interpreted as statistical comparisons across experiments. ns, not statistically significant. **, P < 0.01; ***, P < 0.001.

Sensitivity to FEN1 inhibition extends to TMZ sensitization. A, Cell viability assays of GSC and NSC lines exposed to increasing concentrations of FEN1 inhibitor over 7 days. Data are represented as mean ± SE; n = 4 experimental replicates. B, Cell viability assays of NSC, FEN1i-resistant, and FEN1i-sensitive GSC lines exposed to DMSO (control), 25 μmol/L TMZ, 30 μmol/L FEN1i, or combination (TMZ and FEN1i). Dashed lines represent expected viability under combination treatment. Data are represented as mean ± SE; n = 4 experimental replicates. C and D, Synergy assays between TMZ and FEN1 inhibitor in (C) FEN1i-resistant and (D) FEN1i-sensitive GBM models. Colors correspond to Bliss excess synergy scores, wherein more positive values indicate stronger synergy; n = 4 experimental replicates. ns, not statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Sensitivity to FEN1 inhibition is associated with tumor cell stemness and proliferation. A, Immunofluorescence staining for Ki67 and DAPI in FEN1i-sensitive (BT972, BT935, BT241) and FEN1i-resistant (MBT357, MBT373, BT594) GSCs. B, Secondary sphere formation assay in FEN1i-sensitive and FEN1i-resistant GSCs. Data are represented as mean ± SE; n = 4 experimental replicates. C, Cell viability assays of FEN1i-sensitive GSC lines cultured with or without PTX (cell-cycle stalling agent) for 6 days, exposed to increasing concentrations of FEN1i. Data are represented as mean ± SE; n = 3 experimental replicates. D, Cell viability assay of FEN1i-resistant GSCs with TP53 or AAVS1 (control) knockout, exposed to increasing concentrations of FEN1i. Data are represented as mean ± SE; n = 5 experimental replicates. ns, not statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Single-cell analysis reveals that FEN1 expression correlates with key genes and gene programs involved in GBM proliferation, stemness, and DNA damage repair. A, Codependency analysis of FEN1 expression, with coexpression represented by nCDI. Key genes are highlighted and labeled, indicating strong coexpression with FEN1. B, Gene program analysis showing correlation between FEN1 expression and program activity. Dot size corresponds to −log10 (adjusted P value), with larger dots indicating more statistically significant results. Dots are color-coded according to gene program category, and key programs are highlighted.

Quality control analysis of CRISPR-Cas9 dropout and chemoradiogenetic screens in GBM model BT935, related to figure 1B and 2B. A) Gene-level LFC of CEGs and nonessential genes across treatment arms. All data presented from n = 3 biologically independent replicates. P values are from unpaired Student’s t tests. B) Precision-recall plots indicate high performance of screens across treatment arms. C) Number of essential genes (FDR < 0.05) identified in screens across treatment arms. D) Number of conditional genetic interactions identified in screens across treatment arms (FDR < 0.05).

A) Pearson correlation of genetic interaction (drugZ) scores from all treated screens, presented with (right) and without (left) filtering for genes with false discovery rate (FDR) < 0.05 in at least one screen. B) ClusterProfiler analysis of cGIs (drugZ FDR < 0.05) from all screens. Reactome gene sets enriched at an adjusted P < 0.05 are shown in the dot plot, with dot size corresponding to gene ratio and dot color indicating adjusted P values. Positive and negative cGIs responsible for the only overlapping enriched pathway (DNA Repair) are listed for each model.

A) Methylation-specific PCR (MSP) gel showing MGMT promoter methylation status across GBM cell lines. Methylated (M) and unmethylated (U) primer sets were used following bisulfite conversion. Commercial methylated and unmethylated DNA controls and a no-template water control (H2O) were included as assay controls. B) Cell viability assays of GSC lines exposed to increasing concentrations of TMZ inhibitor over 7 days, related to figure 3. Data are represented as mean ± s.e.; n = 4 experimental replicates. C) Plot of TMZ and FEN1i IC50 values, related to figure 3.

A) Normalized intensities of FEN1 protein from immunoblotting related to figure 4A. B) Representative images of comet assays of FEN1i-resistant (BT594) and sensitive (BT972) GSCs exposed to DMSO alone (control), TMZ (200 µM), FEN1i (30 µM) or combination (TMZ/FEN1i, 200 µM/ 30 µM). C) Normalized intensities of phospho-Chek1, total Chek1, phospho-Chek2 and total Chek2 protein from immunoblotting related to figure 4E and 4F. D) Immunoblotting for phospho-ATM, total ATM, phospho-DNAPK and total DNAPK protein normalized to vinculin control.