Withers, H. | Search bioRxiv

bioRxiv2025

A Sequential Triple-Drug Strategy for Selective Targeting of p53-Mutant Cancers

The tumor suppressor TP53 gene (p53) is mutated in most human malignancies; however, existing treatment options are largely ineffective, lack selectivity, and cause toxic side effects. To address these clinical problems, we developed a sequential triple-drug strategy for p53 mutant cancer cells. Here we show that a combination of a thymidine analogue (TAS102) plus PARP inhibitor (PARPi) promotes formation of DNA double-strand breaks (DSBs) and G2-arrest specifically in p53 mutant cancer cells. Transcriptome analysis revealed that TAS102-PARPi treatment of p53 mutant cells did not repress DNA replication but activated DSB repair and blocked the mitotic program, consistent with G2-arrest. In contrast, TAS102-PARPi treatment of normal p53 wild-type cells resulted in a temporal G1-arrest and rapid recovery of cell cycle capacity after drug withdrawal. In p53 mutant cancer cells, subsequent blocking of a G2-checkpoint kinase, such as WEE1, released these G2-arrested cells into mitosis, leading to massive cell death. Delayed administration of a G2-kinase inhibitor provides time for p53 wild-type cells to repair DNA, thereby minimizing toxicity to normal tissues. This sequential triple-drug strategy exhibited robust efficacy in preclinical models of colorectal and pancreatic cancers and was well tolerated in mice. Together, our findings illustrate a promising triple-drug strategy for targeting p53 mutant malignancies.

cancer biology↗

bioRxiv2024

Image-Based Quantitative Single-Cell Method Showed Increase of Global Chromatin Accessibility in Tumor Compared to Normal Cells

The phenotypic plasticity of cancer cells has recently emerged as an important factor of treatment failure. The mechanisms of phenotypic plasticity are not fully understood. One of the hypotheses is that the degree of chromatin accessibility defines the easiness of cell transitions between different phenotypes. To test this, a method to compare overall chromatin accessibility between cells in a population or between cell populations is needed. We propose to measure the chromatin accessibility of a cell by total fluorescence signal from nuclei stained with DNA-binding fluorescent molecules. This method is based on the existing data that some small molecules bind nucleosome-free DNA more easily than nucleosomal DNA. Thus, nuclear fluorescence of these molecules is proportional to the amount of nucleosome-free DNA, serving as a measure of chromatin accessibility. We optimized the method using several DNA binding molecules and known chromatin modulating agents. Using a set of tumor and non-tumor cells of different origins we observed the tendency to the higher chromatin accessibility of tumor versus non-tumor cells. Chromatin accessibility was also increased upon oncogene-induced transformation of mouse and human cells.

cancer biology↗

bioRxiv2017

The miR-96/RARγ signaling axis governs androgen signaling and PCa progression

Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RAR{gamma}), are commonly reduced in prostate cancer (PCa). Therefore we sought to establish the cellular and gene regulatory consequences of reduced RAR{gamma} expression, and determine RAR{gamma} regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RAR{gamma} levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RAR{gamma} cistrome which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RAR{gamma} to regulate androgen signaling, RAR{gamma} knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RAR{gamma} down-regulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RAR{gamma} expression and function Capture of the miR-96 targetome by biotin-miR96 identified that RAR{gamma} and a number of RAR{gamma} interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RAR{gamma} target genes (e.g. SOX15) that significantly associated with worse disease free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p=0.015). In summary, miR-96 targets a RAR{gamma} network to govern AR signaling, PCa progression and disease outcome.\n\nConflict of interestThe authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.\n\nFUNDINGLESC acknowledges support, in part, of Roswell Park Comprehensive Cancer Center-University of Pittsburg Cancer Institute Ovarian Cancer Specialized Program of Research Excellence National Institutes of Health [P50CA159981-01A1].\n\nMDL acknowledges support of Molecular Pharmacology and Experimental Therapeutics NRSA T32 program [T32CA009072] held at Roswell Park Comprehensive Cancer Center.\n\nMJC and DJS acknowledges support in part from the Prostate program of the Department of Defense Congressionally Directed Medical Research Programs [W81XWH-14-1-0608, W81XWH-11-2-0033] and the National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Centers Genomic Shared Resource.\n\nMJC, GL, AR, HW and PvdB acknowledges support from the European Union-United States Atlantis Program [P116J090011].\n\nMJC and LESC acknowledge support from the National Cancer Institute (NCI) grant P30CA016056 involving the use of OSUCCC The James, CCSG P30CA016058

cancer biology↗