The Hhex gene encodes a transcription factor that is important for both embryonic and post-natal development, especially of hematopoietic tissues. Hhex is one of the most common sites of retroviral integration in mouse models. We found the most common integrations in AKXD (recombinant inbred strains) T-ALLs occur 57-61kb 3' of Hhex and activate Hhex gene expression. The genomic region of murine leukemia virus (MLV) integrations has features of a developmental stage-specific cis regulatory element (CRE), as evidenced by ATAC-seq in murine progenitor cells and high H3K27 acetylation at the syntenic CRE in human hematopoietic cell lines. With ChIP-exonuclease, we describe occupancy of LIM domain binding protein 1 (LDB1), the constitutive partner of the LIM Only-2 (LMO2), GATA1, and TAL1 transcription factors at GATA sites and at a composite GATA-E box within the CRE. With virtual 4C analysis, we observed looping between this +65kb CRE and the proximal intron one enhancer of HHEX in primary human ETP-ALLs and in normal progenitor cells. Our results show that retroviral integrations at intergenic sites can mark and take advantage of CREs. Specifically, in the case of HHEX activation, this newly described +65kb CRE is co-opted in the pathogenesis of ETP-ALL by the LMO2/LDB1 complex.

To identify genes important for colorectal cancer (CRC) development and metastasis, we established a new metastatic mouse organoid model using Sleeping Beauty (SB) transposon mutagenesis. Intestinal organoids derived from mice carrying actively mobilizing SB transposons, an activating KrasG12D, and an inactivating ApcΔ716 allele, were transplanted to immunodeficient mice. While 66.7% of mice developed primary tumors, 7.6% also developed metastatic tumors. Analysis of SB insertion sites in tumors identified numerous candidate cancer genes (CCGs) identified previously in intestinal SB screens performed in vivo, in addition to new CCGs, such as Slit2 and Atxn1. Metastatic tumors from the same mouse were clonally related to each other and to primary tumors, as evidenced by the transposon insertion site. To provide functional validation, we knocked out Slit2, Atxn1, and Cdkn2a in mouse tumor organoids and transplanted to mice. Tumor development was promoted when these gene were knocked out, demonstrating that these are potent tumor suppressors. Cdkn2a knockout cells also metastasized to the liver in 100% of the mice, demonstrating that Cdkn2a loss confers metastatic ability. Our organoid model thus provides a new approach that can be used to understand the evolutionary forces driving CRC metastasis and a rich resource to uncover CCGs promoting CRC.

MITF, a basic Helix-Loop-Helix Zipper (bHLHZip) transcription factor, plays vital roles in melanocyte development and functions as an oncogene. We perform a genetic screen for suppressors of the Mitf-associated pigmentation phenotype in mice and identify an intragenic Mitf mutation that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). The resulting protein is more nuclear but less stable than wild-type MITF and retains DNA-binding ability. As a dimer, it can translocate wild-type and mutant MITF partners into the nucleus, improving its own stability thus ensuring nuclear MITF supply. smFRET analysis shows interactions between K316 SUMOylation and S409 phosphorylation sites across monomers; these interactions largely explain the observed effects. The recurrent melanoma-associated E318K mutation in MITF, which affects K316 SUMOylation, also alters protein regulation in concert with S409. This suggests that residues K316 and S409 of MITF are impacted by SUMOylation and phosphorylation, respectively, mediating effects on nuclear localization and stability through conformational changes. Our work provides a novel mechanism of genetic suppression, and an example of how apparently deleterious mutations lead to normal phenotypes.

Supplementary Table 1 from Whole-Body <i>Sleeping Beauty</i> Mutagenesis Can Cause Penetrant Leukemia/Lymphoma and Rare High-Grade Glioma without Associated Embryonic Lethality

Supplementary Tables 1-3 from A Modified Sleeping Beauty Transposon System That Can Be Used to Model a Wide Variety of Human Cancers in Mice

Supplementary Figures 1-8 from A Modified Sleeping Beauty Transposon System That Can Be Used to Model a Wide Variety of Human Cancers in Mice

