Summary of histopathological diagnoses.

<p>Chc1L+/- and Chc1L-/- mice developed HS and HAL or HS co-occurring with BCL. Chc1L-/- mice developed more cases of HS, while Chc1L+/- mice had more diagnoses of HAL/HS+BCL than HS.</p

Chromosome Condensation 1-Like (<i>Chc1L</i>) Is a Novel Tumor Suppressor Involved in Development of Histiocyte-Rich Neoplasms

<div><p>Human chromosomal region 13q14 is a deletion hotspot in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. This region is believed to host multiple tumor suppressors. Chromosome Condensation 1-like (<i>CHC1L</i>) is located at 13q14, and found within the smallest common region of loss of heterozygosity in prostate cancer. Decreased expression of <i>CHC1L</i> is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. However, there is no direct evidence for <i>CHC1L</i>’s putative tumor suppressing role in current literature. Presently, we describe the generation and characterization of <i>Chc1L</i> knockout mice. <i>Chc1L</i>^-/- mice do not develop cancer at a young age, but bone marrow and spleen cells from 8–12 week-old mice display an exaggerated proliferative response. By approximately two years of age, knockout and heterozygote mice have a markedly increased incidence of tumorigenesis compared to wild-type controls, with tumors occurring mainly in the spleen, mesenteric lymph nodes, liver and intestinal tract. Histopathological analysis found that most heterozygote and knockout mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. Our study suggests that <i>Chc1L</i> is involved in suppression of these two histiocyte-rich neoplasms in mice and supports clinical data suggesting that <i>CHC1L</i> loss of function is an important step in the pathogenesis of cancers containing 13q14 deletion.</p></div

Generation of <i>Chc1L</i>^-/- knockout mice by gene targeting.

<p>A) Gene targeting strategy. (a) The start codon is located in exon 4 of murine <i>Chc1L</i>, and a second ATG codon is in exon 5. EcoRI digestion produces a gene fragment of 20 kb. Deleting 6kb of intron 4, a gene targeting vector was constructed, with unidirectional loxP sites flanking exons 4 and 5. (b) The gene-targeted locus produces a 14 kb fragment upon digestion with EcoRI. (c) Exon 4, intron 4 and exon 5 are lost following Cre-mediated recombination. B) Confirmation of gene targeting in ES cells. Successful knockin of neomycin-selected ES cells was detected by Southern Blot using a 5’ probe, visualizing a 20kb WT locus fragment, and a 14kb gene-targeted fragment. C) Confirmation of gene targeting in mice. Gene knockin was confirmed in mice again using Southern Blot. D) Confirmation of loss of Chc1L expression. Loss of Chc1L expression was confirmed by RT-PCR.</p

<i>Chc1L</i>^+/- and <i>Chc1L</i>^-/- mice have elevated tumor incidence.

<p>A) Splenocyte and bone marrow cell viability. <i>Chc1L</i>^-/- splenocytes and bone marrow cells have increased viability following LPS-stimulation, compared wild-type controls (splenocyte fold-survival <i>Chc1L</i>^-/-/ <i>Chc1L</i>^+/+ = 1.45±0.29, p<0.05, n = 3; bone marrow fold-survival <i>Chc1L</i>^-/-/<i>Chc1L</i>^+/+ = 1.26±0.11, p<0.01, n = 3). B) Tumor incidence. Tumor incidence by genotype. Incidence of observable tumors was highest in <i>Chc1L</i>^+/- and <i>Chc1L</i>^-/- mice (<i>Chc1L</i>^+/+: 26%, <i>Chc1L</i>^+/-: 56%, <i>Chc1L</i>^-/-: 80%). C) Tumor distribution. Tumors were found most often in the spleen, mesenteric lymph nodes and liver. D) Incidence of multiple organs being affected. <i>Chc1L</i>^+/- mice typically had multiple tumor-bearing organs.</p

<i>Chc1L</i>^+/- and <i>Chc1L</i>^-/- mice develop HS and HAL.

