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Intraductal papillary neoplasm of the bile duct (IPNB) is a precursor lesion of cholangiocarcinoma. (1-3) Since the concept of this neoplasm was proposed almost 20 years ago, (4) it has [...], * Context.--Intraductal papillary neoplasm of the bile duct (IPNB) is classified into types 1 and 2 based on criteria proposed in 2019. Recent studies investigated the clinicopathologic and molecular features of IPNB, which contributed to a more detailed understanding of this undercharacterized neoplasm. Objective.--To summarize driver gene mutations, radiologic tumor evolution, and a potentially unique pattern of tumor progression in IPNB. Data Sources.--Data were derived from a literature review and personal clinical and research experiences. Conclusions.--In contrast to de novo cholangiocarcinoma, type 1 IPNB often has mutations in APC, CTNNB1, STK11, and GNAS. These molecular features are shared with intraductal papillary mucinous neoplasm of the pancreas; however, the frequencies of individual gene abnormalities differ between these 2 neoplasms. A radiologic review of sequential images suggested that type 1 IPNB is a slow-growing neoplasm, with an ~1-cm increase in size every 2 to 3 years, and remains in a noninvasive state for many years. A similar papillary neoplasm may develop in the biliary tree years after the complete surgical resection of IPNB. The second neoplasm has the same genetic abnormalities as the first neoplasm, indicating intrabiliary implantation rather than multifocal lesions. In contrast to type 1 IPNB, most cases of type 2 IPNB have invasive malignancy at the initial presentation. Type 2 IPNB shares many clinicopathologic and molecular features with de novo cholangiocarcinoma, questioning the distinctness of this tumor entity. The molecular mechanisms underlying malignant transformation in IPNB warrant further study. (Arch Pathol Lab Med. 2024;148:989-996; doi: 10.5858/arpa.2022-0407-RA)

Germ cell tumors (GCTs) are the most common cancer in men between the ages of 15 and 40. Although most patients are cured, those with disease arising in the mediastinum have distinctly poor outcomes. One in every 17 patients with primary mediastinal nonseminomatous GCTs develop an incurable hematologic malignancy and prior data intriguingly suggest a clonal relationship exists between hematologic malignancies and GCTs in these cases. To date, however, the precise clonal relationship between GCTs and the diverse additional somatic malignancies arising in such individuals have not been determined. Here, we traced the clonal evolution and characterized the genetic features of each neoplasm from a cohort of 15 patients with GCTs and associated hematologic malignancies. We discovered that GCTs and hematologic malignancies developing in such individuals evolved from a common shared precursor, nearly all of which harbored allelically imbalanced p53 and/or RAS pathway mutations. Hematologic malignancies arising in this setting genetically resembled mediastinal GCTs rather than de novo myeloid neoplasms. Our findings argue that this scenario represents a unique clinical syndrome, distinct from de novo GCTs or hematologic malignancies, initiated by an ancestral precursor that gives rise to the parallel evolution of GCTs and blood cancers in these patients., Introduction Germ cell tumors (GCTs) are a model of curable cancer, as most patients with metastatic GCTs are successfully treated with cisplatin-based chemotherapy (1). However, up to 30% of patients [...]

BACKGROUND. Patients with diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), have dismal outcomes. We previously described the H3.3K27M mutation as a shared neoantigen in [HLA-A.sup.*][02.01.sup.+], [H3.3K27M.sup.+] DMGs. Within the Pacific Pediatric Neuro-Oncology Consortium, we assessed the safety and efficacy of an H3.3K27M-targeted peptide vaccine. METHODS. Newly diagnosed patients, aged 3-21 years, with [HLA-A.sup.*][02.01.sup.+] and [H3.3K27M.sup.+] status were enrolled in stratum A (DIPG) or stratum B (nonpontine DMG). Vaccine was administered in combination with polyinosinic-polycytidylic acid-poly-Ilysine carboxymethylcellulose (poly-ICLC) every 3 weeks for 8 cycles, followed by once every 6 weeks. Immunomonitoring and imaging were performed every 3 months. Imaging was centrally reviewed. Immunological responses were assessed in PBMCs using mass cytometry. RESULTS. A total of 19 patients were enrolled in stratum A (median age, 11 years) and 10 in stratum B (median age, 13 years). There were no grade-4 treatment-related adverse events (TRAEs). Injection site reaction was the most commonly reported TRAE. Overall survival (OS) at 12 months was 40% (95% CI, 22%-73%) for patients in stratum A and 39% (95% CI, 16%-93%) for patients in stratum B. The median OS was 16.1 months for patients who had an expansion of H3.3K27M-reactive [CD8.sup.+] T cells compared with 9.8 months for their counterparts (P = 0.05). Patients with DIPG with below-median baseline levels of myeloid-derived suppressor cells had prolonged OS compared with their counterparts (P < 0.01). Immediate pretreatment dexamethasone administration was inversely associated with H3.3K27M-reactive [CD8.sup.+] T cell responses. CONCLUSION. Administration of the H3.3K27M-specific vaccine was well tolerated. Patients with H3.3K27M-specific [CD8.sup.+] immunological responses demonstrated prolonged OS compared with nonresponders. TRIAL REGISTRATION. ClinicalTrials.gov NCT02960230. FUNDING. The V Foundation, the Pacific Pediatric Neuro-Oncology Consortium Foundation, the Pediatric Brain Tumor Foundation, the Mithil Prasad Foundation, the MCJ Amelior Foundation, the Anne and Jason Farber Foundation, Will Power Research Fund Inc., the Isabella Kerr Molina Foundation, the Parker Institute for Cancer Immunotherapy, and the National Institute of Neurological Disorders and Stroke (NINDS), NIH (R35NS105068)., Introduction Diffuse midline gliomas (DMGs) are uniformly fatal pediatric brain cancers, and despite maximal therapy, survival outcomes remain dismal, with less than 10% of patients surviving beyond 2 years (1). [...]

