Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.

Alpha-synuclein (αS) is causally involved in the development of Parkinson disease (PD); however, its role in normal vertebrate physiology has remained unknown. Recent studies demonstrate that αS is induced by noroviral infection in the enteric nervous system of children and protects mice against lethal neurotropic viral infection. Additionally, αS is a potent chemotactic activator of phagocytes. In this report, using both wild-type and αS knockout mice, we show that αS is a critical mediator of inflammatory and immune responses. αS is required for the development of a normal inflammatory response to bacterial peptidoglycan introduced into the peritoneal cavity as well as antigen-specific and T cell responses following intraperitoneal immunization. Furthermore, we show that neural cells are the sources of αS required for immune competence. Our report supports the hypothesis that αS accumulates within the nervous system of PD individuals because of an inflammatory/immune response.

The immune response to radiofrequency ablation (RFA) and cryoablation (CRA) was characterized and compared in a colon cancer mouse model. All studies were conducted under a research protocol approved by the National Institutes of Health, Clinical Center, Animal Care and Use Committee. BALB/cJ mice were inoculated with CT26 cells, and randomized to RFA, CRA, or sham treatment. Mice were sacrificed 3 days post-treatment, and tumor, spleen, and serum were harvested. Cell death was determined by Caspase-3 immunohistochemical and TUNEL stains. Immune response was analyzed using flow cytometry, serum cytokine assay and immunohistochemistry. Cell death, necrosis, and apoptosis induced by ablation were comparable in RFA and CRA. Decreased frequency of systemic T-regulatory cells was found in the CRA group. Both RFA and CRA reduced frequencies of several myeloid-derived suppressor cell (MDSC) subpopulations. RFA induced pro-inflammatory cytokine secretion including TNF-α and IL-12 as well as anti-inflammatory cytokines IL-5, and IL-10. CRA augmented secretion of a wider array of cytokines compared to RFA with both pro- and anti-inflammatory properties including IL-1β, IL-5, IL-6, IL-10, and KC GRO. In the tumor microenvironment, RFA reduced the number of T-regulatory cells, a finding not observed with CRA. Reduction of immune suppression via decreases in T-regulatory cells and MDSC was found to be induced by RFA or CRA. CRA augmented a wider range of cytokines than RFA, which were mainly pro-inflammatory, but also anti-inflammatory. In the tumor microenvironment, RFA demonstrated more pronounced anti-tumoral immunity. Further delineation of specific immunomodulation induced by ablation could inform drug-device development and may play a role in future hypothesis-driven immunomodulatory paradigms that combine immunotherapy drugs with tumor destruction for the treatment of metastatic colon cancer.

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.

Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed.

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.

Woodchucks chronically infected with woodchuck hepatitis virus (WHV), which resembles human hepatitis B virus, develop spontaneous hepatic tumors and may be an important biological and immunological model for human HCC. Nonetheless, this model requires further validation to fully realize its translational potential.

The azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model is commonly used to study colitis-associated cancer. The human commensal bacterium, enterotoxigenic Bacteroides fragilis (ETBF) secretes the Bacteroides fragilis toxin (BFT) which is necessary and sufficient to cause colitis. We report that BALB/c mice infected with WT-ETBF and administered three cycles of AOM/DSS developed numerous, large-sized polyps predominantly in the colorectal region. In addition, AOM/DSS-treated BALB/c mice orally inoculated with wild-type nontoxigenic Bacteroides fragilis (WT-NTBF) overexpressing bft (rETBF) developed numerous polyps whereas mice infected with WT-NTBF overexpressing a biologically inactive bft (rNTBF) did not promote polyp formation. Unexpectedly, the combination of AOM+ETBF did not induce polyp formation whereas ETBF+DSS did induce polyp development in a subset of BALB/c mice. In conclusion, WT-ETBF promoted polyp development in AOM/DSS murine model with increased colitis in BALB/c mice. The model described herein provides an experimental platform for understanding ETBF-induced colonic tumorigenesis and studying colorectal cancer in wild-type mice.

