MRNIP limits ssDNA gaps during replication stress.

Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

Identification of a class of human cancer germline genes with transcriptional silencing refractory to the hypomethylating drug 5-aza-2'-deoxycytidine.

Bona fide germline genes have expression restricted to the germ cells of the gonads. Testis-specific germline development-associated genes can become activated in cancer cells and can potentially drive the oncogenic process and serve as therapeutic/biomarker targets; such germline genes are referred to as cancer/testis genes. Many cancer/testis genes are silenced via hypermethylation of CpG islands in their associated transcriptional control regions and become activated upon treatment with DNA hypomethylating agents; such hypomethylation-induced activation of cancer/testis genes provides a potential combination approach to augment immunotherapeutics. Thus, understanding cancer/testis gene regulation is of increasing clinical importance. Previously studied cancer/testis gene activation has focused on X chromosome encoded cancer/testis genes. Here we find that a sub-set of non-X encoded cancer/testis genes are silenced in non-germline cells via a mechanism that is refractory to epigenetic dysregulation, including treatment with the hypomethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor tricostatin A. These findings formally indicate that there is a sub-group of the clinically important cancer/testis genes that are unlikely to be activated in clinical therapeutic approaches using hypomethylating agents and it indicates a unique transcriptional silencing mechanism for germline genes in non-germline cells that might provide a target mechanism for new clinical therapies.

The parathyroid hormone-responsive B1 gene is interrupted by a t(1;7)(q42;p15) breakpoint associated with Wilms' tumour.

Wilms' tumour (WT) has a diverse and complex molecular aetiology, with several different loci identified by cytogenetic and molecular analyses. One such locus is on chromosome 7p, where cytogenetic abnormalities and loss of heterozygosity (LOH) indicate the presence of a Wilms' tumour suppressor gene. In order to isolate a candidate gene for this locus, we have characterized the breakpoint regions at a novel constitutional chromosome translocation (t(1;7)(q42;p15)), found in a child with WT and skeletal abnormalities. We identified two genes that were interrupted by the translocation: the parathyroid hormone-responsive B1 gene (PTH-B1) at 7p and obscurin at 1q. With no evidence for LOH at 1q42, we focused on the characterization of PTH-B1. We detected novel alternately spliced isoforms of PTH-B1, which were expressed in a wide range of adult and foetal tissues. Importantly, expression of two isoforms were disrupted in the WT of the t(1;7) patient. We also identified an additional splice isoform expressed only in 7p LOH tumours. The disruption of PTH-B1 by the t(1;7), together with aberrant splicing in sporadic WTs, suggests that PTH-B1 is a candidate for the 7p Wilms' tumour suppressor gene.