Some germ cell tumors (GCTs) in men develop into hematologic malignancies; however, the clonal origins of such malignancies remain unknown. In this issue of the JCI, Taylor, Donoghue, et al. unravel the clonal relationship between primary mediastinal nonseminomas (PMNs) and hematologic somatic-type malignancies (HSTMs). Whole-exome sequencing was used to construct phylogenetic trees of the PMNs and the ensuing HSTM clones. HSTMs were derived from multiple distinct clones not detected within the PMNs. Clones from PMNs and HSTMs shared a common precursor, arguably an embryonal carcinoma cell resulting from a reprogrammed primordial germ cell from the thymus. Mutational and copy number variation analysis of a large cohort of patients with PMNs also demonstrated a high prevalence of TP53 mutations not found in testicular nonseminomas. These data likely explain why patients with PMNs are frequently resistant to platinum-based chemotherapy and provide TP53 mutations as potential targets., Germ cell tumors Earlier studies determined that hematologic somatic-type malignancies (HSTMs) and the primary mediastinal nonseminomas (PMNs) from which they originate (1) may share an isochromosome 12p (duplication of the [...]

Identifying genes that result in monogenic diabetes can provide insights that can build a scientific foundation for precision medicine. At present, nearly 20% of neonatal diabetes cases have unknown causes. In this issue of the JCI, De Franco and Lytrivi et al. sequenced the genome of two probands with a rare neonatal diabetes subtype that also associated with microcephaly and epilepsy. The authors revealed mutations in the YIPF5 gene. YIPF5 resides in the Golgi apparatus and is thought to play a critical role in vesicular trafficking. Notably, disrupting YIPF5 in [beta] cell-based models induced ER stress signaling and resulted in the accumulation of intracellular proinsulin. We believe that utilizing registries and biobanks to reveal other monogenic atypical forms of diabetes is an important approach to gaining insight and suggest that an insulin sensitizer may alleviate ER stress associated with YIPF5 disruption by decreasing the demand for insulin secretion., Monogenic diabetes genes Precision medicine for diabetes is often said to be on the horizon. However, as noted in the recent joint American Diabetes Association (ADA)/European Association for the Study [...]

Hemotropic mycoplasmas (or hemoplasmas; size We identified and characterized Candidatus M. haemohominis infections in a patient with pyrexia of unknown origin. The patient had various life-threatening symptoms that were not [...]

Genetic instability, a heritable increase in the rate of genetic mutation, accelerates evolutionary adaptation.sup.1 and is widespread in cancer.sup.2,3. In mammals, instability can arise from damage to both copies of genes involved in DNA metabolism and cell cycle regulation.sup.4 or from inactivation of one copy of a gene whose product is present in limiting amounts (haploinsufficiency.sup.5); however, it has proved difficult to determine the relative importance of these two mechanisms. In Escherichia coli.sup.6, the application of repeated, strong selection enriches for genetic instability. Here we have used this approach to evolve genetic instability in diploid cells of the budding yeast Saccharomyces cerevisiae, and have isolated clones with increased rates of point mutation, mitotic recombination, and chromosome loss. We identified candidate, heterozygous, instability-causing mutations; engineering these mutations, as heterozygotes, into the ancestral diploid strain caused genetic instability. Mutations that inactivated one copy of haploinsufficient genes were more common than those that dominantly altered the function of the mutated gene copy. The mutated genes were enriched for genes functioning in transport, protein quality control, and DNA metabolism, and have revealed new targets for genetic instability.sup.7-11, including essential genes. Although only a minority (10 out of 57 genes with orthologues or close homologues) of the targets we identified have homologous human genes that have been implicated in cancer.sup.2, the remainder are candidates to contribute to human genetic instability. To test this hypothesis, we inactivated six examples in a near-haploid human cell line; five of these mutations increased instability. We conclude that single genetic events cause genetic instability in diploid yeast cells, and propose that similar, heterozygous mutations in mammalian homologues initiate genetic instability in cancer.Repeated selection for adaptive mutations in a diploid yeast model results in increased genetic instability and sheds light on mechanisms of genetic instability that might contribute to tumorigenesis., Author(s): Miguel C. Coelho [sup.1] [sup.2] [sup.3] , Ricardo M. Pinto [sup.3] [sup.4] , Andrew W. Murray [sup.1] [sup.2] Author Affiliations:(1) FAS Center for Systems Biology, Harvard University, Cambridge, USA(2) [...]

Context.--Mutations within the same signature transduction pathway are redundant and, therefore, most are mutually exclusive. Laboratory errors, however, may introduce unexpected coexisting mutations. Objective.--To validate coexisting mutations within epidermal growth factor receptor (EGFR), mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. Design.--In this retrospective study for quality assessment of next-generation sequencing in a clinical diagnostics setting, coexisting mutations within EGFR, KRAS, NRAS, BRAF, AKT1, and PIK3CA genes were examined in 1208 non-small cell lung cancers. Results.--EGFR mutations did not coexist with BRAF mutations, neither kinase-activated nor kinase-impaired mutations. There was a low but similar incidence (3.3%5.1%) of PIK3CA mutations in BRAF-, EGFR-, and KRAS-mutated lung cancers and a rare incidence of coexisting KRAS and EGFR mutations detected in 1 of 1208 lung cancers (0.08%) or 1 of 226 EGFR-mutated lung cancers (0.4%). Coexisting BRAF p.V600E mutation was observed in 3 of 4 AKT1 p.E17K-mutated lung cancers. Mutational profiling of DNA reisolated from subareas with the same or different histomorphology, using an alternative assay, confirmed that coexisting mutations might present within the same (whole or subclonal) population or different populations and clarified that the so-called coexisting activating KRAS and BRAF mutations originally reported in a specimen were indeed present in separate lung nodules submitted in the same block. Conclusions.--The results supported that EGFR and BRAF mutations are early driver mutations in lung cancers. Guidelines from official organizations to establish standard operating procedures are warranted to validate unexpected coexisting mutations and, if clinically indicated, to determine their presence in the same or different tumor populations. doi: 10.5858/arpa.2017-0495-OA, Several therapeutic agents have been approved by the US Food and Drug Administration in the United States for targeted therapy of metastatic non-small cell lung cancers (NSCLCs). (1) These include [...]

Influenza A(H9N2) virus circulates in domestic poultry, and outbreaks have been recorded since the early 1990s in China (1). In Pakistan, H9N2 virus was first detected in a poultry outbreak [...]

