👤 C F Singer

Approximately 40% of patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-), advanced breast cancer (ABC) have PIK3CA alterations, which contributes to endocrine therapy resistance. Alpelisib, an α-selective phosphatidylinositol 3-kinase inhibitor and degrader, given in combination with fulvestrant, is approved for the treatment of PIK3CA-mutated, HR+, HER2- ABC, based on the SOLAR-1 trial. Aside from PIK3CA, other gene alterations are associated with poor prognosis and limited response to treatment in this patient population. In this retrospective analysis, we performed tissue-based next-generation sequencing of 398 patients (237 PIK3CA-altered, 161 PIK3CA-wild type) from SOLAR-1. Progression-free survival (PFS) correlative analysis was performed in the PIK3CA-altered cohort. PIK3CA-altered and PIK3CA-wild type tumors had distinct genomic profiles. In the PIK3CA-altered cohort, patients who received alpelisib plus fulvestrant had a median PFS (mPFS) of 11.01 months versus 5.55 months for those receiving placebo plus fulvestrant (P=0.0004). Patients in the lowest tumor mutational burden quartile as well as those with FGFR1 or FGFR2 alterations derived greater PFS benefit from alpelisib plus fulvestrant versus placebo plus fulvestrant (18.5 versus 3.22 months; HR 0.38; 95% CI 0.21-0.68. FGFR1 12.71 versus 3.75 months; HR 0.38; 95% CI 0.17-0.81; P=0.32. FGFR2: 9.63 versus 2.78 months; HR 0.31; 95% CI 0.1-0.94; P=0.29); patients with MYC or RAD21 alterations derived limited PFS benefit. Cox and multi-task machine learning models identified lower Eastern Cooperative Oncology Group performance status, prior cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) treatment, and PTEN or TP53 alterations among the most deleterious factors for PFS in the PIK3CA-altered cohort. Alpelisib plus fulvestrant provides clinical benefit for patients with PIK3CA-altered, HR+, HER2- ABC across a range of concomitant alterations, including those previously implicated in endocrine therapy or CDK4/6i resistance. Machine learning models identified factors including gene mutations that influenced PFS. Show less

Infiltration of adipocytes into the pancreatic parenchyma has been linked to impaired insulin secretion in individuals with increased genetic risk of T2D and prediabetic conditions. However, the study of this ectopic fat depot has been limited by the lack of suitable in vitro models. Here, we developed a novel 3D model of functionally mature human pancreatic adipose tissue organoids by aggregating human pancreatic adipose tissue-derived stromal vascular fraction (SVF) cells into organoids and differentiating them over 19 days. These organoids carry biological properties of the in situ pancreatic fat, presenting levels of adipogenic markers comparable to native pancreatic adipocytes and improved lipolytic and anti-lipolytic response compared to conventional 2D cultures. The organoids harbour a small population of immune cells, mimicking in vivo adipose environment. Furthermore, they express GIPR, allowing investigation of incretin effects in pancreatic fat. In accordance, GIP and the dual GLP1R/GIPR agonist tirzepatide stimulate lipolysis but had distinct effects on the expression of proinflammatory cytokines. This novel adipose organoid model is a valuable tool to study the metabolic impact of incretin signalling in pancreatic adipose tissue, revealing potential therapeutic targets of incretins beyond islets. The donor-specific metabolic memory of these organoids enables examination of the pancreatic fat-islet crosstalk in a donor-related metabolic context. Show less

The GramAdapt Social Contact Dataset is a curated dataset of 34 language pairs with qualitative and quantifiable data on social interaction and aspects of societal multilingualism. The language pairs were sampled globally to represent the world's linguistic diversity. The dataset can be used to interrogate the social dimensions of language contact independently or in conjunction with appropriate linguistic data. The data were collected by distributing a questionnaire to experts who have experience with either one or both of the language communities of a pair. The data represent subjective expert assessments based on choices from predetermined answers which can be quantified. Authors 1, 2 and 3 manually checked the response to identify possible misjudgments or misunderstandings. This results in a dataset containing 13,493 data points. This dataset is a first of its kind in the field of linguistics, built upon wide findings from sociolinguistics, historical linguistics, psycholinguistics, and linguistic anthropology. Show less

