Browsing by Author "Alruwaili, Mohammed Muharrab"

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Development of a novel therapeutic intervention for p53-mutant advanced cancers leveraging their DNA damage response liabilities
(University at Buffalo, 2025-05) Alruwaili, Mohammed Muharrab; Andrei, Bakin
Colorectal Cancer (CRC) and Pancreatic Ductal Adenocarcinoma (PDAC) are among most lethal cancers worldwide. Despite initial response to standard-of-care therapy, a significant proportion of CRC/PDAC cancers relapse and progress to metastatic disease with poor overall survival (OS). Thus, better treatment options are urgently needed. Genetic alterations in the tumor suppressor p53 gene (TP53) are found in most CRC and PDAC cases and contribute to cancer relapse, progression, and metastasis. Even though the functional consequences of p53 mutations have been extensively studied, there are no FDA-approved drug or their combination targeting p53-mutant cancers. This thesis work is aimed at the development of effective therapeutic approaches for p53-mutant cancers. The initial concept of the two-drug strategy, working as an inducer-amplifier pair, was suggested for selectively targeting p53-mutant triple-negative breast cancer (Zonneville at al., Commun. Biology, 2021). The current thesis research investigated the mechanisms underlying the efficacy and selectivity of our two-drug strategy using p53-mutant CRC and PDAC models. Our two-drug strategy utilizes thymidine analogue TAS102, acting as an inducer of single-strand DNA breaks through a post-replicative DNA repair, and poly (ADP-ribose) polymerase (PARP) inhibitor, acting by delaying repair of single-strand breaks in DNA. This two-drug concept is based on the following rationale. Our analysis of the genomic data from the Cancer Genome Atlas (TCGA) showed high expression levels of Base-Excision Repair (BER) and elevated tumor mutational burden (TMB) in p53-mutant CRC and PDAC, suggesting impairment in BER. The BER activity in CRC and PADC cells was examined by a new methodology with thymidine analogues (ethynyl-deoxyuridine (EdU) or trifluorothymidine (TFT, a component of TAS102)). The analysis revealed a significant delay in the removal of genomic EdU or TFT in p53-mutant cells compared to isogenic p53 wildtype (p53WT) cells. We also noted that p53-mutant cells accumulated in the late S/G2 phase while p53WT accumulated in G1. Thymidine analogues (EdU, TFT) induced buildup of DNA breaks in p53-mutant cancer cells, while non-tumor p53WT cells showed only a transient DNA damage. Further, addition of a PARP inhibitor (PARPi) increased DNA double-strand breaks (DSBs) and cell death selectively in p53-mutant cells, while PARPi alone did not induce DNA damage. Cytotoxicity analysis showed that thymidine analogues synergized with PARP inhibitors selectively in p53-mutant cells. In preclinical vivo models, the TAS102-PARPi combination was far more effective than either drug alone in p53-mutant Cell-Derived Xenograft (CDX) and Patient-Derived Xenograft (PDX) models. Immunohistochemistry data showed that the two-drug treatment increased DNA damage and cell death while decreasing cell proliferation. The two-drug combination and TAS102 exhibited comparable tumor control in the p53WT PDX model. Importantly, the TAS102-PARPi therapy did not exhibit major side toxic effects in mice even after prolonged drug administration. Our two-drug TAS102-PARPi strategy is now being tested in the first-in-human phase I study with TAS102 and talazoparib in refractory CRC (NCT04514497, PI Dr. Fountzilas). The dose-escalation part of the study showed that the TAS102-talazoparib treatment is well-tolerated, and no major dose-limiting toxicities were observed. The analysis showed that the addition of talazoparib to TAS102 increased TFT-positive cells in peripheral blood mononuclear cells (PBMCs) and in tumor tissues. The median progression-free survival (mPFS) was 5.7 months in the DL3 group compared to 1.9 months for historical TAS102 alone. Investigation of the mechanism behind the two-drug strategy showed that incorporation of thymidine analogues into DNA provoked post-replicative BER-mediated repair, generating DNA single-strand breaks (SSBs). As SSB repair is assisted by PARP, the inhibition of PARP increased more lethal DSBs. Non-tumor p53WT cells responded with activation of the p53-p21 axis, leading to G1-arrest and efficient removal of thymidine analogues. In contrast, p53-mutant cells, lacking the G1 checkpoint, accumulated DNA damage and were arrested in G2 for a prolonged time. Investigation of DNA damage response (DDR) revealed that TAS102-PARPi induced signaling pathways mediated by ATM (Ataxia-Telangiectasia Mutated) and ATR (Ataxia-Telangiectasia Rad3-related) kinases. Our study showed that ATM kinase-controlled activation of the p53-p21 axis, whereas ATR kinase-controlled activation of CHK1 and WEE1 kinases, which mediate induction of the G2-checkpoint, blocking the entrance to mitosis. Furthermore, the cell-cycle analysis demonstrated that the TAS102-PARPi treatment induced G2-arrest and high levels of DSBs selectively in p53-mutant cells while p53WT cells were transiently arrested in G1 and resumed the cell cycle after drug withdrawal. This finding suggested that blockade of ATR kinase or its downstream effectors may release p53-mutant cells with unrepaired DNA into mitosis, leading to cell death. This hypothesis was examined using several approaches, including cytotoxicity, immunofluorescence microscopy, and flow cytometry. We found that the blockade of ATM reduced sensitivity to TAS102-talazoparib in all tested cell lines. In contrast, blockade of ATR kinase markedly enhanced the cytotoxicity of TAS102-talazoparib in p53-mutant cells and increased levels of γH2AX and phospho-H3 in cells treated with TAS102-talazoparib, indicating increased DNA damage and entrance to mitosis. Similar effects were observed by blockade of downstream effector kinase WEE1. Furthermore, blockade of WEE1 markedly increased cell death in p53-mutant cells treated with TAS102-talazoparib. Based on these data, we developed a triple-drug strategy combining the TAS102-PARPi treatment with a sequential delayed application of the G2 checkpoint kinase inhibitors. This sequential triple-drug combination works as an inducer–amplifier–terminator trio to induce cell death in p53-mutant cells selectively. The in vivo studies showed that this sequential triple-drug strategy was more effective in tumor growth control compared to the TAS102-PARPi treatment or WEE1i alone. In the CRC HT29 tumor xenograft model, the triple-drug treatment reduced tumor growth by 63%, compared to 18% by WEE1i or 43% by TAS102-talazoparib alone. Likewise, the triple-drug regimen showed greater tumor control in the CRC-PDX-03-26-RP model carrying p53H178T/Ter67fs and KRAS-G12D. In the PDAC PDX-14312 model, carrying p53R175H, a triple-drug treatment reduced tumor growth by ~80%, whereas WEE1i monotherapy by ~30% or TAS102-talazoparib by ~43%. Importantly, neither of the treatments exhibited major side toxic effects or caused changes in major organs (kidney, liver, lungs, heart) based on the pathology analysis of mice. These preclinical data provide strong support for a sequential triple-drug treatment strategy that works as an inducer–amplifier–terminator trio to induce cell death in p53-mutant cancers. This novel triple-drug therapeutic strategy for p53-mutant cancers has strong potential to improve the management of CRC and PDAC and meaningfully impact the health of cancer patients.
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