Pediatric brain tumors are highly associated with epileptic seizures.sup.1. However, their epileptogenic mechanisms remain unclear. Here, we show that the oncogenic BRAF somatic mutation p.Val600Glu (V600E) in developing neurons underlies intrinsic epileptogenicity in ganglioglioma, one of the leading causes of intractable epilepsy.sup.2. To do so, we developed a mouse model harboring the BRAF.sup.V600E somatic mutation during early brain development to reflect the most frequent mutation, as well as the origin and timing thereof. Therein, the BRAF.sup.V600E mutation arising in progenitor cells during brain development led to the acquisition of intrinsic epileptogenic properties in neuronal lineage cells, whereas tumorigenic properties were attributed to high proliferation of glial lineage cells. RNA sequencing analysis of patient brain tissues with the mutation revealed that BRAF.sup.V600E-induced epileptogenesis is mediated by RE1-silencing transcription factor (REST), which is a regulator of ion channels and neurotransmitter receptors associated with epilepsy. Moreover, we found that seizures in mice were significantly alleviated by an FDA-approved BRAF.sup.V600E inhibitor, vemurafenib, as well as various genetic inhibitions of Rest. Accordingly, this study provides direct evidence of a BRAF somatic mutation contributing to the intrinsic epileptogenicity in pediatric brain tumors and suggests that BRAF and REST could be treatment targets for intractable epilepsy. In pediatric brain tumors that are accompanied by epileptic seizures, the BRAF somatic mutation V600E contributes to intrinsic epileptic properties in neurons, which can be suppressed by vemurafenib in mice., Author(s): Hyun Yong Koh [sup.1] , Se Hoon Kim [sup.2] , Jaeson Jang [sup.3] , Hyungguk Kim [sup.4] , Sungwook Han [sup.5] , Jae Seok Lim [sup.1] , Geurim Son [...]

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNA.sup.Ala mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin. Mitochondrially targeted zinc-finger nucleases reduce mutational burden and correct biochemical defects in a mouse model of mitochondrial disease., Author(s): Payam A. Gammage [sup.1] , Carlo Viscomi [sup.1] , Marie-Lune Simard [sup.2] , Ana S. H. Costa [sup.3] , Edoardo Gaude [sup.3] , Christopher A. Powell [sup.1] , Lindsey [...]

Prostate cancer is an androgen-dependent disease subject to interactions between the tumor epithelium and its microenvironment. Here, we found that epigenetic changes in prostatic cancer- associated fibroblasts (CAF) initiated a cascade of stromal-epithelial interactions. This facilitated lethal prostate cancer growth and development of resistance to androgen signaling deprivation therapy (ADT). We identified a Ras inhibitor, RASAL3, as epigenetically silenced in human prostatic CAF, leading to oncogenic Ras activity driving macropinocytosis-mediated glutamine synthesis. Interestingly, ADT further promoted RASAL3 epigenetic silencing and glutamine secretion by prostatic fibroblasts. In an orthotopic xenograft model, subsequent inhibition of macropinocytosis and glutamine transport resulted in antitumor effects. Stromal glutamine served as a source of energy through anaplerosis and as a mediator of neuroendocrine differentiation for prostate adenocarcinoma. Antagonizing the uptake of glutamine restored sensitivity to ADT in a castration-resistant xenograft model. In validating these findings, we found that prostate cancer patients on ADT with therapeutic resistance had elevated blood glutamine levels compared with those with therapeutically responsive disease (odds ratio = 7.451, P = 0.02). Identification of epigenetic regulation of Ras activity in prostatic CAF revealed RASAL3 as a sensor for metabolic and neuroendocrine reprogramming in prostate cancer patients failing ADT., Introduction Despite improved detection and therapies, nearly 30,000 men continue to die of prostate cancer (PCa) in the United States and over 300,000 globally every year (1, 2). Currently, all [...]

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end- stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re- expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/ Cas9 knockout of nup107or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in- frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype., Introduction Steroid-resistant nephrotic syndrome (SRNS), a disease of the renal glomerular filter, is characterized by proteinuria, edema, and hypoalbuminemia. In contrast to other forms of nephrotic syndrome, SRNS does not [...]

Nucleophosmin (NPM1) is among the most frequently mutated genes in acute myeloid leukemia (AML). It is not known, however, how the resulting oncoprotein mutant NPM1 is leukemogenic. To reveal the cellular machinery in which NPM1 participates in myeloid cells, we analyzed the endogenous NPM1 protein interactome by mass spectrometry and discovered abundant amounts of the master transcription factor driver of monocyte lineage differentiation PU.1 (also known as SPI1). Mutant NPM1, which aberrantly accumulates in cytoplasm, dislocated PU.1 into cytoplasm with it. CEBPA and RUNX1, the master transcription factors that collaborate with PU.1 to activate granulomonocytic lineage fates, remained nuclear; but without PU.1, their coregulator interactions were toggled from coactivators to corepressors, repressing instead of activating more than 500 granulocyte and monocyte terminal differentiation genes. An inhibitor of nuclear export, selinexor, by locking mutant NPM1/PU.1 in the nucleus, activated terminal monocytic fates. Direct depletion of the corepressor DNA methyltransferase 1 (DNMT1) from the CEBPA/RUNX1 protein interactome using the clinical drug decitabine activated terminal granulocytic fates. Together, these noncytotoxic treatments extended survival by more than 160 days versus vehicle in a patient-derived xenotransplant model of NPM1/FLT3-mutated AML. In sum, mutant NPM1 represses monocyte and granulocyte terminal differentiation by disrupting PU.1/CEBPA/RUNX1 collaboration, a transforming action that can be reversed by pharmacodynamically directed dosing of clinical small molecules., Introduction Nucleophosmin (NPM1) is one of the most frequently mutated genes in de novo acute myeloid leukemia (AML) (~30% of cases) (1). Only approximately 50% of patients with NPM1-mutated AML [...]