Hematopoietically-expressed Homeobox ( HHEX) is required for the maintenance of hematopoietic stem cells (HSPCs) and common lymphoid progenitor cells. HHEX is also the second most frequent integration site in retroviral insertional mutagenesis screens of leukemias and lymphomas arising in AKXD recombinant inbred mouse strains, implying that it is an important oncogene. Enforced expression of HHEX induces T-cell acute lymphoblastic leukemias (T-ALL) in murine bone marrow transplantation models. In human studies, HHEXmRNA expression is upregulated in human T-ALL studies, especially in Early T-cell Precursor (ETP-) ALL subtypes where it is concordantly expressed with LMO2. The HHEX locus is not rearranged in ETP-ALL where it is upregulated so the mechanism of HHEX activation is not clear. We analyzed T-ALL induced by retroviral mutagenesis and found an intergenic site of frequent integration, 65kb 3‘ of the Hhex coding exons. Integrations clustered within a 3 kb genomic area that is also an open chromatin region (OCR) by ATAC-seq analysis and that induced Hhex upregulation. The syntenic human region was highly enriched for H3K27 acetylation and for occupancy by multiple transcription factors, notably GATA1 and TAL1, per ENCODE. Our prior data had shown that HHEX was a downstream target of the LMO2 complex comprised of GATA1/2, TAL1 or LYL1, and nucleated by the scaffolding protein, LIM domain binding protein 1 (LDB1). We had previously shown by ChIP-PCR that the LMO2 complex was bound to the intron 1 enhancer. Thus, we performed ChIP-exonuclease analysis of LDB1 in the human ETP-ALL model cell line, LOUCY. We confirmed its occupancy at the intron 1 enhancer but we also observed LDB1 occupancy 68kb 3‘ of the HHEX coding exons, in the exact region syntenic to the murine common insertion sites. ChIP-exo analysis allowed us to pinpoint a core element with composite GATA/E box sites that was highly conserved across multiple mammalian species. LDB1 occupancy at intron 1 and at +68kb of the HHEX locus was reminiscent of LDB1's occupancy at the beta globin locus where LDB1's occupancy and homodimerization mediates chromatin looping between composite E box/GATA sites in the locus control region (LCR) and the beta globin proximal promoters. To test whether a similar interaction was occurring at the HHEX locus, we analyzed virtual 4C (chromatin conformation capture) data. We confirmed looping between the +68kb element and the proximal promoter of HHEX in human ETP-ALL primary samples and in human HSPCs. The experiments showed that looping occurs in certain developmental contexts in HSPCs and is recapitulated in ETP-ALL and mediated by the LMO2/LDB1 protein complex. HHEX is a downstream oncogene of the LMO2/LDB1 complex and is activated through this distal +68kb regulatory element. Our studies raise the possibility that dissection of this regulatory element and interference with chromatin looping are potential therapeutic mechanisms that could disrupt oncogene expression.

Figure S1. Expression of ZNF326 a in human breast cancer tissues. The images show a representative scoring intensities following immunostaining with anti-ZNF326 antibody (Range of low, moderate and high). Figure S2. RT-PCR analysis of the candidate cancer genes downregulated by shRNA-pools in the (A) HCC70 and (B) MDA-MB-231 breast cancer cell lines. Figure S3. Stable ZNF326 knockdown in (A) HCC70, (B) HCC1569 and (C) MDA-MB-231 TNBC cell lines using two independent shRNAs was confirmed by qRT-PCR. (D) Ectopic expression of ZNF326 in MDA-MB-231 cells transduced with lentivirus particles expressing ZNF326 cDNA and empty vector (control) respectively. ZNF326 mRNA levels were quantified by qRT-PCR. Figure S4. Western blot analysis of mammosphere cell lysates of HCC70 (A) and MDA-MB-231 (B) with stable shRNA (left panel) and ectopic overexpressing ZNF326 (right panel). Figure S5. Gene expression-based outcome for breast cancer online (GOBO) was used to obtain the expression analysis of (A) DACH1 and (B) GATA3 in different breast cancer subtypes.

Supplementary Table 3 from Whole-Body <i>Sleeping Beauty</i> Mutagenesis Can Cause Penetrant Leukemia/Lymphoma and Rare High-Grade Glioma without Associated Embryonic Lethality

Supplementary Figure 4 from Whole-Body Sleeping Beauty Mutagenesis Can Cause Penetrant Leukemia/Lymphoma and Rare High-Grade Glioma without Associated Embryonic Lethality

Table S1. siRNA oligos of the candidate cancer genes. Table S2. List of lentivirus shRNA clones. Table S3. Primer sets used in the ChIP assay. Table S4. List of TaqMan probes. Table S5. Candidate cancer genes were screen in four TNBC cell lines using siRNA oligos.

