Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL

Abstract

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

Figure 1.. Molecular and functional profiling identifies CFLAR, the CAK complex, and the transcriptional kinase CDK13 as potential therapeutic targets in KRAS TP53 mutant PancVH1 PDCL.

A. Schematic overview of molecular and functional profiling of PancVH1 cells. B. PancVH1 immunoblot characterization of KRAS signaling - KRAS-GTP, MEK activation (MEK Ser217/221), truncation of the p53 protein, and reduction in expression of CDNK2A (p16Ink4a), and SMAD4 (DPC4). HPNE cell line used as a non-tumorigenic control. C. PancVH1 candidate driver genes as defined by cross reference to cancer landscapes. D. Left panel: schematic of druggable genome RNAi arrayed screen; 93 cell seeded plates; 31 siRNA library plates (hDG1-hDG31) with transfection reagents arrayed in triplicate on 93 cell seeded plates. Right panel: PancVH1 Druggable Genome RNAi screen results as mean (n=3) percent viability (y -axis) and library index of 6659 targeted genes (x-axis) with 327 (5%) RNAi selected hits (red). Lower panel; pancreatic pathway siRNA controls sublibrary to KRAS (n=108), TP53 (n=93), CDKN2A (n=93), SMAD4 (n=93) and 46 genes (n=18–21) compiled from the literature, KIF11 positive control (n= 744). E. PancVH1 RNAi secondary screen significant hits (-log₁₀P-value > 1; dotted line denotes - log₁₀P-value=1); color coded by MSigDB significantly overlapping genesets (FDR <0.05): KRAS_SHP2 (PID) (red), MYC_Targets_V1 (green), cell cycle (blue), and apoptosis (orange); CCNH (CyclinH); also includes MAPK and TNF signaling. F. Differential analysis of PancVH1 vs. HPNE secondary RNAi screen results; preferential lethality (P-value <0.05) and cross reference to KRAS mutant PDAC cell line RNAi screens (JHU) differential with HPNE (red) and PancVH1-HPNE (black).

Figure 2.. Drug profiling identifies the TFIIH complex as a potential therapeutic target in KRAS mutant PDAC PDCLs.

A. CNIS drug screen of PancVH1 and HPNE; heatmap of 318 drug sensitivities shown as log₁₀IC50; Bivariate plot of PancVH1 log₁₀IC50 vs. potency fold change (HPNE_IC50/PancVH1_IC50); SI Dataset S4A–E. B. Bivariate plot of PancVH1 log₁₀IC50 vs. potency fold change (HPNE_IC50/PancVH1_IC50) from low-throughput drug screen with 50 selected drugs targeting RNAi targets nominated from druggable genome RNAi screen and associated PDAC biology; SI Dataset S4F–G. C. Heatmap of log₁₀IC50 values from fitted dose-response curves of 22 drugs on 14 TKCC PDAC PDCLs; 72hr; 10-point; singleton; normalized to vehicle (DMSO) condition; AUC values and additional data in SI Dataset S6; TKCC PDCL structural variant (SV) subtypes (Stable, Focal, Scattered, Unstable); molecular subtypes: classical (orange), squamous (blue); and KRAS, TP53, CDKN2A, and SMAD4 status (top) are shown. D. Heatmap of log₁₀IC50 values from fitted dose-response curves of 7 drugs including THZ1, SNS-032, and Triptolide on 11 TKCC PDAC PDCLs; 72hr assay; 10-point; 4 technical replicates; normalized to vehicle (DMSO) condition; AUC values and additional data in SI Dataset S6. E. Fitted dose response curves of THZ1, SNS-032, and Triptolide on PancVH1 and HPNE cells; 72hr assay; 10-point; 3 technical replicates; range; 50μM −1.4nM; response data are presented as a fitted curve to the mean fractional viability of vehicle condition (DMSO); AUC (area under the curve) values were calculated via trapezoid method with n-parameter logistic regression (nplr); R² value indicates goodness of fit (0–1).

Figure 3.. TFIIH inhibition initiates a CFLAR-mediated caspase-8 dependent apoptotic response in KRAS TP53 mutant PancVH1 PDCL.

A. Immunoblot of dose dependent response to THZ1, SNS-032, THZ531, and Triptolide on CFLAR (cFLIP_L / cFLIP_S) expression in PancVH1 and HPNE cells; drug concentrations (nM); 24hr; GAPDH loading control. B. Corresponding immunoblot probed for caspase-8, caspase-3 (cleaved), PARP, H2A.X Ser139, p53, and p21; drug concentrations (nM); 24hr; GAPDH loading control. C. Caspase-8 inhibition rescues cell death/apoptotic phenotype upon treatment with THZ1, SNS-032, THZ531, and Triptolide in PancVH1 with no significant effect on HPNE; bar graphs of relative cellular viability at 24 hrs post-treatment with vehicle (solvent), drugs [500nM], drugs with Z-FA-FMK (selective inhibitor of caspases-2,−3,−6,−7 as control), and drugs with Z-IETD-FMK (specific inhibitor of caspase-8); mean ± s.d., n=3; unpaired t-test; significance: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. D. Caspase-8 activity measured in PancVH1 and HPNE at 24 hrs post-treatment with vehicle (solvent) and THZ1, SNS-032, THZ531, and Triptolide at 500nM; bar graph, mean ± s.d., n=3; unpaired t-test; significance: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05.