Mutations underlie all cancers, and their identification and study are the foundation of cancer biology. We describe what we believe to be a novel approach to mutagenesis and cancer studies based on the DNA polymerase s (POLE) ultramutator phenotype recently described in human cancers, in which a single amino acid substitution (most commonly P286R) in the proofreading domain results in error-prone DNA replication. We engineered a conditional [Pole.sup.P286R] allele in mice. [Pole.sup.P286R/+] embryonic fibroblasts exhibited a striking mutator phenotype and immortalized more efficiently. [Pole.sup.P286R/+] mice were born at Mendelian ratios but rapidly developed lethal cancers of diverse lineages, yielding the most cancer-prone monoallelic model described to date, to our knowledge. Comprehensive whole-genome sequencing analyses showed that the cancers were driven by high base substitution rates in the range of human cancers, overcoming a major limitation of previous murine cancer models. These data establish polymerase-mediated ultramutagenesis as an efficient in vivo approach for the generation of diverse animal cancer models that recapitulate the high mutational loads inherent to human cancers., Introduction Eukaryotic DNA replication is astoundingly accurate, with an error rate of only 1 per [10.sup.9] to [10.sup.10] bases, or approximately 1 mutation per cell division. Replication proceeds at DNA [...]

The host immune system plays a pivotal role in the emergence of tumor cells that are refractory to multiple clinical interventions including immunotherapy, chemotherapy, and radiotherapy. Here, we examined the molecular mechanisms by which the immune system triggers cross-resistance to these interventions. By examining the biological changes in murine and tumor cells subjected to sequential rounds of in vitro or in vivo immune selection via cognate cytotoxic T lymphocytes, we found that multimodality resistance arises through a core metabolic reprogramming pathway instigated by epigenetic loss of the ATP synthase subunit ATP5H, which leads to ROS accumulation and HIF-1[alpha] stabilization under normoxia. Furthermore, this pathway confers to tumor cells a stem-like and invasive phenotype. In vivo delivery of antioxidants reverses these phenotypic changes and resensitizes tumor cells to therapy. ATP5H loss in the tumor is strongly linked to failure of therapy, disease progression, and poor survival in patients with cancer. Collectively, our results reveal a mechanism underlying immune-driven multimodality resistance to cancer therapy and demonstrate that rational targeting of mitochondrial metabolic reprogramming in tumor cells may overcome this resistance. We believe these results hold important implications for the clinical management of cancer., Introduction The adaptation of tumor cells to immune surveillance as well as the emergence of refractory tumor cells after initial therapy are responsible for eventual disease relapse, progression, and metastasis. [...]

Anaplastic thyroid carcinomas (ATCs) have a high prevalence of BRAF and TP53 mutations. A trial of vemurafenib in nonmelanoma [BRAF.sup.V600E]-mutant cancers showed significant, although short- lived, responses in ATCs, indicating that these virulent tumors remain addicted to BRAF despite their high mutation burden. To explore the mechanisms mediating acquired resistance to BRAF blockade, we generated mice with thyroid-specific deletion of p53 and dox-dependent expression of [BRAF.sup.V600E], 50% of which developed ATCs after dox treatment. Upon dox withdrawal there was complete regression in all mice, although recurrences were later detected in 85% of animals. The relapsed tumors had elevated MAPK transcriptional output, and retained responses to the MEK/RAF inhibitor CH5126766 in vivo and in vitro. Whole-exome sequencing identified recurrent focal amplifications of chromosome 6, with a minimal region of overlap that included Met. Met- amplified recurrences overexpressed the receptor as well as its ligand Hgf. Growth, signaling, and viability of Met-amplified tumor cells were suppressed in vitro and in vivo by the Met kinase inhibitors PF-04217903 and crizotinib, whereas primary ATCs and Met-diploid relapses were resistant. Hence, recurrences are the rule after BRAF suppression in murine ATCs, most commonly due to activation of HGF/MET signaling, which generates exquisite dependency to MET kinase inhibitors., Introduction Anaplastic thyroid cancers (ATCs) are extraordinarily virulent tumors that affect mostly elderly patients and have a median survival of 6 months. Cytotoxic chemotherapy with antimicrotubule agents and/or anthracyclines in [...]

Epithelial cell dysfunction is postulated as an important component in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mutations in the surfactant protein C (SP-C) gene (SFTPC), an alveolar type II (AT2) cell-restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knockin mouse model capable of regulated expression of an IPF-associated isoleucine-to-threonine substitution at codon 73 (I73T) in Sftpc ([SP-C.sup.I73T]). Tamoxifen-treated [SP-C.sup.I73T] cohorts developed rapid increases in [Sftpc.sup.I73T] mRNA and misprocessed [proSP- C.sup.I73T] protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA content of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGF-[beta]1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, a-smooth muscle actin- positive ([alpha]-SMA-positive) cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP- C expression in vivo causes spontaneous lung fibrosis, strengthening the role of AT2 cell dysfunction as a key upstream driver of IPF pathogenesis., Introduction Idiopathic pulmonary fibrosis (IPF) represents the most common subtype of a larger family of diffuse parenchymal lung diseases (DPLDs) of unknown etiology in adults (1). The incidence of IPF [...]

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the [p40.sup.phox] subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 [p40.sup.phox]-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMAinduced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited [p40.sup.phox] deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD., Introduction Chronic granulomatous disease (CGD) is a recessive primary immunodeficiency (PID) caused by loss-of-function (LOF) mutations of autosomal or X-linked genes encoding 5 components of the phagocyte-reduced NADPH oxidase complex [...]

