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703 Smarca4 (Brg1) Controls Germinal Center B-Cell Development and Is a Haploinsufficient Tumor Suppressor in Lymphoma

Program: Oral and Poster Abstracts
Type: Oral
Session: 603. Lymphoid Oncogenesis: Basic: Mechanisms in Lymphomagenesis
Hematology Disease Topics & Pathways:
Research, Fundamental Science, immunology, Biological Processes, molecular biology
Monday, December 12, 2022: 10:30 AM

Qing Deng, PhD1*, Cem Meydan, PhD2*, Priya Lakra, PhD3*, Darko Barisic4*, Christopher R Chin5*, Estela Rojas-Neira6*, Vida Ravanmehr6*, Jared Henderson, PhD1*, Panhong Gou7*, Nader Kim El-Mallawany, MD8*, Carl Allen, MD, PhD9*, Branko Cuglievan, MD10*, Francisco Vega, MD, PhD11, Christopher E Mason, PhD12*, Ari Melnick, MD13 and Michael R. Green, PhD14

1Department of Lymphoma & Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX
2Department of Medicine, Division of Hematology & Medical Oncology, Division of Hematology/Oncology, Dept. of Medicine, Weill Cornell Medical College, New York, NY
3Department of Lymphoma & Myeloma, University of Texas MD Anderson Cancer Center, Houston, TX
4Weill Cornell Medicine, New York, NY
5Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY
6University of Texas MD Anderson Cancer Center, Houston, TX
7The University of Texas MD Anderson Cancer Center, Houston, TX
8Section of Hematology/Oncology, Baylor College of Medicine/Texas Children's Hospital, Houston, TX
9Department of Pediatrics, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX
10Division of Pediatric Hematology Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX
11Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX
12Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY
13Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY
14Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Friendswood, TX

SMARCA4 encodes one of two mutually exclusive ATPases present in BAF (aka SWI/SNF) chromatin remodeling complexes. SMARCA4 is dynamically expressed during normal B-cell development, with high expression in germinal center B (GCB)-cells, and is targeted by heterozygous inactivating mutations in GCB-derived lymphomas including 34% of pediatric Burkitt lymphoma (BL), 17% of adult BL, 4% of GCB-DLBCL. However, the functional role of SMARCA4 in GCB cell development and malignant transformation has not been thoroughly investigated.

We generated a novel floxed Smarca4 transgenic mouse model and crossed to the Aid-Cre line for GC-specific conditional knock-out (cKO). Following immunization with sheep red blood cells (SRBCs) to induce a GC reaction, Smarca4+/- mice had a significant increase in GCB-cell abundance compared to Smarca4+/+ littermates (p<0.001) due to both an increased average GC size and number. Notably, Smarca4-/- mice displayed a significantly reduced frequency of GC B-cells (p<0.001) and sorting of residual cells revealed retention of 1 or more copies of the floxed allele, suggesting that biallelic cKO of Smarca4 is lethal to GCB cells. We therefore focused subsequent studies on comparisons between Smarca4+/- and Smarca4+/+ mice. Further analysis revealed significant polarization towards a centroblastic phenotype in Smarca4+/- mice (p<0.001), in line with the centroblastic morphology of human BL. Analysis of competitive advantage with the same GC using bone marrow chimera models showed a significant advantage of Smarca4+/- GCB cells compared to Smarca4+/+, associated with a significant increase in proliferation measured by Edu incorporation and reduction of apoptosis measured by Annexin-B. We extended upon this using single cell RNA-seq (scRNA-seq) of GCB cells from Smarca4+/- and Smarca4+/+ mice, which confirmed an increased centroblastic polarization of Smarca4+/- GCB cells and identified a significant increase in light zone to dark zone recycling cells that may underly this polarization. We validated this using the Myc-GFP allele, which confirmed increased Myc+ recycling cells in GCs from Smarca4+/- mice compared to Smarca4-/- mice.

BAF complexes drive ATP-dependent mobilization and eviction of nucleosomes to convert chromatin from inactive to active states. Brg1 deficiency may therefore alter distinct BAF complexes and transcription factor programs. We therefore explored the role of Smarca4 in regulating chromatin accessibility and gene expression using ATAC-seq and RNA-seq in Smarca4+/- and Smarca4+/+ murine GCB-cells. Supervised analysis revealed predominant loss of chromatin accessibility associated with repression of nearby genes. Smarca4+/- cells showed significant loss of accessibility of genes upregulated in centrocytes, and a preferential loss of NF-ĸB target genes (GSEA FDR<0.01).

MYC translocations are pathognomonic for BL, in which SMARCA4 mutations are most frequent. We therefore crossed our Smarca4 cKO mice with a cre-inducible Myc knock-in (Myc-KI) allele to mimic combined Smarca4 loss-of-function and Myc activation. The addition of Myc-KI did not lead to a significant increase in GCB cell frequency following a single SRBC immunization. However, serial re-challenge of mice with SRBCs led to lymphadenopathy, splenomegaly and infiltration of highly proliferative B-cells into the major organs (lung, liver, and kidney) in Smarca4+/- + Myc-KI mice following 3 immunizations that was not observed with Smarca4+/- or Myc-KI alleles alone. Furthermore, long term follow-up of serially-challenged mice showed a significant reduction in overall survival in Smarca4+/- + Myc-KI mice compared to Smarca4+/- or Myc-KI mice (p=0.010).

Collectively, these data demonstrate a competitive advantage of GCB cells with monoallelic Smarca4 cKO, and a centroblastic GC polarization driven in part by increased recycling. Mechanistically, this is associated with loss of chromatin accessibility and gene expression of genes that are expressed in centrocytes, including NFkB target genes. Monoallelic Smarca4 cKO cooperates with Myc over-expression to drive expansion and extranodal dissemination of GCB cells following serial re-challenge, leading to reduced survival. These data provide the first functional and mechanistic insight into the role of SMARCA4 in GCB cell development and lymphomagenesis.

Disclosures: Vega: Geron Corporation: Research Funding; Allogene: Research Funding; Crisp Therapeutics: Research Funding. Mason: Tempus Labs: Membership on an entity's Board of Directors or advisory committees. Melnick: Astra Zeneca: Consultancy, Research Funding; Canaan Management, LLC: Consultancy; Celgene Corporation: Consultancy; Constellation Pharmaceuticals: Consultancy, Other: Scientific Advisory Board; Daiichi Sankyo: Consultancy, Research Funding; Epizyme: Consultancy, Other: Scientific Advisory Board, Research Funding; Exo Therapeutics: Other: Scientific Advisory Board; Janssen: Research Funding; Janssen Global Advisory: Other: Scientific Advisory Board; KDAC Therapeutics: Current holder of stock options in a privately-held company; Treeline Biosciences: Consultancy; CASMA Therapeuctics: Consultancy. Green: Kite/Gilead: Research Funding; Tessa Therapeutics: Honoraria; Allogene: Research Funding; Abbvie: Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Monte Rosa Therapeutics: Honoraria; Sanofi: Research Funding; KDAc Therapeutics: Current holder of stock options in a privately-held company.

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