Abstract
DNA replicative stress is associated with malignant transformation. Mini-chromosome maintenance 2 (hereafter Mcm2) is a component of the DNA helicase complex that plays a pivotal role in DNA replication. The Mcm2-7 complex is loaded onto chromatin during the G1-phase of the cell cycle and is required for initiation of DNA replication in the subsequent S-phase.
Mice with a Cre cassette inserted into the 3'UTR of the Mcm2 gene (designated Mcm2 Cre) were generated as a tool to assess Mcm2 expression. Unexpectedly, mice with two copies of this allele (Mcm2 Cre/Cre) had decreased expression of Mcm2 protein (~20-30% of the wild type (WT) expression level), likely due to decreased RNA stability caused by insertion of the Cre cassette. Surprisingly, all Mcm2 Cre/Cre mice developed a lethal pre-T lymphoblastic leukemia/lymphoma (pre-T LBL) with a median survival of under 3 months. These pre-T LBL were characterized by 10-20 interstitial deletions of approximately 50-1000 kb, often involving genes known to be mutated or deleted in human pre-T LBL, such as Notch1, Pten, Tcf3, and Cdkn2a. Thus, Mcm2 Cre/Cre mice display a novel mutator/deletor phenotype that results in malignant transformation.
Although the Mcm2 hypomorph is a germline defect, the malignancies we identified in Mcm2 Cre/Cre mice were restricted to thymocytes. We hypothesized that Mcm2 Cre/Cre mice were susceptible to non-thymocyte malignancies, but due to the early onset of pre-T LBL (within 4 months of age), these putative non-thymocyte malignancies did not have an opportunity to develop. We transplanted Mcm2 Cre/Cre Lin-Sca-1+Kit+ (LSK) hematopoietic stem/progenitor cells (HSPCs) isolated from 5-week-old Mcm2 Cre/Cre bone marrow into WT recipients, which led to marked anemia and thrombocytopenia without evidence of leukemic transformation at 5 months post-transplant. Consistent with this finding, bone marrow from 5-week-old Mcm2 Cre/Cre mice also showed decreased LSK and Lin-Sca-1-Kit+ (LK) cells compared to WT mice, suggesting that the bone marrow failure was uncovered by the more prolonged survival of Mcm2 Cre/Cre LSK recipients vs. non-transplanted Mcm2 Cre/Cre mice (5 mos. vs. <3 mos.) .
As an alternate approach to preventing pre-T LBL, we crossed the Mcm2 Cre allele onto a nude (nu/nu) background, as nude mice lack thymic epithelial cells, preventing T cell development. Mcm2 Cre/Cre nu/nu mice had markedly prolonged survival compared to Mcm2 Cre/Cre (median 296.5 vs. 80.5 days, respectively; P < 0.0001). Most of the Mcm2 Cre/Cre nu/nu mice succumbed to B-cell precursor acute lymphoblastic leukemia (BCP-ALL) within 10 months of life. To determine whether Mcm2 Cre/Cre nu/nu would develop acute myeloid leukemia (AML) if placed on myeloid leukemia sensitized background, we crossed Mcm2 Cre/Cre nu/nu mice with mice expressing a NUP98-PHF23 (NP23) transgene, as the NP23 fusion gene predisposes mice to develop AML. All Mcm2 Cre/Cre nu/nu NP23 mice died within 5 months of age, primarily due to BCP-ALL. Sparse whole-genome sequencing (WGS) was used to detect copy number aberration (CNA), and we identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Ikzf3, Il7r, and Bcor. Whole-exome sequencing identified recurrent mutations, Jak1/Jak3, Ptpn11, and Kras.
In an effort to identify 100-1000 kb interstitial deletions in non-hematopoietic tissue, we used sparse WGS to identify CNA in single-cell cloned mouse embryo fibroblasts (MEF) from Mcm2 Cre/Cre, Mcm2 Cre/Wt, and Mcm2 Wt/Wt mice. Very few CNAs were identified in any of the MEFs. We conclude that this unique deletor phenotype found in Mcm2 Cre/Cre mice is a powerful tool for the identification of tumor suppressor genes in lymphoid leukemia, and that B- and T- lymphocytes are uniquely susceptible to this deletor phenotype.
No relevant conflicts of interest to declare.