Although the pathogenesis of Parkinson's disease (PD) remains unclear, mutations in leucine-rich repeat kinase 2 (Lrrk2) are among the major causes of familial PD. Most of these mutations disrupt Lrrk2 kinase and (or) GTPase domain function, resulting in neuronal degeneration. However, the signal pathways underlying Lrrk2-induced neuronal degeneration are not fully understood. There is an expanding body of evidence that suggests a link between Lrrk2 function and MAP kinase (MAPK) cascades. To further investigate this link in vivo, genetic RNAi screens of the MAPK pathways were performed in a Drosophila model to identify genetic modifier(s) that can suppress G2019S-Lrrk2-induced PD-like phenotypes. The results revealed that the knockdown of hemipterous (hep, or JNKK) increased fly survival time, improved locomotor function, and reduced loss of dopaminergic neurons in G2019S-Lrrk2 transgenic flies. Expression of the dominant-negative allele of JNK (JNK-DN), a kinase that is downstream of hep in G2019S-Lrrk2 transgenic flies, elicited a similar effect. Moreover, treatment with the JNK inhibitor SP600125 partially reversed the G2019S-Lrrk2-induced loss of dopaminergic neurons. These results indicate that the hep pathway plays an important role in Lrrk2-linked Parkinsonism in flies. These studies provide new insights into the molecular mechanisms underlying Lrrk2-linked PD pathogenesis and aid in identifying potential therapeutic targets. Key words: Lrrk2, Parkinson's disease, JNK, neuronal degeneration, hep, dopamine neuron, Drosophila model. Meme si la pathogenese de la maladie de Parkinson (MP) demeure floue, des mutations de la proteine Lrrk2 (leucine riche repeat kinase 2) sont parmi les principales causes de la MP hereditaire. La plupart de ces mutations dereglent la fonction des domaines kinase ou GTPase de Lrrk2, resultant en une degeneration neuronale. Toutefois, les voies de signalisation qui sous-tendent la degeneration neuronale induite par Lrrk2 ne sont pas completement comprises. Ilyadeplus en plus de donnees qui suggerent qu'il existe un lien entre la fonction de Lrrk2 et les cascades des MAP kinases (MAPK). Afin d'approfondir ce lien in vivo, des criblages genetiques des voies des MAPK a l'aide d'ARNi ont ete realises chez la drosophile afin d'identifier les modificateurs genetiques qui peuvent supprimer les phenotypes de type parkinsonien induits par G2019S-Lrrk2. Les resultats ont revele que le knockdown de hemipterous (hep ou JNKK) accroissait la duree de vie des mouches, ameliorait la fonction locomotrice et reduisait la perte de neurones dopaminergiques chez les mouches transgeniques G2019S-Lrrk2. L'expression de l'allele dominant negatif de JNK (JNK-DN), une kinase qui agit en aval de hep chez les mouches transgeniques G2019S-Lrrk2, provoquait un effet similaire. De plus, un traitement avec l'inhibiteur de JNK SP600125 renversait partiellement la perte de neurones dopaminergiques induite par G2019S-Lrrk2. Ces resultats indiquent que la voie de hep joue un role important dans le parkinsonisme lie a Lrrk2 chez les mouches. Ces etudes apportent un eclairage nouveau sur les mecanismes moleculaires qui sous-tendent la pathogenese de la MP liee a Lrrk2 et contribuent a identifier des cibles therapeutiques potentielles. [Traduit par la Redaction] Mots-cles : Lrrk2, maladie de Parkinson, JNK, degeneration neuronale, hep, neurone a dopamine, modele de la drosophile., Introduction Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. The clinical [...]

Lamins A and C are involved in many cellular functions, owing to its ability to bind chromatin and transcription factors and affect their properties. Mutations of the LMNA gene encoding lamin A/C affect differentiation capacity of stem cells. However, the signaling pathways involved in interactions with lamins during cellular differentiation remain unclear. Lipodystrophy associated with LMNA mutation R482L causes loss of fat tissue. In this study we investigated the role of LMNA mutation R482L in modulating Notch signaling activity in the adipogenic differentiation of mesenchymal stem cells. Notch was activated using lentiviral Notch intracellular domain. Activation of Notch was estimated through the expression of Notch-responsive genes by qPCR and by activation of a luciferase CSL-reporter construct. The effect of LMNA mutation on Notch activation and adipogenic differentiation was analyzed in cells bearing lentiviral LMNA WT or LMNA R482L. We show that, when Notch is activated, LMNA R482L contributes to down-regulation of Notch activation in undifferentiated and differentiated cells, and decreases adipogenic differentiation. Thus, lamin A/C interacts with Notch signaling, thereby influencing cellular differentiation, and point mutation in LMNA could halt this interaction. Key words: lamin A/C, Notch signaling, Dunnigan-type familial partial lipodystrophy, adipogenic differentiation. La lamine A/C intervient dans plusieurs fonctions cellulaires a cause de sa capacite a lier la chromatine et des facteurs de transcription et a affecter leurs proprietes. Des mutations du gene LMNA qui code la lamine A/C affectent les capacites de differenciation des cellules souches. Toutefois, les voies de signalisation qui interagissent avec les lamines lors de la differenciation cellulaire demeurent peu claires. La lipodystrophie associee a la mutation R482L de LMNA provoque une perte de tissu graisseux. Dans cette etude, les auteurs ont examine le role de la mutation R482L de LMNA dans la modulation de l'activite de la signalisation de Notch dans la differenciation adipogenique des cellules souches mesenchymateuses. Notch a ete active a l'aide du domaine intracellulaire de Notch exprime dans un lentivirus. L'activation de Notch a ete estimee par l'expression de genes repondant a Notch par qPCR et par l'activation d'un rapporteur CSL-luciferase. L'effet de la mutation de LMNA sur l'activation de Notchetladifferenciation adipogeniqueaete analyse dans des cellules portantunlentivirus comprenantLMNAsauvage ouLMNA R482L. Les auteurs montrent que LMNA R482L contribue a reguler a la baisse l'activation de Notch dans les cellules non differenciees et differenciees et qu'il diminue la differenciation adipogenique lorsque Notch est active. Ainsi, la lamine A/C interagit avec la signalisation de Notch influencant par consequent la differenciation cellulaire, et une mutation ponctuelle de LMNA pourrait interrompre cette interaction. [Traduit par la Redaction] Mots-cles : lamine A/C, signalisation de Notch, lipodystrophie partielle familiale de type Dunnigan, differenciation adipogenique., Introduction The nuclear lamina consists of intermediate filament proteins called lamins and primarily localizes at the inner side of the nuclear envelope (Aebi et al. 1986). Most differentiated somatic cells [...]

Somatic mutations in tet methylcytosinedioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies.sup.1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage.sup.1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2.sup.-/- mice.sup.8,9 and humans with TET2 mutations.sup.1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2.sup.-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2.sup.-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2.sup.-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.Microbial signals are crucial to the development of pre-leukaemic myeloproliferation, which can be induced by disrupting the intestinal barrier or by introducing systemic bacterial stimuli in Tet2-deficient mice., Author(s): Marlies Meisel [sup.1] [sup.2] , Reinhard Hinterleitner [sup.1] [sup.2] , Alain Pacis [sup.3] [sup.4] , Li Chen [sup.1] [sup.2] , Zachary M. Earley [sup.1] [sup.2] , Toufic Mayassi [sup.1] [...]