We conducted research on CDK4/6 inhibitors (CDK4/6i) simultaneously in the preclinical and clinical spaces to gain a deeper understanding of how senescence influences tumor growth in humans. We coordinated a first-in-kind phase II clinical trial of the CDK4/6i abemaciclib for patients with progressive dedifferentiated liposarcoma (DDLS) with cellular studies interrogating the molecular basis of geroconversion. Thirty patients with progressing DDLS enrolled and were treated with 200 mg of abemaciclib twice daily. The median progression-free survival was 33 weeks at the time of the data lock, with 23 of 30 progression-free at 12 weeks (76.7%, two-sided 95% CI, 57.7%-90.1%). No new safety signals were identified. Concurrent preclinical work in liposarcoma cell lines identified ANGPTL4 as a necessary late regulator of geroconversion, the pathway from reversible cell-cycle exit to a stably arrested inflammation-provoking senescent cell. Using this insight, we were able to identify patients in which abemaciclib induced tumor cell senescence. Senescence correlated with increased leukocyte infiltration, primarily CD4-positive cells, within a month of therapy. However, those individuals with both senescence and increased TILs were also more likely to acquire resistance later in therapy. These suggest that combining senolytics with abemaciclib in a subset of patients may improve the duration of response. Abemaciclib was well tolerated and showed promising activity in DDLS. The discovery of ANGPTL4 as a late regulator of geroconversion helped to define how CDK4/6i-induced cellular senescence modulates the immune tumor microenvironment and contributes to both positive and negative clinical outcomes. See related commentary by Weiss et al., p. 649. Show less

There is an incomplete understanding of the burden of splice-disrupting variants in definitively associated inherited heart disease genes and whether these genes can amplify from blood RNA to support functional confirmation of splicing outcomes. We performed burden testing of rare splice-disrupting variants in people with inherited heart disease and sudden unexplained death compared to 125,748 population controls. ClinGen definitively disease-associated inherited heart disease genes were amplified using RNA extracted from fresh blood, derived cardiomyocytes, and myectomy tissue. Variants were functionally assessed and classified for pathogenicity. We found 88 in silico-predicted splice-disrupting variants in 128 out of 1242 (10.3%) unrelated participants. There was an excess burden of splice-disrupting variants in PKP2 (5.9%), FLNC (2.7%), TTN (2.8%), MYBPC3 (8.2%) and MYH7 (1.3%), in distinct cardiomyopathy subtypes, and KCNQ1 (3.6%) in long QT syndrome. Blood RNA supported the amplification of 21 out of 31 definitive disease-associated inherited heart disease genes. Our functional studies confirmed altered splicing in six variants. Eleven variants of uncertain significance were reclassified as likely pathogenic based on functional studies and six were used for cascade genetic testing in 12 family members. Our study highlights that splice-disrupting variants are a significant cause of inherited heart disease, and that analysis of blood RNA confirms splicing outcomes and supports variant pathogenicity classification. Show less

In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis. Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues. Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor. The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma. Show less

Transcriptome sequencing can improve genetic diagnosis of Mendelian diseases but requires access to tissue expressing disease-relevant transcripts. We explored genetic testing of hypertrophic cardiomyopathy using transcriptome sequencing of patient-specific human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). We also explored whether antisense oligonucleotides (AOs) could inhibit aberrant mRNA splicing in hiPSC-CMs. We derived hiPSC-CMs from patients with hypertrophic cardiomyopathy due to Transcriptome sequencing of hiPSC-CMs confirmed aberrant splicing in 2 people with previously identified Transcriptome sequencing of patient specific hiPSC-CMs solved a previously undiagnosed genetic cause of hypertrophic cardiomyopathy and may be a useful adjunct approach to genetic testing. Antisense oligonucleotide inhibition of cryptic exon splicing is a potential future personalized therapeutic option. Show less

Copy-number variant (CNV) analysis is increasingly performed in genetic diagnostics. We leveraged recent gene curation efforts and technical standards for interpretation and reporting of CNVs to characterize clinically relevant CNVs in patients with inherited heart disease and sudden cardiac death. Exome sequencing data were analyzed for CNVs using eXome-Hidden Markov Model tool in 48 established disease genes. CNV breakpoint junctions were characterized. CNVs were classified using the American College of Medical Genetics and Genomics technical standards. We identified eight CNVs in 690 unrelated probands (1.2%). Characterization of breakpoint junctions revealed nonhomologous end joining was responsible for four deletions, whereas one duplication was caused by nonallelic homologous recombination between duplicated sequences in MYH6 and MYH7. Identifying the precise breakpoint junctions determined the genomic involvement and proved useful for interpreting the clinical relevance of CNVs. Three large deletions involving TTN, MYBPC3, and KCNH2 were classified as pathogenic in three patients. Haplotype analysis of a deletion in ACTN2, found in two families, suggests the deletion was caused by an ancestral event. CNVs infrequently cause inherited heart diseases and should be investigated when standard genetic testing does not reveal a genetic diagnosis. Show less

