Biology subjects

Humphrey, T. C.

Publications and source records attributed to Humphrey, T. C..

3 recordsLinked to original sources

bioRxiv2021

R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

WEE1 inhibitors have now advanced into clinical studies as monotherapy or in combination with chemoradiotherapy in TP53, RAS, BRAF, and SETD2 mutation carriers across several tumour types, yet mechanisms of resistance are still poorly understood. Here, we further elucidate the mechanisms by which AZD1775, the most potent WEE1 inhibitor, kills cells and reveal additional genetic interactions that can result in resistance, but could be used to optimise its clinical utility. We identified RNA Polymerase II-associated factor 1 (PAF1) complex members, CDC73, CTR9, and PAF1 as major determinants of WEE1-inhibitor sensitivity in isogenic SETD2-positive and negative cell lines. PAF1-knockdown cells resist higher doses of the WEE1 inhibitor, which we show is due to reduced DNA damage induction ({gamma}H2AX) and delayed G1 checkpoint activation, ultimately protecting cells against replicative catastrophe. Investigations into the molecular mechanisms responsible for PAF1-mediated resistance identify involvement of R-loops and subsequent activation of the cyclin-dependent kinase inhibitor p21Cip1/Waf1, which in addition to causing prolonged G1 arrest in the following cell cycle, also regulates CDK activity, therefore limiting replication. These results provide evidence that the PAF1 complex and p21 are important regulators of proliferation under increased DNA replication stress and their expression levels might be useful biomarkers to predict clinical response to WEE1 inhibitors and other ribonucleotide reductase inhibitors.

cancer biology↗

bioRxiv2021

Therapeutic targeting of SETD2-deficient cancer cells with the small-molecule compound RITA

The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in certain cancer types, identifying SETD2 as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to the compound RITA, resulting in significant p53 induction and apoptosis. This is further associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. RITA sensitivity is linked to the phenol sulphotransferase SULT1A1, which we find to be highly upregulated in cells that lack SETD2. Depletion of SULT1A1 or addition of the phenol sulphotransferase inhibitor DCNP abolishes these phenotypes and suppresses the sensitivity of SETD2-deficient cancer cells, identifying SULT1A1 activity to be critical in mediating the potent cytotoxicity of RITA against SETD2-deficient cells. These findings define a novel therapeutic strategy for targeting the loss of SETD2 in cancer. Significance StatementThe histone-modifying enzyme SETD2 has emerged as an important tumour suppressor in a number of different cancer types, identifying it as a promising therapeutic target. The concept of synthetic lethality, a genetic interaction in which the simultaneous loss of two genes or pathways that regulate a common essential process renders the cell nonviable, is a valuable tool for killing cancer cells that have known mutations. In this study, we conducted a synthetic lethality screen for compounds that specifically target SETD2-deficient cancer cells. The top hit, a compound called RITA, reduces cell viability and induces cell death only in the context of SETD2 loss, thereby highlighting a potential novel therapeutic strategy for treating SETD2-deficient cancers.

cell biology↗

bioRxiv2020

Persistent broken chromosome inheritance drives genome instability

Persistent DNA damage arising from unrepaired broken chromosomes or telomere loss can promote DNA damage checkpoint adaptation, and cell cycle progression, thereby increasing cell survival but also genome instability. However, the nature and extent of such instability is unclear. We show, using Schizosaccharomyces pombe, that inherited broken chromosomes, arising from failed homologous recombination repair, are subject to cycles of segregation, DNA replication and extensive end-processing, termed here SERPent cycles, by daughter cells, over multiple generations. Following Chk1 loss these post-adaptive cycles continue until extensive processing through inverted repeats promotes annealing, fold-back inversion and a spectrum of chromosomal rearrangements, typically isochromosomes, or chromosome loss, in the resultant population. These findings explain how persistent DNA damage drives widespread genome instability, with implications for punctuated evolution, genetic disease and tumorigenesis. One Sentence SummaryReplication and processing of inherited broken chromosomes drives chromosomal instability.

molecular biology↗