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Unlabelled: The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice). Nbs1(ΔB/ΔB) Atm(-/-) hematopoietic cells derived by crossing to vav(cre) were viable in vivo. Nbs1(ΔB/ΔB) Atm(-/-) (VAV) mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress., Implications: A novel mouse model was developed, by combining a vav(cre)-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis., (©2015 American Association for Cancer Research.)

Unlabelled: Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis, with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small-cell cancer to combined checkpoint kinase 1 (CHK1) inhibition and DNA-damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of CHK1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy in which checkpoint inhibition in combination with DNA-damaging chemotherapy is synthetically lethal in tumor cells but not normal cells with somatic mutations that impair Mre11 complex function., Significance: Strategies to effect deep and lasting responses to cancer therapy in patients with metastatic disease have remained difficult to attain, especially in early-phase clinical trials. Here, we present an in-depth genomic and functional genetic analysis identifying RAD50 hypomorphism as a contributing factor to a curative response to systemic combination therapy in a patient with recurrent, metastatic small-cell cancer., (©2014 American Association for Cancer Research.)

C57BL/6J mice carrying the Min allele of Adenomatous polyposis coli (Apc) develop numerous adenomas along the entire length of the intestine and consequently die at an early age. This short lifespan would prevent the accumulation of somatic genetic mutations or epigenetic alterations necessary for tumor progression. To overcome this limitation, we generated F(1) Apc(Min/+) hybrids by crossing C57BR/cdcJ and SWR/J females to C57BL/6J Apc(Min/+) males. These hybrids developed few intestinal tumors and often lived longer than 1 year. Many of the tumors (24-87%) were invasive adenocarcinomas, in which neoplastic tissue penetrated through the muscle wall into the mesentery. In a few cases (3%), lesions metastasized by extension to regional lymph nodes. The development of these familial cancers does not require chromosomal gains or losses, a high level of microsatellite instability, or the presence of Helicobacter. To test whether genetic instability might accelerate tumor progression, we generated Apc(Min/+) mice homozygous for the hypomorphic allele of the Nijmegen breakage syndrome gene (Nbs1(DeltaB)) and also treated Apc(Min/+) mice with a strong somatic mutagen. These imposed genetic instabilities did not reduce the time required for cancers to form nor increase the percentage of cancers nor drive progression to the point of distant metastasis. In summary, we have found that the Apc(Min/+) mouse model for familial intestinal cancer can develop frequent invasive cancers in the absence of overt genomic instability. Possible factors that promote invasion include age-dependent epigenetic changes, conservative somatic recombination, or direct effects of alleles in the F(1) hybrid genetic background.

The Mre11 complex promotes DNA double-strand break repair and regulates DNA damage signaling via activation of the ataxia-telangiectasia mutated (ATM) kinase. The hypermorphic Rad50(S) allele encodes a variant of Rad50, a member of the Mre11 complex. Cells expressing Rad50(S) experience constitutive ATM activation, which leads to precipitous apoptotic attrition in hematopoietic cells. In this study, we show that ATM activation by the Rad50S-containing Mre11 complex enhances the proliferation of LSK cells, a population consisting of hematopoietic stem cells and multipotent progenitor cells. In Rad50(S/S) mice, enhanced LSK proliferation triggers apoptotic attrition. This phenotype is mitigated when Rad50(S/S) is combined with mutations that alter either LSK cell quiescence (myeloid elf-1-like factor/ELF4-deficient mice) or hematopoietic differentiation (p21- and p27-deficient mice), indicating that the LSK population is a primary target of Rad50(S) pathology. We show that cells from Rad50(S/S) mice are hypersensitive to camptothecin, a topoisomerase I inhibitor that causes DNA damage primarily during DNA replication. On this basis, we propose that apoptotic attrition of Rad50(S/S) hematopoietic cells results from enhanced proliferation in the context of topoisomerase-associated DNA damage. Impairment of apoptosis in Rad50(S/S) mice promotes hematopoietic malignancy, suggesting that primitive hematopoietic cells serve as a reservoir of potentially oncogenic lesions in Rad50(S/S) mice. These data provide compelling evidence that the Mre11 complex plays a role in the metabolism of topoisomerase lesions in mammals, and further suggest that such lesions can accumulate in primitive hematopoietic cells and confer significant oncogenic potential.

The Mre11 complex undergoes dramatic relocalization in the nuclei of gamma-irradiated and replicating human cells. In this study, we examined Mre11 complex localization and chromatin association in synchronous cultures to examine the molecular determinants of relocalization. The data indicate that the complex is deposited on chromatin in an S phase-specific manner. Mre11 complex chromatin association in S phase was resistant to detergent extraction, in contrast to that in gamma-irradiated cells. The complex exhibits extensive colocalization with proliferating cell nuclear antigen throughout S phase, and chromatin loading is enhanced by replication fork stalling, suggesting that the replication fork is a site of Mre11 complex chromatin loading. This is supported by the observation that the complex localized to single-stranded DNA arising in hydroxyurea-treated cells. Although the Mre11 complex appears to function as a DNA damage sensor, limited colocalization with Brca1 or gamma-H2AX was observed, arguing that neither DNA damage nor gamma-H2AX is required for Mre11 complex chromatin loading. These data provide a potential molecular basis for promotion of sister chromatid association and recombination by the Mre11 complex as well as for ATM-Mre11 complex-dependent activation of cell cycle checkpoints.