Mice with a Unique Mutator Phenotype Allow Detection of Lymphoid Leukemia Tumor Suppressor Genes
Authors: Yin Mianmian , Baslan Timour , Freeland Amy , Matsukawa Toshihiro , Pruitt Steven C. , Aplan Peter D. 
Published: October 2019

Abstract
Mice that are homozygous for a hypomorphic allele of the DNA replication factor minichromosome maintenance protein 2 (designated Mcm2hypo) on a C57Bl/6 background are born viable and are healthy for the first 2 months of life. Despite the fact that this is a germline mutation, all mice develop a specific malignancy-- precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), beginning at 3 months of age. Copy number aberration (CNA) analysis showed that these pre-T LBL samples had 8-14 small (100-1000 kb) interstitial deletions per sample. Remarkably, all mice had two or more deletions, often bi-allelic, that encompassed genes known to be relevant for human pre-T LBL, including Pten, Cdkn1a, Tcf3, and Tcf12.
Mice that express a NUP98-HOXD13 (NHD13) transgene develop myelodysplastic syndrome, followed by leukemic transformation in the majority of mice. The acute leukemias that develop in the NHD13 mice are most commonly myeloid, less commonly T-cell, and, rarely, B-lineage.
In an effort to identify myeloid tumor suppressor genes, we crossed NHD13 transgene with Mcm2hypo mice (both on C57Bl/6 backgorund), reasoning that bi-allelic mutations of tumor suppressor genes in myeloid cells that collaborated with the NHD13 transgene could be selected due to a growth advantage in vivo. However, all Mcm2hypo:NHD13+ mice developed a pre-T LBL by 3 months of age, reflecting the highly penetrant nature of the Mcm2hypo phenotype, and none of the Mcm2hypo:NHD13+ mice developed myeloid leukemia. Surprisingly, approximately 30% of the Mcm2hypo:NHD13+ mice developed concurrent B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and pre-T LBL. In these animals, the thymus was typically infiltrated with pre-T LBL cells, whereas the bone marrow and spleen were infiltrated with BCP-ALL cells, characterized by Igh clonal VDJ rearrangement and CD19 staining. Parenchymal organs (lung, kidney, liver) were variably infiltrated with pre-T LBL, BCP-ALL, or both. CNA analysis showed that the pre-T LBL were characterized by short (100-1000 kb) deletions including Pten, CDkn1a, Tcf3, and Tcf12 deletions, similar to the Mcm2hypo pre-T LBL, whereas the BCP-ALL were characterized by homozygous or heterozygous deletions including Pax5 and a 400 kb region encompassing Cebpb and Ptpn1. There were no shared deletions present in both BCP-ALL and pre-T LBL from the same mouse, indicating that the BCP-ALL and pre-T LBL arose independently, and not from a common precursor. In addition to the Pax5 and Cebpb/Ptpn1 deletions, most of the BCP-ALL displayed a focal copy number gain of 400kb bounded by Abl1 and Nup214. We hypothesized that this copy number gain may lead to a Nup214-Abl1 fusion gene, via generation of an extrachromosomal episome or tandem duplication, as has been reported for BCP-ALL and pre-T LBL patients. RT-PCR confirmed that these mice expressed a Nup214-Abl1 fusion gene. Finally, in an attempt to determine if Mcm2hypo mice would develop non-T cell malignancy if they were protected from pre-T LBL, we crossed the Mcm2hypo allele onto an athymic nude (nu/nu; NU/J) background. Although this study is still in progress, Mcm2hypo:nu/nu mice have not developed pre-T LBL as of 5 months. However, crossing the Mcm2hypo:nu/nu with a NUP98-PHF23 (NP23) transgene generated Mcm2hypo:nu/nu:NP23+ mice; these mice have developed BCP-ALL similar to the Mcm2hypo:NHD13+ mice. In sum, these studies show that an Mcm2 "deletor" phenotype can induce gross chromosomal rearrangements, including both deletions and amplifications, that delete tumor suppressor genes (e.g. Pten) and activate proto-oncogenes (Abl1), in both T and B lymphocytes. We find it intriguing that both types of malignancy characterized in Mcm2hypo mice occur in cells (pre-B or pre-T) that are programmed to undergo DNA rearrangements, and tolerate DNA double strand breaks (DSBs). Ongoing studies are focused on determining the mechanism(s) that lead to these deletions, and whether the Mcm2 "deletor" phenotype can produce malignancy in non-lymphoid hematopoietic cells.

Disclosures
Aplan: NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.