Supplementary Figure 1 from Whole-Body <i>Sleeping Beauty</i> Mutagenesis Can Cause Penetrant Leukemia/Lymphoma and Rare High-Grade Glioma without Associated Embryonic Lethality

Supplementary Figure 2 from Whole-Body Sleeping Beauty Mutagenesis Can Cause Penetrant Leukemia/Lymphoma and Rare High-Grade Glioma without Associated Embryonic Lethality

Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is a deadly but poorly understood disease, and its treatment options are very limited. The aim of this study was to identify the molecular drivers of ICC and search for therapeutic targets. Approach and Results: We performed a Sleeping Beauty transposon‐based in vivo insertional mutagenesis screen in liver‐specific Pten‐deficient mice and identified TNF receptor–related factor 3 (Traf3) as the most significantly mutated gene in murine ICCs in a loss‐of‐function manner. Liver‐specific Traf3 deletion caused marked cholangiocyte overgrowth and spontaneous development of ICC in Pten knockout and Kras G12D mutant mice. Hepatocyte‐specific, but not cholangiocyte‐specific, Traf3‐deficient and Pten‐deficient mice recapitulated these phenotypes. Lineage tracing and single‐cell RNA sequencing suggested that these ICCs were derived from hepatocytes through transdifferentiation. TRAF3 and PTEN inhibition induced a transdifferentiation‐like phenotype of hepatocyte‐lineage cells into proliferative cholangiocytes through NF‐κB‐inducing kinase (NIK) up‐regulation in vitro. Intrahepatic NIK levels were elevated in liver‐specific Traf3‐deficient and Pten‐deficient mice, and NIK inhibition alleviated cholangiocyte overgrowth. In human ICCs, we identified an inverse correlation between TRAF3 and NIK expression, with low TRAF3 or high NIK expression associated with poor prognosis. Finally, we showed that NIK inhibition by a small molecule inhibitor or gene silencing suppressed the growth of multiple human ICC cells in vitro and ICC xenografts in vivo. Conclusions: TRAF3 inactivation promotes ICC development through NIK‐mediated hepatocyte transdifferentiation. The oncogenic TRAF3–NIK axis may be a potential therapeutic target for ICC.

Supplementary Figure 3 from Whole-Body <i>Sleeping Beauty</i> Mutagenesis Can Cause Penetrant Leukemia/Lymphoma and Rare High-Grade Glioma without Associated Embryonic Lethality

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide and the only cancer with an increasing incidence in the United States. Recent advances in sequencing technology have enabled detailed profiling of liver cancer genomes and revealed extensive inter- and intra-tumor heterogeneity, making it difficult to identify driver genes for HCC. To identify HCC driver genes, we performed transposon mutagenesis screens in a mouse HBV model of HCC and discovered many candidate cancer genes (SB/HBV-CCGs). Here, we show that one of these genes, RNF125 is a potent anti-proliferative tumor suppressor gene in HCC. RNF125 is one of nine CCGs whose expression was >3-fold downregulated in human HCC. Depletion of RNF125 in immortalized mouse liver cells led to tumor formation in transplanted mice and accelerated growth of human liver cancer cell lines, while its overexpression inhibited their growth, demonstrating the tumor-suppressive function of RNF125 in mouse and human liver. Whole-transcriptome analysis revealed that RNF125 transcriptionally suppresses multiple genes involved in cell proliferation and/or liver regeneration, including Egfr, Met, and Il6r. Blocking Egfr or Met pathway expression inhibited the increased cell proliferation observed in RNF125 knockdown cells. In HCC patients, low expression levels of RNF125 were correlated with poor prognosis demonstrating an important role for RNF125 in HCC. Collectively, our results identify RNF125 as a novel anti-proliferative tumor suppressor in HCC.

