Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal.

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Of the 1352 samples in the cohort, which were provided by multiple collaborators, four samples of breast tissue for which DNA was analyzed were a part of another study. Since these samples did not go through the same rigorous screening process, including pathology review and extraction of DNA from paraffin tissues, that was applied to all other samples, the authors are less confident in their exact identity and have decided to retract them from the study. This does not change the message of the paper, but results in changes in and in one pie chart in Figure 2E, that for breast cancer. The corrected figure parts are below. In addition, the supplemental material containing sample information has been updated online.

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In urothelial bladder cancer (UBC), risk stratification remains an important unmet need. Limitless self‐renewal, governed by TERT expression and telomerase activation, is crucial for cancer progression. Thus, telomerase activation through the interplay of mutations ( TERT p Mut ) and epigenetic alterations in the TERT promoter may provide further insight into UBC behavior. Here, we investigated the combined effect of TERT p Mut and the TERT Hypermethylated Oncological Region (THOR) status on telomerase activation and patient outcome in a UBC international cohort ( n = 237). We verified that TER Tp Mut were frequent (76.8%) and present in all stages and grades of UBC. Hypermethylation of THOR was associated with higher TERT expression and higher‐risk disease in nonmuscle invasive bladder cancers (NMIBC). TERT p Mut alone predicted disease recurrence (HR: 3.18, 95%CI 1.84 to 5.51, p < 0.0001) but not progression in NMIBC. Combined THOR high / TER Tp Mut increased the risk of disease recurrence (HR 5.12, p < 0.0001) and progression (HR 3.92, p = 0.025). Increased THOR hypermethylation doubled the risk of stage progression of both TERT p wt and TERT p Mut NMIBC. These results highlight that both mechanisms are common and coexist in bladder cancer and while TERT p Mut is an early event in bladder carcinogenesis THOR hypermethylation is a dynamic process that contributes to disease progression. While the absence of alterations comprises an extremely indolent phenotype, the combined genetic and epigenetic alterations of TERT bring additional prognostic value in NMIBC and provide a novel insight into telomere biology in cancer.

Although replication repair deficiency, either by mismatch repair deficiency (MMRD) and/or loss of DNA polymerase proofreading, can cause hypermutation in cancer, microsatellite instability (MSI) is considered a hallmark of MMRD alone. By genome-wide analysis of tumors with germline and somatic deficiencies in replication repair, we reveal a novel association between loss of polymerase proofreading and MSI, especially when both components are lost. Analysis of indels in microsatellites (MS-indels) identified five distinct signatures (MS-sigs). MMRD MS-sigs are dominated by multibase losses, whereas mutant-polymerase MS-sigs contain primarily single-base gains. MS deletions in MMRD tumors depend on the original size of the MS and converge to a preferred length, providing mechanistic insight. Finally, we demonstrate that MS-sigs can be a powerful clinical tool for managing individuals with germline MMRD and replication repair–deficient cancers, as they can detect the replication repair deficiency in normal cells and predict their response to immunotherapy. Significance: Exome- and genome-wide MSI analysis reveals novel signatures that are uniquely attributed to mismatch repair and DNA polymerase. This provides new mechanistic insight into MS maintenance and can be applied clinically for diagnosis of replication repair deficiency and immunotherapy response prediction. This article is highlighted in the In This Issue feature, p. 995

DNA repair is essential for maintaining genomic stability, and defects in this process significantly increase the risk of cancer. Clear-cell renal cell carcinoma (CCRCC) caused by inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is characterized by high genomic instability. However, the molecular mechanism underlying the association between the loss of VHL and genomic instability remains unclear. Here, we show that suppressor of cytokine signaling 1 (SOCS1) promotes nuclear redistribution and K63-ubiquitylation of VHL in response to DNA double-strand breaks (DSBs). Loss of VHL or VHL mutations that compromise its K63-ubiquitylation attenuates the DNA-damage response (DDR), resulting in decreased homologous recombination repair and persistence of DSBs. These results identify VHL as a component of the DDR network, inactivation of which contributes to the genomic instability associated with CCRCC.

Fanconi anemia and Bloom syndrome are genomic instability syndromes caused by mutations in proteins that participate in overlapping DNA repair and replication pathways. Here, we show that the monoubiquitinated form of the Fanconi Anemia protein FANCD2 acts in opposition to the BLM DNA helicase to restrain telomere replication and recombination in human cells that utilize the Alternative Lengthening of Telomeres (ALT) pathway. ALT relies on exchanges of telomeric DNA to maintain telomeres, a process that we show FANCD2 suppresses. Depletion of FANCD2 results in a hyper-ALT phenotype, including an increase in extrachromosomal telomeric repeat DNAs, putative recombinational byproducts that we show exist as intertwined complexes forming the nucleic acid component of ALT-associated PML bodies. Increases in telomeric DNA are suppressed by loss of BLM but not RAD51, occur without parallel upregulation of shelterin proteins TRF1 and TRF2, and are associated with increased frequencies of deprotected and fragile telomeres. Inactivation of the FA pathway does not trigger ALT, as FANCD2 depleted telomerase positive cells do not acquire ALT-like phenotypes. We observe frequent fragile telomeres in ALT cells, suggesting that telomere sequences are prone to replication problems. We propose that, in ALT cells, FANCD2 promotes intramolecular resolution of stalled replication forks in telomeric DNA while BLM facilitates their resection and subsequent involvement in the intermolecular exchanges that drive ALT.