* Context.--Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective.--To investigate the feasibility of using next-generation sequencing (NgS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000X-500 000X) sequencing (NGS-qNPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design.--A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-housedesigned bioinformatic pipeline. NGS-qNPM1 was com pared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). Results.--The NGS-qNPM1 assay had a sensitivity of between [10.sup.-4] and [10.sup.-5] and showed high concordance and correlation with reference methodologies. Moreover, the NGS-qNPM1 assay was able to be integrated into the laboratory's existing, targeted amplicon-based sequencing workflow. Conclusions.--An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities. (Arch Pathol Lab Med. 2018;142:606-612; doi: 10.5858/arpa.2017-0229-0A), Assessment of the response of acute myeloid leukemia (AML) to intensive therapy is central to therapeutic decision making. It is well-established that detection of measurable residual disease (MRD) after remission-induction [...]

Immune evasion and the suppression of antitumor responses during cancer progression are considered hallmarks of cancer and are typically attributed to tumor-derived factors. Although the molecular basis for the crosstalk between tumor and immune cells is an area of active investigation, whether host-specific germline variants can dictate immunosuppressive mechanisms has remained a challenge to address. A commonly occurring germline mutation (c.1162G>A/rs351855 G/A) in the FGFR4 (CD334) gene enhances signal transducer and activator of transcription 3 (STAT3) signaling and is associated with poor prognosis and accelerated progression of multiple cancer types. Here, using rs351855 SNP-knockin transgenic mice and Fgfr4-knockout mice, we reveal the genotype-specific gain of immunological function of suppressing the CD8/ [CD4.sup.*][FOXP3.sup.+][CD25.sup.*] regulatory T cell ratio in vivo. Furthermore, using knockin transgenic mouse models for lung and breast cancers, we establish the host-specific, tumor-extrinsic functions of STAT3-enhancing germline variants in impeding the tumor infiltration of CD8 T cells. Thus, STAT3-enhancing germline receptor variants contribute to immune evasion through their pleiotropic functions in immune cells., Introduction Immune evasion is considered the hallmark of cancers (1). Strategies that restore the capacity of the immune system to recognize and eliminate malignant cells have produced clinical benefits. However, [...]

In these studies, we evaluated the contribution of the NLRP3 inflammasome to Crohn's disease (CD) in a kindred containing individuals having a missense mutation in CARD8, a protein known to inhibit this inflammasome. Whole exome sequencing and PCR studies identified the affected individuals as having a V44I mutation in a single allele of the T60 isoform of CARD8. The serum levels of IL-1[beta] in the affected individuals were increased compared with those in healthy controls, and their peripheral monocytes produced increased amounts of IL-1[beta] when stimulated by NLRP3 activators. Immunoblot studies probing the basis of these findings showed that mutated T60 CARD8 failed to downregulate the NLRP3 inflammasome because it did not bind to NLRP3 and inhibit its oligomerization. In addition, these studies showed that mutated T60 CARD8 exerted a dominant-negative effect by its capacity to bind to and form oligomers with unmutated T60 or T48 CARD8 that impeded their binding to NLRP3. Finally, inflammasome activation studies revealed that intact but not mutated CARD8 prevented NLRP3 deubiquitination and serine dephosphorylation. CD due to a CARD8 mutation was not effectively treated by anti-TNF-[alpha], but did respond to IL-1[beta] inhibitors. Thus, patients with anti-TNF- [alpha]-resistant CD may respond to this treatment option., Introduction The influence of genetic factors in the pathogenesis of inflammatory bowel disease (IBD) was made dramatically evident by the identification of numerous IBD-associated risk polymorphisms, i.e., SNPs in various [...]

Author(s): Amritha Nair [1, 2]; Hsiang-Ching Chung [1, 2, 3]; Tingting Sun [1, 2]; Siddhartha Tyagi [1, 2]; Lacey E Dobrolecki [4]; Rocio Dominguez-Vidana [1, 2, 3]; Sarah J Kurley [...]

Author(s): Michael Seiler [1]; Akihide Yoshimi [2]; Rachel Darman [1]; Betty Chan [1]; Gregg Keaney [1]; Michael Thomas [1]; Anant A Agrawal [1]; Benjamin Caleb [1]; Alfredo Csibi [1]; Eckley [...]

Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD[sup.+] leukemia cells. This synergized with the allogeneic CD8[sup.+] T cell response, leading to long-term survival in six mouse models of FLT3-ITD[sup.+] AML. Sorafenib-related IL-15 production caused an increase in CD8[sup.+]CD107a[sup.+]IFN-[gamma][sup.+] T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD[sup.+] AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8[sup.+] T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT., Author(s): Nimitha R Mathew [1, 2]; Francis Baumgartner [1]; Lukas Braun [1]; David O'Sullivan [3]; Simone Thomas [4]; Miguel Waterhouse [1]; Tony A Müller [1]; Kathrin Hanke [1, 2]; Sanaz [...]

The nonerythrocytic [alpha]-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein [alpha]II spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of [alpha]II spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that [alpha]II spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that [alpha]II spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate [alpha]II spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5., Introduction Early infantile epileptic encephalopathies (EIEEs) are a devastating group of epilepsies characterized by refractory seizures and cognitive arrest or regression, and they typically carry a poor prognosis (1). Recent [...]

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of [Jak2.sup.V617F] and mutant [IDH1.sup.R132H] or [Idh2.sup.R140Q] induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. [Jak2.sup.V617F] [Idh2.sup.R140Q]-mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both [JAK2.sup.mut] and [IDH2.sup.mut] mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype., Introduction Myeloproliferative neoplasms (MPNs) are clonal myeloid malignancies characterized by somatic mutations acquired in hematopoietic stem/progenitor cells, which drive the expansion of 1 or more myeloid lineages. Although patients with [...]

Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the C-terminally retracted (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding., Author(s): Alexander F. Schubert [1]; Christina Gladkova [1]; Els Pardon [2, 3]; Jane L. Wagstaff [1]; Stefan M. V. Freund [1]; Jan Steyaert [2, 3]; Sarah L. Maslen [1]; David [...]

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at TusTer, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with 10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer., Author(s): Nicholas A. Willis [1]; Richard L. Frock [2]; Francesca Menghi [3]; Erin E. Duffey [1]; Arvind Panday [1]; Virginia Camacho [4]; E. Paul Hasty [5]; Edison T. Liu [3, [...]

