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DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor. However, a p53-independent function of Mdmx that impacts genome stability has been described, but this function is not well understood. In the present study, we determined that of the 13 different cancer types evaluated, 6-90% of those that had elevated levels of Mdmx had concurrent inactivation (mutated or deleted) of p53. We show elevated levels of Mdmx-inhibited double-strand DNA break repair and induced chromosome and chromatid breaks independent of p53, leading to genome instability. Mdmx impaired early DNA damage-response signaling, such as phosphorylation of the serine/threonine-glutamine motif, mediated by the ATM kinase. Moreover, we identified Mdmx associated with Nbs1 of the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and this association increased upon DNA damage and was detected at chromatin. Elevated Mdmx levels also increased cellular transformation in a p53-independent manner. Unexpectedly, all Mdmx-mediated phenotypes also occurred in cells lacking Mdm2 and were independent of the Mdm2-binding domain (RING) of Mdmx. Therefore, Mdmx-mediated inhibition of the DNA damage response resulted in delayed DNA repair and increased genome instability and transformation independent of p53 and Mdm2. Our results reveal a novel p53- and Mdm2-independent oncogenic function of Mdmx that provides new insight into the many cancers that overexpress Mdmx.

Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Emu-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis.