Mcm2 hypomorph leads to acute leukemia or hematopoietic stem cell failure, dependent on genetic context

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2Cre ) have decreased Mcm2 expression and invariably develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), due to 100-1000 kb deletions involving important tumor suppressor genes. To determine whether mice that were protected from pre-T LBL would develop non-T-cell malignancies, we used two approaches. Mice engrafted with Mcm2Cre/Cre Lin- Sca-1+ Kit+ hematopoietic stem/progenitor cells did not develop hematologic malignancy; however, these mice died of hematopoietic stem cell failure by 6 months of age. Placing the Mcm2Cre allele onto an athymic nu/nu background completely prevented pre-T LBL and extended survival of these mice three-fold (median 296.5 vs. 80.5 days). Ultimately, most Mcm2Cre/Cre ;nu/nu mice developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Nf1, Ikzf3, and Bcor. Moreover, whole-exome sequencing identified recurrent mutations of genes known to be involved in BCP-ALL progression, such as Jak1/Jak3, Ptpn11, and Kras. These findings demonstrate that an Mcm2Cre/Cre hypomorph can induce hematopoietic dysfunction via hematopoietic stem cell failure as well as a "deletor" phenotype affecting known or suspected tumor suppressor genes

Mice with a Unique Mutator Phenotype Allow Detection of Lymphoid Leukemia Tumor Suppressor Genes

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 &amp; Royalties: NUP98-HOXD13 mice. </jats:sec

MicroRNA-224 Is Involved in Transforming Growth Factor-β-Mediated Mouse Granulosa Cell Proliferation and Granulosa Cell Function by Targeting Smad4

Abstract Many members of the TGF-β superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF-β1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF-β-mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF-β1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFβ superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF-β1/Smads pathway. The ectopic expression of miR-224 can enhance TGF-β1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-β1. In addition, both miR-224 and TGF-β1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-β1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders.</jats:p

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model.

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina

Correlation of autoantigen-specific Treg frequency with development of spontaneous organ-specific autoimmunity in a mouse model of uveitis

Abstract About 50% of AireGW/+Lyn−/− mice developed autoimmune uveitis, and this was accompanied by the expansion of interphotoreceptor retinoid-binding protein (IRBP) specific CD4+T cells. Moreover, the response to IRBP was necessary for disease initiation, as deletion of IRBP prevented uveitis. Mass cytometry (CyTOF) analysis of CD4+T cells in the retina of the mice with uveitis identified activated T cell subsets characterized as Ly6Chi effectors, PD-1hi effectors, and Treg. To study cellular mechanisms that promoted development of uveitis, we did single-cell RNA sequencing (scRNA-seq) of eye-draining lymph node (LN) CD4+T cells that recognize the P2 epitope of IRBP (amino acid 271–290). Uniform manifold approximation and projection (UMAP) analysis showed that these CD4+T cells included distinct subsets that were Ly6c1hi , Pdcd1hi and Treg, with the Treg population being overrepresented in the mice without disease. Pseudotime analysis indicated that the Ly6c1hi T cell subset represented an earlier stage in activation, whereas the Pdcd1hi subset represented a later stage. Similar analysis of P2–specific CD4+T cells from the retina of mice with uveitis identified 6 subsets of P2-specific T cells, and was largely consistent with the CyTOF analysis of total CD4+ T cells from the retina. Pseudotime analysis indicated that toward the earlier time were cells characterized by being proliferative (Mki67hi), followed by a Lag3hi subset and at other end of the pseudotime axis by a Lag3hi / Pdcd1hi subset. Our results are consistent with the hypothesis that the fraction of Treg within the IRBP-specific CD4+ T cells in the draining LN determines whether these genetically susceptible mice develop eye-specific autoimmunity or are protected.</jats:p

Engineered Bcor Mutations Lead to Acute Lymphoblastic Leukemia of Progenitor B-1 Lymphocyte Origin in a Sensitized Background