Introduction:Sarcoidosis is a chronic multisystem inflammatory disease which may affect any organ. Also bone can be involved both directly and indirectly. Data on BMD values and fragility fractures in sarcoidosis patients are few and heterogeneous. This study aimed to characterized the presence of fracture and the relative risk factors in patients with sarcoidosis.Materials and methods:In this single center cross-sectional study we evaluated 252 sarcoidosis patients (54.7 ± 12.1 years) compared to sex-and age matched healthy controls. We measured BMD at lumbar spine, at femoral neck and at total hip. Moreover, the presence of fragility fractures was collected during osteoporosis visit and all radiological images were examined for the presence of any vertebral fracture according to Genant's method's. Lung function measurements, including forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1/FVC, and diffusion capacity for carbon monoxide (DLCO) were assessed.Results:Bone Mineral Density T-scores were lower in patients affected by sarcoidosis with respect to those obtained in healthy controls, but the difference was statistically significant only for BMD-LS (p < 0.01) and BMD-TH (p < 0.05). Moreover, BMD values at all skeletal sites were significantly associated with DLCO (%) (p < 0.05). The prevalence of fragility fracture was higher in patients with sarcoidosis than in healthy controls (30.6 vs. 12.3%). The patients with ≥3 vertebral fracture had lower values of FVC (%), FEV1 (%), and DLCO (%). Multiple regression analyses showed that BMI was positively associated with fragility fracture, while BMD-TH, DLCO(%) and therapy use was negatively associated.Conclusions:Vertebral fractures represent a frequent complication in patients with sarcoidosis. Furthermore, the number of vertebral fractures was linked with a worsening in pulmonary functional tests. Therefore, the degree of severity of the sarcoidosis disease appears to be the main determinant of bone fragility.

AbstractUterine leiomyosarcoma (ULMS) is a malignancy, which arises from the uterine smooth muscle. Because of its rarity, aggressive nature, and extremely poor prognosis, the molecular mechanisms driving ULMS remain elusive. To identify candidate cancer genes (CCG) driving ULMS, we conducted an in vivo Sleeping Beauty (SB) transposon mutagenesis screen in uterine myometrium–specific, PTEN knockout, KRAS mutant (PTEN KO/KRAS) mice. ULMS quickly developed in SB PTEN KO/KRAS mice, but not in PTEN KO/KRAS mice, demonstrating the critical importance of SB mutagenesis for driving ULMS in this model. Subsequent sequencing of SB insertion sites in these tumors identified 19 ULMS CCGs that were significantly enriched in known cancer genes. Among them, Zfp217 and Sfmbt2 functioned at early stages of tumor initiation and appeared to be oncogenes. Expression of ZNF217, the human homolog of ZFP217, was shown to be elevated in human ULMS compared with paired normal uterine smooth muscle, where it negatively correlated with patient prognosis. Inhibition of ZNF217 suppressed, whereas overexpression induced, proliferation, survival, migration, and stemness of human ULMS. In a second ex vivo ULMS SB metastasis screen, three CCGs were identified that may drive ULMS metastasis to the lung. One of these CCGs, Nrd1 (NRDC in humans), showed stronger expression in human metastatic tumors compared with primary ULMS and negatively associated with patient survival. NRDC knockdown impaired migration and adhesion without affecting cell proliferation, whereas overexpression had the opposite effect. Together, these results reveal novel mechanism driving ULMS tumorigenesis and metastasis and identify ZNF217 and NRDC as potential targets for ULMS therapy.Significance:An in vivo Sleeping Beauty transposon mutagenesis screen identifies candidate cancer genes that drive initiation and progression of uterine leiomyosarcoma and may serve as therapeutic targets.

Cancer genome sequencing studies have identified driver genes for a variety of different cancers and helped to understand the genetic landscape of human cancer. It is still challenging, however, to identify cancer driver genes with confidence simply from genetic data alone. In vivo forward genetic screens using Sleeping Beauty (SB) transposon mutagenesis provides another powerful genetic tool for identifying candidate cancer driver genes in wild-type and sensitized mouse tumors. By comparing cancer driver genes identified in human and mouse tumors, cancer driver genes can be identified with additional confidence based upon comparative oncogenomics. This review describes how SB mutagenesis works in mice and focuses on studies that have identified cancer driver genes in the mouse gastrointestinal tract.

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene Prdm16 during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. To investigate the role of Prdm16 in adult HSCs, we generated a novel conditional knockout mouse model and deleted Prdm16 in adult mouse hematopoietic system using the IFNinducible Mx1-Cre. Our results show that Prdm16 deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of Prdm16 significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which Cdkn1a and Egr1 were identified as direct targets of Prdm16. Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence.