Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.

Rapid development of myeloproliferative neoplasm in mice with Ptpn11D61Y mutation and haploinsufficient for Dnmt3a.

PTPN11 gain-of-function mutation is the most common mutation found in patients with juvenile myelomonocytic leukemia and DNMT3A loss occurs in over 20% of acute myeloid leukemia patients. We studied the combined effect of both Ptpn11 gain-of-function mutation (D61Y) and Dnmt3a haploinsufficiency on mouse hematopoiesis, the presence of which has been described in both juvenile myelomonocytic leukemia and acute myeloid leukemia patients. Double mutant mice rapidly become moribund relative to any of the other genotypes, which is associated with enlargement of the spleen and an increase in white blood cell counts. An increase in the mature myeloid cell compartment as reflected by the presence of Gr1+Mac1+ cells was also observed in double mutant mice relative to any other group. Consistent with these observations, a significant increase in the absolute number of granulocyte macrophage progenitors (GMPs) was seen in double mutant mice. A decrease in the lymphoid compartment including both T and B cells was noted in the double mutant mice. Another significant difference was the presence of extramedullary erythropoiesis with increased erythroid progenitors in the spleens of Dnmt3a+/-;D61Y mice relative to other groups. Taken together, our results suggest that the combined haploinsufficiency of Dnmt3a and presence of an activated Shp2 changes the composition of multiple hematopoietic lineages in mice relative to the individual heterozygosity of these genes.

Pharmacologic inhibition of PI3K p110δ in mutant Shp2E76K-expressing mice.

Juvenile myelomonocytic leukemia is a childhood malignancy that lacks effective chemotherapies and thus has poor patient outcomes. PI3K p110δ has been found to promote hyperproliferation of cells expressing mutant Shp2. In this study, we tested the efficacy of a PI3Kδ inhibitor in mice expressing the Shp2 gain-of-function mutation, E76K. We found that in vivo treatment of mice led to significantly decreased splenomegaly, reduced frequency of bone marrow progenitor cells, and increased terminally differentiated peripheral blood myeloid cells. The survival of drug-treated mice was significantly prolonged compared to vehicle-treated controls, although mice from both groups ultimately succumbed to a similar myeloid cell expansion. PI3Kδ inhibitors are currently used to treat patients with relapsed lymphoid malignancies, such as chronic lymphocytic leukemia. The current findings provide evidence for using PI3Kδ inhibitors as a treatment strategy for JMML and potentially other myeloid diseases.

Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene.

BACKGROUND: Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. OBJECTIVE: Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. METHODS: Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. RESULTS: Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2-M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2-RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA-BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. CONCLUSIONS: XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins.

Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Signaling.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. EXPERIMENTAL DESIGN: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. RESULTS: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. CONCLUSIONS: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.

Innate immune induction and influenza protection elicited by a response-selective agonist of human C5a.

The anaphylatoxin C5a is an especially potent mediator of both local and systemic inflammation. However, C5a also plays an essential role in mucosal host defense against bacterial, viral, and fungal infection. We have developed a response-selective agonist of human C5a, termed EP67, which retains the immunoenhancing activity of C5a at the expense of its inflammatory, anaphylagenic properties. EP67 insufflation results in the rapid induction of pulmonary cytokines and chemokines. This is followed by an influx of innate immune effector cells, including neutrophils, NK cells, and dendritic cells. EP67 exhibits both prophylactic and therapeutic protection when tested in a murine model of influenza A infection. Mice treated with EP67 within a twenty-four hour window of non-lethal infection were significantly protected from influenza-induced weight loss. Furthermore, EP67 delivered twenty-four hours after lethal infection completely blocked influenza-induced mortality (0% vs. 100% survival). Since protection based on innate immune induction is not restricted to any specific pathogen, EP67 may well prove equally efficacious against a wide variety of possible viral, bacterial, and fungal pathogens. Such a strategy could be used to stop the worldwide spread of emergent respiratory diseases, including but not limited to novel strains of influenza.

Age-associated changes in miRNA expression profiles in thymopoiesis.

During adult life, the thymus involutes and thymic output of mature T cells drastically declines. The molecular events underlying this process are not well understood. Here, we present evidence of the importance of miRNAs in regulating T cell differentiation in the aged. miRNAs are a wide-ranging regulatory element influencing gene expression throughout the lifetime of the organism. To establish whether they play a role in the age-specific thymic decline, the miRNA expression pattern was examined in TN subsets of young and aged mice. Fifty-two percent of the miRNAs exhibited elevated expression levels in the aged TN1 cells. This expression profile leads us to hypothesize that the large number of highly expressed miRNAs, indicative of rigidly controlled protein expression, limits the developmental potential of this population and results in the age-induced decline in thymopoiesis.

A novel approach to thymic rejuvenation in the aged.

With advancing age, the mammalian thymus undergoes involution, a progressive loss of architectural integrity and lymphoid cellularity that results in reduced T lymphopoiesis. Thymic involution also is associated with extreme malnutrition and states of immune deficiency, such as active HIV infection, after chemotherapy, or during pregnancy. Immune recovery appears to require restoration of normal thymopoiesis. Although several means are known to increase thymic cellularity in the aged, including systemic administration of hormones, androgen ablation, and thymic tissue transplantation, each suffers from specific limitations that prevent widespread application. This paper presents a novel approach to rejuvenate T cell differentiation in the aged that employs intrathymic implantation of engineered stromal cells. Two different proteins have been examined for their impact on thymopoiesis after delivery by somatic cell implantation. Intrathymic injection of IL-7-producing stromal cells enhances the earliest specification steps of T cell development, resulting in the increased representation of pro-T cells in the aged thymus. In contrast, increasing the intrathymic levels of sonic hedgehog diminishes this aspect of T cell poiesis.

IL-7 gene therapy in aging restores early thymopoiesis without reversing involution.

Thymic involution begins early in life and continues throughout adulthood, resulting in a decreased population of naive T cells in the periphery and a reduced ability to fight off newly encountered infectious diseases. We have previously shown that the first step of thymopoiesis is specifically blocked in aging. This block at the DN1 to DN2 transition and the subsequent loss of thymic output in old age mirrors the changes seen in IL-7-deficient mice, and it is hypothesized that decreased intrathymic IL-7 is involved in age-related thymic involution. To separate the effect of IL-7 on thymic involution from its function as a peripheral lymphocyte growth cofactor, we injected IL-7-secreting stromal cells into the thymi of recipient mice. The increased local concentration of IL-7 maintained the first step of thymopoiesis at a level far higher than was seen in age-matched controls. However, despite this success, there was no decrease in thymic involution or increase in T cell output. The inability of IL-7 to prevent involution led us to the discovery of an additional age-sensitive step in thymopoiesis, proliferation of the DN4 population, which is unaffected by IL-7 expression.

A CD45 minigene restores regulated isoform expression and immune function in CD45-deficient mice: therapeutic implications for human CD45-null severe combined immunodeficiency.

Transgenic mice have been generated that carry a CD45 minigene under control of the human leukocyte function-associated antigen (LFA-1, CD11a) promoter. CD45-null mice carrying the transgene exhibit the lymphocyte lineage-specific isoform expression patterns of wild-type mice. Furthermore, these mice have normal thymocyte development and peripheral T-cell numbers. The proliferative ability of T cells in response to mitogens and antigen also is regained, as is B-cell responsiveness to anti-IgM. The antibody response to antigen is also restored and is similar to that of normal mice. Therefore, introduction of a functional CD45 minigene is sufficient to overcome the principal severe combined immunodeficiency (SCID)-associated defects and represents a potential route to a gene therapy for human CD45-deficent SCID.