The tumour suppressor complex BRCA1BARD1 functions in the repair of DNA double-stranded breaks by homologous recombination. During this process, BRCA1BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumour suppressor complex, BRCA2PALB2, and the recombinase RAD51. Here, by examining purified wild-type and mutant BRCA1BARD1, we show that both BRCA1 and BARD1 bind DNA and interact with RAD51, and that BRCA1BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. We provide evidence that BRCA1 and BARD1 are indispensable for RAD51 stimulation. Notably, BRCA1BARD1 mutants with weakened RAD51 interactions show compromised DNA joint formation and impaired mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1BARD1 in homologous recombination, an attribute of the tumour suppressor complex that could be targeted in cancer therapy., Author(s): Weixing Zhao (corresponding author) [1]; Justin B. Steinfeld [2]; Fengshan Liang [1, 3, 4]; Xiaoyong Chen [3, 4]; David G. Maranon [5]; Chu Jian Ma [2]; Youngho Kwon [1]; [...]

Author(s): Rebekah Tillotson [1]; Jim Selfridge [1]; Martha V. Koerner [1]; Kamal K. E. Gadalla [2, 3]; Jacky Guy [1]; Dina De Sousa [1]; Ralph D. Hector [2]; Stuart R. [...]

Mutations in MECP2 cause Rett syndrome (RTT), an X-linked neurological disorder characterized by regressive loss of neurodevelopmental milestones and acquired psychomotor deficits. However, the cellular heterogeneity of the brain impedes an understanding of how MECP2 mutations contribute to RTT. Here we developed a Cre-inducible method for cell-type-specific biotin tagging of MeCP2 in mice. Combining this approach with an allelic series of knock-in mice carrying frequent RTT-associated mutations (encoding T158M and R106W) enabled the selective profiling of RTT-associated nuclear transcriptomes in excitatory and inhibitory cortical neurons. We found that most gene-expression changes were largely specific to each RTT-associated mutation and cell type. Lowly expressed cell-type-enriched genes were preferentially disrupted by MeCP2 mutations, with upregulated and downregulated genes reflecting distinct functional categories. Subcellular RNA analysis in MeCP2-mutant neurons further revealed reductions in the nascent transcription of long genes and uncovered widespread post-transcriptional compensation at the cellular level. Finally, we overcame X-linked cellular mosaicism in female RTT models and identified distinct gene-expression changes between neighboring wild-type and mutant neurons, providing contextual insights into RTT etiology that support personalized therapeutic interventions., Author(s): Brian S Johnson [1]; Ying-Tao Zhao [1]; Maria Fasolino [1]; Janine M Lamonica [1]; Yoon Jung Kim [2]; George Georgakilas [1]; Kathleen H Wood [1]; Daniel Bu [1]; Yue [...]

Author(s): Elodie Bal (corresponding author) [1, 2]; Hyun-Sook Park [3]; Zakia Belaid-Choucair [1, 2, 4]; Hülya Kayserili [5, 6]; Magali Naville [7, 8, 9]; Marine Madrange [1, 2]; Elena Chiticariu [...]

Acute myelogenous leukemia (AML) frequently relapses after complete remission (CR), necessitating Improved detection and phenotypic characterization of treatment-resistant residual disease. In this work, we have optimized droplet digital PCR to broadly measure mutated alleles of recurrently mutated genes in CR marrows of AML patients at levels as low as 0.002% variant allele frequency. Most gene mutations persisted in CR, albeit at highly variable and gene-dependent levels. The majority of AML cases demonstrated residual aberrant oligoclonal hematopoiesis. Importantly, we detected very rare cells (as few as 1 in 15,000) that were genomically similar to the dominant blast populations at diagnosis and were fully clonally represented at relapse, identifying these rare cells as one common source of AML relapse. Clinically, the mutant allele burden was associated with overall survival in AML, and our findings narrow the repertoire of gene mutations useful in minimal residual disease-based prognostication in AML. Overall, this work delineates rare cell populations that cause AML relapse, with direct implications for AML research directions and strategies to improve AML therapies and outcome., Introduction Acute myelogenous leukemia (AML) affects 21,000 new patients yearly in the US and causes 10,000 premature deaths. While most patients with AML achieve complete remission (CR) with induction chemotherapy, [...]

Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in PKD1 and PKD2 genes. Recent work suggests that epigenetic modulation of gene expression and protein function may play a role in ADPKD pathogenesis. In this study, we identified SMYD2, a SET and MYND domain protein with lysine methyltransferase activity, as a regulator of renal cyst growth. SMYD2 was upregulated in renal epithelial cells and tissues from Pkd1-knockout mice as well as in ADPKD patients. SMYD2 deficiency delayed renal cyst growth in postnatal kidneys from Pkd1 mutant mice. Pkd1 and Smyd2 double-knockout mice lived longerthan Pkd1-knockout mice. Targeting SMYD2 with its specific inhibitor, AZ505, delayed cyst growth in both early-and later-stage Pkd1 conditional knockout mouse models. SMYD2 carried out its function via methylation and activation of STAT3 and the p65 subunit of NF-[kappa]B, leading to increased cystic renal epithelial cell proliferation and survival. We further identified two positive feedback loops that integrate epigenetic regulation and renal inflammation in cyst development: SMYD2/IL-G/STAT3/SMYD2 and SMYD2/TNF-[alpha]/NF-[kappa]B/SMYD2. These pathways provide mechanisms by which SMYD2 might be induced by cyst fluid IL-6 and TNF-[alpha] in ADPKD kidneys. The SMYD2 transcriptional target gene Ptpn13 also linked SMYD2 to other PKD-associated signaling pathways, including ERK, mTOR, and Alct signaling, via PTPN13 mediated Phosphorylation., Introduction Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in one of two genes, PKD1 or PKD2, which encode the proteins polycystin-1 (PCI) and polycystin-2 (PC2), respectively (1,2). [...]

