Chu, Liang

Abstract KaLwRij/C57Bl (KaLwRij, “Kal-ridge”) is a spontaneous mutant mouse strain predisposed to monoclonal gammopathy of uncertain significance (MGUS) and parallels many features of human pathology including progression to multiple myeloma (MM). In African Americans, an increased prevalence of MM is due to an increased risk of MGUS development (rather than increased rate of progression from MGUS) and is likely therefore mediated by germline MGUS risk alleles. In both humans and mice, the genetic basis for MGUS/MM risk is unknown. We sought to identify the genetic basis for MGUS/MM risk in KaLwRij mice to shed light on genes or pathways that might mediate risk in humans. We employed a novel integrative computational analysis, combining KaLwRij/B6 modified haplotype block analysis and low-power human MM/control single nucleotide polymorphism (SNP) data. We used high-density SNP arrays to establish genetic distances of KaLwRij from 11 another diverse inbred strains and confirmed close homology to parent C57Bl/6. Haplotype block analysis identified genetic loci that diverged from C57Bl/6 and included over 500 genes. We filtered the haplotype list using previously published RNA expression data from the bone marrow of C57Bl/6 and KaLwRij mice, to identify 71 differentially expressed genes. As a final filter, we used SNP data from a genome wide association study (GWAS) of human Caucasian MM case and normal volunteer samples (∼300 each) to identify SNPs differentially represented in MM patients. Combining these sets yielded four (4) candidate genes. Our top candidate, Samsn1 (Hacs1), is a negative regulator of B-cell activation and interestingly, was first identified as a gene differentially expressed in MM. Strikingly, we found that KaLwRij mice had a complete germline deletion of the Samsn1 gene. Samsn1 is expressed within the hematopoetic compartment, including B-cells, macrophages, and osteoclasts (OC), key cell types that participate in MM pathology. Consistent with the reported inhibitory function of SAMSN1, KaLwRij had enhanced naïve B-cell response and constitutive Lyn phosphorylation. Further, we found that KaLwRij macrophages and OC also have constitutive Lyn activation consistent activation of cytokine signaling pathways in stromal cells. Absence of Samsn1 may lead to unregulated B-cell and macrophage/OC activation, priming both a pro-tumor bone microenvironment and B-cells for development of MGUS and predisposition to MM. Our approach focused on germline loci rather than malignant cell genetic alterations, advancing our understanding of host predisposition. Identification of the gene or genes predisposing patients to MGUS/MM will provide novel targets for identifying at-risk individuals and will potentially allow the develop of preventative therapeutics for MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Multiple myeloma (MM) is a malignancy of plasma B-cells that is invariably preceded by monoclonal gammopathy of undetermined significance (MGUS), a pre-neoplastic plasma cell proliferative disorder. C57Bl/KaLwRij (KaLwRij) is a spontaneous inbred mouse strain predisposed to benign idiopathic paraproteinemia (BIP), a plasma cell dyscrasia that parallels many features of human pathology including progression to myeloma. In both humans and mice, the genetic basis for MGUS/MM risk is unknown. We sought to identify the genetic basis for MGUS/MM risk in KaLwRij mice to shed light on genes or pathways that mediate disease risk in humans. Using an integrative approach, we combined KaLwRij x B6 haplotype analysis (418 genes) and a MM patient association study (180 genes) to identify five candidate genes likely to contribute to both murine BIP-susceptibility and human MM risk. Surprisingly, we found KaLwRij mice had homozygous germline deletion of one of the five candidate genes, Samsn1, a putative negative regulator of B-cell activation. Consistent with the reported inhibitory function of SAMSN1 in B-cells, KaLwRij mice had enhanced naïve B-cell response in vitro following stimulation with IL4 and LPS and in vivo following immunization. We found that re-expression of Samsn1 in KaLwRij-derived myeloma cell line 5TGM1 decreased MM tumor growth in vitro, suggesting that loss of Samsn1 contributes to tumor growth in part through a cell intrinsic mechanism. Importantly, germline risk alleles may also influence cell types beyond the pre-malignant B-cells. It is established that the host environment also plays a critical role in supporting myeloma growth in KaLwRij mice. We found that Samsn1 is highly expressed in macrophages, but not in other MM-supportive cell types including osteoclasts and bone marrow stromal cells. Myeloma-associated macrophages play critical roles in supporting tumor growth and are broadly classified as anti-tumor M1 macrophages and tumor-promoting M2 macrophages. Microarray analysis of macrophages from B6 and KaLwRij mice prior to onset of BIP revealed three differentially expressed genes, but only one, Samsn1, is conserved in the human genome. We found that both unpolararized primary peritoneal and MCSF cultured bone marrow macrophages from healthy KaLwRij mice had markedly elevated expression of pro-tumorigenic M2 macrophage markers FIZZ1 and YM1 compared to B6 controls, despite the absence of activating cytokines. To confirm that loss of Samsn1 influenced macrophage activation and M2 polarization, we obtained bone marrow macrophages from mice with a targeted deletion of Samsn1 and found that unpolarized Samsn1-/- cells had similarly increased pro-tumor M2 macrophage marker expression. To evaluate the biologic significance of Samsn1 loss in macrophages on MM tumor growth, we established 5TGM1 MM tumors and injected M2 polarized macrophages from B6, KaLwRij, or Samsn1-/- mice. We found that MM tumor burden was markedly enhanced by the addition of a Samsn1-/- or KaLwRij macrophages compared to wild type macrophages. Thus, we found that loss of Samsn1 in macrophages enhances M2 polarization, prior to MGUS/MM development and can promote established myeloma tumor growth. Together, this data suggests that Samsn1 is a gene contributing to BIP-susceptibility in KaLwRij mice through mutually exclusive functions in multiple cell types including B-cells and macrophages. It is likely that SAMSN1 and its pathway members contribute to human MM progression, and may represent a point for preventative therapeutic intervention. Disclosures No relevant conflicts of interest to declare.