Abstract Chromosomal translocations resulting in NUP98 fusion genes have been associated with a wide spectrum of hematologic malignancies, including MDS, AML, T-ALL, and B cell precursor (BCP) ALL. Based on gene expression profiles and murine transplantation experiments, it is thought that NUP98 fusions can confer aberrant self-renewal potential to hematopoietic cells. Approximately 90% of mice that express a NUP98-PHF23 (NP23) fusion in the hematopoietic compartment, under the control of Vav1 regulatory elements develop AML and/or T-ALL. However, approximately 10% of NP23 mice develop an aggressive acute lymphoblastic leukemia of B1-lymphocyte progenitor origin (pro B-1 ALL). Whole exome sequencing demonstrated that all NP23 pro-B1-ALL had acquired somatic frameshift mutations of the BCL6 co-repressor (Bcor) gene, and most had acquired mutations in the Jak/Stat pathway. To determine whether experimentally engineered Bcor mutations would lead to pro B-1 ALL, we used CRISPR-Cas9 to introduce Bcor indel mutations into NP23 hematopoietic stem and progenitor cells through the use of Bcor single guide RNAs (Bcor sgRNA). Recipient mice transplanted with NP23 bone marrow (BM) or fetal liver (FL) cells that had been transduced with a Bcor sgRNA developed pro B-1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. In addition, similar to some human BCP ALL, the Bcor sgRNA/NP23 murine pro B-1 ALL had acquired somatic mutations in Jak kinase genes. A distinct subset of pediatric BCP ALL are characterized by rearrangement and overexpression of the CRLF2 gene (designated CRLF2r); the CRLF2 gene is the receptor for thymic stromal lymphopoietin (TSLP), a cytokine that plays a role in normal progenitor B1 cell development. The NP23 pro-B1 ALL are similar to CRLF2r BCP ALL in terms of a preferential V heavy chain (VH) usage, gene expression profile, and propensity for acquired JAK/STAT pathways mutations. JAK inhibitors (ruxolitinib and tofacitinib) induced apoptosis and inhibited the growth of pro B-1 ALL cell lines established from Bcor sgRNA/NP23 recipients, at clinically achievable concentrations (10-100 nM). Taken together, these findings demonstrate that a CRISPR-induced Bcor frameshift collaborates with an NP23 transgene to predispose B-1 progenitors to leukemic transformation. These two events are unlikely to be sufficient for leukemic transformation, as we detected spontaneous Jak pathway mutations that were required for continued growth of the leukemic cells. This constellation of mutations (NP23 expression leading to increased stem cell self-renewal, Bcor frameshift leading to impaired B cell differentiation, and Jak pathway mutations leading to dysregulated proliferation) is similar to that seen in human BCP ALL patients, and suggests that the NP23/Bcor mutant mice and cell lines will be a useful model for human pro-B1 ALL. Disclosures Aplan: NIH Office of Technolgy Transfer: Employment, Patents &amp; Royalties: NUP98-HOXD13 mice. </jats:sec

Autoantigen specific T-cell receptor induces organ-specific autoimmunity by escaping T cell negative selection

Abstract Previously we reported a new model of spontaneous autoimmune uveitis in which mice with a hypomorphic mutation of Aire (AireGW/+) are combined with deletion of Lyn. The key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+Lyn−/−IRBP−/− mice failed to develop uveitis. We performed single-cell RNA-seq with TCR sequencing for IRBP 271–290 (P2)-specific CD4+ T cells from eye-draining lymph nodes (LN) of these mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCRs was expressed in thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR. In addition, we generated TCR transgenic mice with this TCR clonotype. The TCR transgenic mice spontaneously developed uveitis with expansion of P2-specific CD4+ T cells. Combined with AireGW/+ or IRBP−/−, there were more P2-specific CD4+ T cells in the thymus of TCR transgenic mice. Thus, suboptimal Aire function in AireGW/+Lyn−/− mice results in a partial defect in negative selection of T cells recognizing IRBP in the thymus, promoting development of uveitis. This disease is accelerated in mice expressing a TCR transgene recognizing IRBP. Supported by  AI138479</jats:p

Engineered Bcor mutations lead to acute leukemia of progenitor B-1 lymphocyte origin in a sensitized background

Abstract Approximately 10% of NUP98-PHF23 (NP23) mice develop an aggressive acute lymphoblastic leukemia of B-1 lymphocyte progenitor origin (pro-B1 ALL), accompanied by somatic frameshift mutations of the BCL6 interacting corepressor (Bcor) gene, most commonly within a 9-bp “hotspot” in Bcor exon 8. To determine whether experimentally engineered Bcor mutations would lead to pro-B1 ALL, we used clustered, regularly interspaced, short palindromic repeats–associated protein 9 to introduce a Bcor frameshift mutation into NP23 hematopoietic stem and progenitor cells through the use of Bcor small guide RNAs (Bcor sgRNAs). Recipient mice transplanted with NP23 bone marrow or fetal liver cells that had been transduced with a Bcor sgRNA developed pro-B1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. Similar to a subset of human B-cell precursor ALL, the murine pro-B1 ALL had acquired somatic mutations in Jak kinase genes. JAK inhibitors (ruxolitinib and tofacitinib) inhibited the growth of pro-B1 ALL cell lines established from Bcor sgRNA/NP23 recipients at clinically achievable concentrations (100 nM). Our results demonstrate that Bcor mutations collaborate with NP23 to induce pro-B1 ALL, and that JAK inhibitors are potential therapies for pro-B1 ALL.</jats:p