Osteoporosis and other manifestations of bone disease are frequent in patients with systemic mastocytosis (SM) in association with the presence of mast cell infiltrates in bone marrow, although the mechanisms behind bone disease remain poorly understood. We find that extracellular vesicles (EVs) released by neoplastic mast cells and present in the serum of patients with SM (SM-EVs) block osteoblast differentiation and mineralization in culture, and when injected into mice diminish the expression of osteoblast markers, and trabecular bone volume and microarchitecture. We demonstrate that miRNA-30a and miRNA-23a, increased in SM-EVs and neoplastic mast cell-derived EVs, attenuate osteoblast maturation by suppressing expression of RUNX2 and SMAD1/5, essential drivers of osteogenesis. Thus, SM-EVs carry and deliver miRNAs that epigenetically interfere with bone formation and can contribute to bone mass reduction in SM. These findings also suggest possibilities for novel approaches to the management of bone disease in mast cell proliferative disorders.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell lymphoproliferative malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL is an orphan disease with no curative drug treatment regimens urgently needing new combination therapy. HTLV-1-infected cells rely on viral proteins, Tax and HBZ (HTLV-1-b-ZIP factor), to activate the transcription of various host genes that are critical for promoting leukemic transformation. Inhibition of bromodomain and extraterminal motif (BET) protein was previously shown to collapse the transcriptional network directed by BATF3 super-enhancer and thereby induced ATL cell apoptosis. In the current work, by using xenograft, ex vivo, and in vitro models, we demonstrated that I-BET762 (BETi) synergized with copanlisib (PI3Ki) and bardoxolone methyl (NF-κBi) to dramatically decrease the growth of ATL cells. Mechanistically, the triple combination exhibited synergistic activity by down-regulating the expression of c-MYC while upregulating the level of the glucocorticoid-induced leucine zipper (GILZ). The triple combination also enhanced apoptosis induction by elevating the expression of active caspase-3 and cleaved PARP. Importantly, the triple combination prolonged the survival of ATL-bearing xenograft mice and inhibited the proliferation of ATL cells from peripheral blood mononuclear cells (PBMCs) of both acute and smoldering/chronic ATL patients. Therefore, our data provide the rationale for a clinical trial exploring the multiagent combination of BET, PI3K/AKT, and NF-κB inhibitors for ATL patients and expands the potential treatments for this recalcitrant malignancy.

Tumours growing in a sheet-like manner on the surface of organs and tissues with complex topologies represent a difficult-to-treat clinical scenario. Their complete surgical resection is difficult due to the complicated anatomy of the diseased tissue. Residual cancer often responds poorly to systemic therapy and locoregional treatment is hindered by the limited accessibility to microscopic tumour foci. Here we engineered a peptide-based surface-fill hydrogel (SFH) that can be syringe- or spray-delivered to surface cancers during surgery or used as a primary therapy. Once applied, SFH can shape change in response to alterations in tissue morphology that may occur during surgery. Implanted SFH releases nanoparticles composed of microRNA and intrinsically disordered peptides that enter cancer cells attenuating their oncogenic signature. With a single application, SFH shows efficacy in four preclinical models of mesothelioma, demonstrating the therapeutic impact of the local application of tumour-specific microRNA, which might change the treatment paradigm for mesothelioma and possibly other surface cancers.

Despite promising preclinical studies, toxicities have precluded combinations of chemotherapy and DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted chemotherapy delivery might enable clinical translation of such combinations.

Retroviruses cause cancers in animals by integrating in or near oncogenes. Although HIV-1 infection increases the risk of cancer, most of the risk is associated with immunodeficiency and coinfection by oncogenic virus (Epstein-Barr virus, Kaposi sarcoma herpesvirus, and human papillomavirus). HIV-1 proviruses integrated in some oncogenes cause clonal expansion of infected T cells in vivo; however, the infected cells are not transformed, and it is generally believed that HIV-1 does not cause cancer directly. We show that HIV-1 proviruses integrated in the first introns of signal transducer and activator of transcription 3 (STAT3) and lymphocyte-specific protein tyrosine kinase (LCK) can play an important role in the development of T cell lymphomas. The development of these cancers appears to be a multistep process involving additional nonviral mutations, which could help explain why T cell lymphomas are rare in persons with HIV-1 infection.

Breast cancer is a heterogenous disease with several histological and molecular subtypes. Models that represent these subtypes are essential for translational research aimed at improving clinical strategy for targeted therapeutics.

The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.