<p>A) Representative H+E staining. Panel 1) <i>Chc1L</i>^+/- and <i>Chc1L</i>^-/- spleens were often enlarged, with normal structure obliterated by proliferation of tumor cells with abundant, eosinophilic cytoplasm, and irregular nuclei with open chromatin and prominent nucleoli (scale bars are 4 mm and 50 μm). Panel 2) <i>Chc1L</i>^+/- and <i>Chc1L</i>^-/- lymph nodes were also enlarged, with normal structure displaced by tumor cells with morphology as described in the spleen (scale bars are 1 mm and 50 μm). Panel 3) Frequently, multifocal areas of tumor cell infiltration that destroy the hepatic parenchyma were observed in <i>Chc1L</i>^+/- and <i>Chc1L</i>^-/- mice (scale bars are 1 mm and 50 μm). Panel 4) Peyer’s patch is severely enlarged by tumor cells which have destroyed the submucosa (scale bars are 1 mm and 50 μm). Panel 5) HS cells have pleiomorphic morphology, varying from spindle shaped (wide arrows) to round (thin arrows) (scale bar is 100 μm). Panel 6) Multinucleated giant cells (arrow) have collected in this proliferation of HS cells. Mitotic figures are abundant (scale bar is 100 μm). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135755#pone.0135755.s001" target="_blank">S1 Fig</a> for H+E controls. B) Immunohistochemical staining confirms diagnosis. Panels 1–3) A lymph node differentially diagnosed with HS co-occurring with B cell lymphoma or HAL. The abnormally structured lymph node is shown, with accumulation of Mac2+ (panel 1), F4/80+ (panel 2) histiocytes admixed with B220+ (panel 3) B lymphocytes (scale bars are 50 μm). Panels 4–6) A lymph node diagnosed with HS is shown. The enlarged lymph node has abundant Mac2+ (panel 4) and F4/80+ (panel 5) histiocytes, and only the occasional B220+ (panel 6) B cell (scale bars are 50 μm).</p

KaLwRij novel germline missense, stoploss, and stopgain mutations.

<p>KaLwRij novel germline missense, stoploss, and stopgain mutations.</p

Whole Genome Sequence of Multiple Myeloma-Prone C57BL/KaLwRij Mouse Strain Suggests the Origin of Disease Involves Multiple Cell Types

<div><p>Monoclonal gammopathy of undetermined significance (MGUS) is the requisite precursor to multiple myeloma (MM), a malignancy of antibody-producing plasma B-cells. The genetic basis of MGUS and its progression to MM remains poorly understood. C57BL/KaLwRij (KaLwRij) is a spontaneously-derived inbred mouse strain with a high frequency of benign idiopathic paraproteinemia (BIP), a phenotype with similarities to MGUS including progression to MM. Using mouse haplotype analysis, human MM SNP array data, and whole exome and whole genome sequencing of KaLwRij mice, we identified novel KaLwRij gene variants, including deletion of <i>Samsn1</i> and deleterious point mutations in <i>Tnfrsf22</i> and <i>Tnfrsf23</i>. These variants significantly affected multiple cell types implicated in MM pathogenesis including B-cells, macrophages, and bone marrow stromal cells. These data demonstrate that multiple cell types contribute to MM development prior to the acquisition of somatic driver mutations in KaLwRij mice, and suggest that MM may an inherently non-cell autonomous malignancy.</p></div

Loss of Samsn1 enhanced pro-tumor macrophage function.

<p>(a) Microarray analysis of gene expression in C57BL/6 and KaLwRij primary bone marrow macrophages. (b) Proliferation of C57BL/6 and KaLwRij macrophages was measured by MTT assay. (c) Macrophage M2 polarization marker Chi3l3 in bone marrow macrophages. (d) Wild-type or <i>Samsn1</i>^<i>-/-</i> M2 macrophages polarized ex vivo were injected directly into established 5TGM1 tumors and tumor burden was monitored by bidirectional caliper measurement.</p

The KaLwRij strain was predisposed to BIP and intersecting mouse and human genetic analyses identified candidate genes that may influence murine BIP risk and human MM risk.

<p>(a) Phylogenetic tree demonstrating genetic distances of 12 inbred strains of mice. (b) Number of C57BL/6 and KaLwRij mice with positive M-spike by SPEP. (c) Haplotype analysis identified contiguous regions of non-shared polymorphic alleles between KaLwRij and C57BL/6 mice (red bars) in 419 genes. (D) GWAS between MM patients and healthy volunteers. SNPs in the 99th percentile (dashed line) fell in 178 genes. (e) Venn diagram representing combined analysis of (c) and (d), resulting in a candidate gene list of 5 genes.</p

KaLwRij bone marrow stromal cells (BMSCs) have altered gene expression profiles independent of <i>Samsn1</i>.

<p>(a) Microarray analysis of B6 and KaLwRij primary BMSCs. (b-d) RT-qPCR analysis of <i>Tnfrsf22</i>, <i>Tnfrsf23</i>, <i>Tnfrsf26</i>, <i>Adipoq</i>, and <i>Fstl4</i> mRNA levels in B6 and KaLwRij BMSCs. * P >0.05, ** P > 0.005, *** P < 0.0005.</p