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The Novel Immunocompetent Eµ-SOX11CCND1 Mouse Model Molecularly and Phenotypically Mimics Human Mantle Cell Lymphoma

ABSTRACT: Mantle Cell Lymphoma (MCL) is an incurable blood cancer affecting older adults with a median survival of 80 months. There is a great need for novel therapies and immunocompetent pre-clinical models that effectively mimic the tumor micro-environment (TME). Most MCL murine models are patient derived xenografts (PDXs), which are immunocompromised, making them suboptimal to test immunotherapies. Previous attempts at an immunocompetent model were less than optimal with limited pathology, narrow phenotype, or the use of genes not implicated in MCL. Here, we present a novel transgenic, immunocompetent mouse model for MCL by expressing two key MCL genes, SRY-Box Transcription Factor 11 (SOX11), and Cyclin D1 (CCND1) under the Eµ enhancer in C57BL/6 mice. The resulting double transgenic mice showed lethal expansion of B1a (CD5+CD19+CD23-) sub-B cell population in lymphatic organs, as well as the liver, lung, and kidney. We subsequently show this population can further adoptively transferred to healthy mice, which develop aggressive MCL. Passage one was profiled and found to express key human MCL driver genes, as well as cell surface marks, including the immune suppressive PD-1 and PDL-1. This model is resistant to ibrutinib and venetoclax, medications currently used for MCL, but highly responsive to a protein arginine methyltransferase 5 (PRMT5) inhibitor (OS 18 vs. 67 days p<0.01), supporting our previous work showing PRMT5 as a promising target for MCL. This study provides the pathological and genetic characterization needed to utilize the Eµ-SOX11CCND1 model of MCL to improve the preclinical research of immunotherapeutic and immunomodulatory agents.

ORGANISM(S): Mus musculus

PROVIDER: GSE281125 | GEO | 2025/07/25

REPOSITORIES: GEO

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Project description:Purpose: Karyotype screening in patients with chronic lymphocytic leukemia (CLL) has identified a rare chromosome translocation, t(X;14)(q28;q32), placing the mature T cell proliferation 1 (MTCP1) gene under transcriptional control of the immunoglobulin (IGH) locus. To establish the role of MTCP1 as a driving gene in CLL, we generated a novel mouse model (Eµ-MTCP1) which recapitulates overexpression of MTCP1 in murine immature and mature B cells. Eµ-MTCP1 mice developed a CLL-like disease found in the blood, spleen, lymph nodes, and other tissues. As the MTCP1 protein shares significant homology with the TCL1A oncoprotein, and the Eµ-TCL1 mouse model is widely used as an preclinical tool in CLL due to the development of a CLL-like disease in the blood, spleen, and other tissues, we aimed to evaluate the transcriptome between the CLL-like disease of Eµ-MTCP1 and Eµ-TCL1 mice. Methods: Transgenic Eµ-MTCP1 mice were generated on a C57BL/6NTac background at The Ohio State University Comprehensive Cancer Center’s Transgenic Mouse Facility via pronuclear injection of linear constructs derived from a plasmid vector encoding murine VH promoter-IgH-Eμ enhancer elements followed by human cDNA encoding the p13 kDa MTCP1 protein. Eµ-MTCP1 and Eµ-TCL1 mice were aged and monitored for development of CLL-like disease my monthly flow cytometry assessment of the blood. Upon reaching significant accumulation in the blood (>60%), mice were randomly chosen for euthanization and cell populations were isolated from the spleen according to previously described protocols. From this suspension, B cells were isolated using EasySepTM mouse pan B cell isolation kit (STEMCELL Technologies; Cat #19844). Cell pellets were captured and washed in PBS on ice prior to resuspension in TRIzol reagent and stored at -80°. Total RNA was isolated from TRIzol suspensions using a chloroform/ethanol extraction method and quantified via Qubit RNA HS Assay kit (Invitrogen). The Clontech SMARTer v4 kit (Takara Bio USA, Inc.) was used for global preamplification. Illumina sequencing libraries were derived from the resultant cDNA using the Illumina Nextera XT DNA Library Prep Kit following manufacturer’s instructions. RNA-sequencing libraries were prepared with the Illumina Tru-Seq stranded kit and sequenced on a Hiseq 4000 targeting 40x10E6 fragments per sample. Transcript-level abundances were estimated using Salmon with the genocode mouse release 23, imported using tximport, with normalization and differential expression computed with DESeq2. Data processing was performed according to the CLEAR workflow, which identifies reliably quantifiable transcripts in low-input RNA-seq for differentially expressed gene (DEG) transcripts using gene coverage profiles. MiXCR (v3.0.5) was used with default parameters except the rnaseq alignment was replaced with kaligner2 to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of unique CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. MiXCR then generates a list of unique CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. To verify expression of the human MTCP1 and TCL1 transgene (hMTCP1 & hTCL1) in mice, transcript level abundances were estimated using Salmon with a modified gencode mouse reference that contained sequences from human MTCP1 and human TCL1 genes extracted from the grch38 human reference. Results & Conclusion: Across splenic B cells isolated from both Eµ-MTCP1 and Eµ-TCL1 mice there was prominent usage of distinct heavy chain BCR gene loci in each mouse analyzed. In contrast, wildtype mice exhibited high degree of variability in heavy chain V gene usage without dominant emergence of any gene, indicative of a heterogeneous mixture of splenic B cells. Similar patterns suggesting clonal leukemic populations were observed by kappa and lamba light chain usage. The complexity and aggressive nature of human CLL is often stratified by IGHV mutation status of the malignant B cell population. The most abundant tumorigenic clones isolated from spleens of Eµ-MTCP1 and Eµ-TCL1 mice both exhibited low mutational burden in IGHV, well below the threshold for classification as “mutated." The low mutational burden in this region is consistent with the aggressive, IGHV-unmutated, subtype of human CLL. Moreover, we aimed to determine the overall transcriptional profile of these monoclonal tumor cells and to establish relation to that of the CLL-like cells in Eµ-TCL1 mice. Principal component analysis of the global transcription profile revealed distinct clustering of Eµ-MTCP1 and Eµ-TCL1 tumor cell transcriptomes along PC2, with significant segregation from wildtype littermates across PC1. Analysis of the overall gene expression pattern between Eµ-MTCP1 and Eµ-TCL1 transgenic strains revealed a significant overlap in genes most variable from B cells of wildtype animals. A considerable degree of overlap in gene expression was noted when directly comparing Eµ-MTCP1 and Eµ-TCL1 expression profiles, with a total of only 92 of 27,054 analyzed genes having significant variation (p<0.001) from one transgenic model to the other.

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The Novel Immunocompetent Eµ-SOX11CCND1 Mouse Model Molecularly and Phenotypically Mimics Human Mantle Cell Lymphoma

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