Figure 4.. TFIIH inhibition disrupts the protein stability of RPB1 resulting in the downregulation of IAPs and transcriptional effectors of oncogenic signaling in KRAS TP53 mutant PDCLs.

A. Immunoblots of dose dependent TFIIIH inhibition on the expression of RPB1, CFLAR, and IAPs: MCL1, and XIAP in PDCLs: PancVH1, TKCC18-LO (Stable), TKCC16-LO (Scattered), TKCC27-LO (Unstable); drug concentrations (μM); 24hr; GAPDH loading control. B. Immunoblots of dose dependent TFIIIH inhibition on the expression of RPB1, FOSL1, and STAT3 in PDCLs; drug concentrations (μM); 24hr; GAPDH loading control. C. Proposed model of TFIIH inhibition on RNA polymerase II transcription. TFIIH complex: GTF2H1 (p62), GTF2H2 (p44), GTF2H3 (p34), GTF2H4 (p52), GTF2H5 (p8), ATP-dependent DNA helicase subunits XPD (ERCC2), and XPB (ERCC3), and the CAK that includes CDK7, Cyclin H, and MAT1, and inhibition via the covalent CDK7/12/13 inhibitor THZ1, the dual kinase CDK7/CDK9 inhibitor SNS-032, and XPB (subunit of TFIIH) inhibitor Triptolide. In this model, TFIIH inhibition results in the disruption of the spatiotemporal phosphorylation of the RNA polymerase II Subunit B1 carboxy terminal domain (CTD) heptapeptide repeat as well as the stability of the RPB1 protein. RNA polymerase II transcription initiation is therefore arrested leading to the downregulation of CFLAR, IAPs and transcriptional effectors of the KRAS mutant signaling cascade; RPB1 also acts as a substrate for transcriptional kinases CDK9/CyclinT (P-TEFb), essential to RNA polymerase II transcription elongation, and CDK13/CyclinK, involved in the coordination of RNA splicing.

Figure 5.. TRAIL in combination with TFIIH inhibitors demonstrate synergy of response in KRAS mutant PDAC PDCLs.

A. Proposed model for combination therapeutic strategy where TFIIH/RPB1 inhibition decreases the expression of CFLAR, the negative regulator that competes with caspase-8 for binding to the death inducing signaling complex (DISC) and inhibits the execution of the extrinsic apoptotic pathway. TFIIH inhibition also decreases expression of IAPs MCL1 and XIAP, both with key roles in the execution of mitochondrial-mediated apoptosis. In this model, TRAIL could potentially synergize with TFIIH inhibition to optimize the extrinsic and intrinsic mitochondrial-mediated apoptotic pathway in KRAS mutant PDAC. B. Public datasets (QCMG, ICGC, UTSW) genomic data on PDAC patients (cBioPortal). C. izTRAIL dose response curves on KRAS mutant PDCLs and HPNE; izTRAIL concentrations range (0–400 ng/ml); fitted dose response curve, nonlinear regression; PDCL izTRAIL sensitivity divided into two groups (red < 50% at 100ng/ml < blue). HPNE cell line (black). D. Immunoblot of combination treatment with TFIIH inhibitors (THZ1 (500nM), SNS-032 (500nM), Triptolide (25nM)) and izTRAIL(25 ng/ml); 24hr assay; PARP, BID, TRAIL-R1 (DR4), TRAIL-R2 (DR5) expression via immunoblot in both PancVH1 and HPNE; GAPDH loading control; vehicle (DMSO); gemcitabine (25nM). E. Immunoblot of combination treatment with izTRAIL(25ng/ml) and TFIIH inhibitor Triptolide (25nM); 24hr assay; PARP, BID, TRAIL-R1 (DR4), and TRAIL-R2 (DR5) protein expression in PDCLs; GAPDH loading control. F. Bar graphs of Bliss sum demonstrating drug synergy of response to TFIIH inhibition (THZ1, SNS-032, and Triptolide) in combination with izTRAIL in KRAS mutant PDCLs (10/11); 72 hr post-treatment assay endpoint; Bliss sum calculated by excess over Bliss independence model across the combination matrix: synergistic>0, additive=0, antagonistic <0.