Huntington's disease is a neurodegenerative disorder caused by a polyglutamine repeat in the Huntingtin gene (HTT). Although suppressing the expression of mutant HTT (mHTT) has been explored as a therapeutic strategy to treat Huntington's disease, considerable efforts have gone into developing allele-specific suppression of mHTT expression, given that loss of Htt in mice can lead to embryonic lethality. It remains unknown whether depletion of HTT in the adult brain, regardless of its allele, could be a safe therapy. Here, we report that permanent suppression of endogenous mHTT expression in the striatum of mHTT-expressing mice (HD140Q-knockin mice) using CRISPR/Cas9-mediated inactivation effectively depleted HTT aggregates and attenuated early neuropathology. The reduction of mHTT expression in striatal neuronal cells in adult HD140Q-knockin mice did not affect viability, but alleviated motor deficits. Our studies suggest that non-allele-specific CRISPR/Cas9-mediated gene editing could be used to efficiently and permanently eliminate polyglutamine expansion-mediated neuronal toxicity in the adult brain., Introduction Expansion of a CAG/glutamine repeat in various genes causes at least 9 different neurodegenerative diseases, including Huntington's disease (HD). In HD, the expanded CAG repeat encodes a polyglutamine (polyQ) [...]

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double -strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNAPK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA-and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients., Introduction Currently available antileukemic treatments often fail to eradicate drug-refractory quiescent leukemia stem cells (LSCs) and drug-resistant proliferating LSCs and leukemia progenitor cells (LPCs). Previous reports suggest that altered DNA [...]

Mutations of the splicing factor-encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common [U2AF1.sup.S34F] mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that [U2AF1.sup.S34F] expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that [U2AF1.sup.S34F] induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. [U2AF1.sup.S34F] altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the [U2AF1.sup.S34F] target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor-associated protein, recapitulated phenotypes associated with [U2AF1.sup.S34F] expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in [U2AF1.sup.S34F] MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities., Introduction The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by ineffective hematopoiesis resulting in peripheral blood cytopenias of the myeloid lineage, including anemia [...]

Extensive 3' alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splice variants. However, their behavioral relevance remains unknown. The present study generated 3 mutant mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129). One mouse truncated all C termini downstream of Oprml exon 3 (mE3M mice), while the other two selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice). Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7-associated truncation diminished morphine tolerance and reward without altering physical dependence, whereas the exon 4-associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement in morphine tolerance. In cell-based studies, exon 7-associated variants shifted the bias of several mu opioids toward [beta]-arrestin 2 over G protein activation compared with the exon 4-associated variant, suggesting an interaction of exon 7-associated C-terminal tails with [beta]-arrestin 2 in morphine-induced desensitization and tolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacological importance of OPRM1 3' alternative splicing., Introduction Most clinically used opiates, including morphine, act through the mu opioid receptor (1), producing both potent analgesia and a number of side effects, such as tolerance, physical dependence, constipation, [...]

Dowling-Degos disease (DDD) is an autosomal-dominant disorder of skin pigmentation associated with mutations in keratin 5 (KRT5), protein O-fucosyltransferase 1 (POFUT1), or protein O-glucosyltransferase 1 (POGLUT1). Here, we have identified 6 heterozygous truncating mutations in PSENEN, encoding presenilin enhancer protein 2, in 6 unrelated patients and families with DDD in whom mutations in KRT5, POFUT1, and POGLUT1 have been excluded. Further examination revealed that the histopathologic feature of follicular hyperkeratosis distinguished these 6 patients from previously studied individuals with DDD. Knockdown of psenen in zebrafish larvae resulted in a phenotype with scattered pigmentation that mimicked human DDD. In the developing zebrafish larvae, in vivo monitoring of pigment cells suggested that disturbances in melanocyte migration and differentiation underlie the DDD pathogenesis associated with PSENEN. Six of the PSENEN mutation carriers presented with comorbid acne inversa (AI), an inflammatory hair follicle disorder, and had a history of nicotine abuse and/or obesity, which are known trigger factors for AI. Previously, PSENEN mutations were identified in familial AI, and comanifestation of DDD and AI has been reported for decades. The present work suggests that PSENEN mutations can indeed cause a comanifestation of DDD and AI that is likely triggered by predisposing factors for AI. Thus, the present report describes a DDD subphenotype in PSENEN mutation carriers that is associated with increased susceptibility to AI., Introduction Skin pigmentation is a complex process in which the location, degree, and type of pigmentation provides a visual manifestation of genetic heterogeneity in pathways regulating the function of melanocytes. [...]

Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer gain-of-function activity by converting [alpha]-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte-associated genes and IFN-[gamma]-inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas., Introduction Gliomas are the most common primary brain tumors and are typically classified on the basis of WHO criteria as grades I-IV, in order of increasing anaplasia (1). Grade IV [...]

Cervical cancer remains one of the leading causes of cancer-related deaths worldwide. Here we report the extensive molecular characterization of 228 primary cervical cancers, one of the largest comprehensive genomic studies of cervical cancer to date. We observed notable APOBEC mutagenesis patterns and identified SHKBP1, ERBB3, CASP8, HLA-A and TGFBR2 as novel significantly mutated genes in cervical cancer. We also discovered amplifications in immune targets CD274 (also known as PD-L1) and PDCD1LG2 (also known as PD-L2), and the BCAR4 long non-coding RNA, which has been associated with response to lapatinib. Integration of human papilloma virus (HPV) was observed in all HPV18-related samples and 76% of HPV16-related samples, and was associated with structural aberrations and increased target-gene expression. We identified a unique set of endometrial-like cervical cancers, comprised predominantly of HPV-negative tumours with relatively high frequencies of KRAS, ARID1A and PTEN mutations. Integrative clustering of 178 samples identified keratin-low squamous, keratin-high squamous and adenocarcinoma-rich subgroups. These molecular analyses reveal new potential therapeutic targets for cervical cancers., Author(s): The Cancer Genome Atlas Research Network; Albert Einstein College of Medicine; Robert D. Burk [1]; Zigui Chen [1]; Analytical Biological Services; Charles Saller [2]; Katherine Tarvin [2]; Barretos Cancer [...]

Drug-resistant tuberculosis (TB) poses a significant threat to global health, with an estimated 480,000 new cases of multidrug-resistant tuberculosis (MDR TB) in 2014; 10% of these cases were classified as [...]

Author(s): Ajit Vikram [1]; Christopher M Lewarchik [2]; Jin-Young Yoon [1]; Asma Naqvi [2]; Santosh Kumar [1]; Gina M Morgan [1]; Julia S Jacobs [1]; Qiuxia Li [1]; Young-Rae Kim [...]