MYBPC3 splicing errors are a common cause of hypertrophic cardiomyopathy (HCM). Variants affecting essential splice-site dinucleotides inhibit splicing, whereas the impact of variants at conserved flanking nucleotides is less clear. We evaluated the contribution of MYBPC3 splice-site variants in a large cohort of patients with HCM and assessed the impact on splicing with RNA analysis. Patients attending a specialized multidisciplinary clinic, with a clinical diagnosis of HCM and genetic testing of at least 46 cardiomyopathy-associated genes, were included. Patients with variants in MYBPC3 splice sites with in silico-predicted effects on splicing were selected. RNA was extracted from fresh venous blood or paraffin-embedded myocardial tissue of the patients, amplified, and sequenced. Variants were classified for pathogenicity using the American College of Medical Genetics and Genomics guidelines. We found 29 rare MYBPC3 splice-site variants in 56 of 557 (10%) unrelated HCM probands. Three variants were not predicted to alter RNA splicing, and 13 essential splice dinucleotide, nonsense, and short insertion or deletion variants were not further assessed. RNA analysis was performed on 9 variants (c.654+5G>C, c.772G>A, c.821+3G>T, c.927-9G>A, c.1090G>A, c.1624G>A, c.1624+4A>T, c.3190+5G>A, and c.3491-3C>G), and RNA splicing errors were confirmed for 7. Four variants in 4 families resulted in clinically meaningful reclassifications. After RNA analysis, 4 of 56 (7%) families with MYBPC3 splice-site variants were reclassified from uncertain clinical significance to likely pathogenic. RNA analysis of splice-site variants can assist in understanding pathogenicity and increase the diagnostic yield of genetic testing in HCM. Show less

Hey2 gene mutations in both humans and mice have been associated with multiple cardiac defects. However, the currently reported localization of Hey2 in the ventricular compact zone cannot explain the wide variety of cardiac defects. Furthermore, it was reported that, in contrast to other organs, Notch doesn't regulate Hey2 in the heart. To determine the expression pattern and the regulation of Hey2, we used novel methods including RNAscope and a Hey2 Show less

The healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs comprises somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition-independent rearrangements in inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2 (also called PSD93), and affect 44-63% of cells of the cells in the healthy brain. Show less

Isoniazid (INH), the mainstay therapeutic for tuberculosis infection, has been associated with rare but serious hepatotoxicity in the clinic. However, the mechanisms underlying inter-individual variability in the response to this drug have remained elusive. A genetically diverse mouse population model in combination with a systems biology approach was utilized to identify transcriptional changes, INH-responsive metabolites, and gene variants that contribute to the liver response in genetically sensitive individuals. Sensitive mouse strains developed severe microvesicular steatosis compared with corresponding vehicle control mice following 3 days of oral treatment with INH. Genes involved in mitochondrial dysfunction were enriched among liver transcripts altered with INH treatment. Those associated with INH treatment and susceptibility to INH-induced steatosis in the liver included apolipoprotein A-IV, lysosomal-associated membrane protein 1, and choline phosphotransferase 1. These alterations were accompanied by metabolomic changes including reduced levels of glutathione and the choline metabolites betaine and phosphocholine, suggesting that oxidative stress and reduced lipid export may additionally contribute to INH-induced steatosis. Finally, genome-wide association mapping revealed that polymorphisms in perilipin 2 were linked to increased triglyceride levels following INH treatment, implicating a role for inter-individual differences in lipid packaging in the susceptibility to INH-induced steatosis. Taken together, our data suggest that INH-induced steatosis is caused by not one, but multiple events involving lipid retention in the livers of genetically sensitive individuals. This work also highlights the value of using a mouse diversity panel to investigate drug-induced responses across a diverse population. Show less

Immune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique proapoptotic protein signature (Puma⁺⁺⁺p-Bim⁺⁺p-JNK⁺⁺DUSP6⁻) and repressed expression of prosurvival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only ∼1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3⁺CD25⁻ Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a proapoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus. Show less

Migration and homing of DCs to lymphoid organs is pivotal for inducing adaptive immunity and tolerance. DC homing depends on the chemokine receptor CCR7. However, expression of CCR7 alone is not sufficient for effective DC migration. A second signal, mediated by prostaglandin E(2) (PGE(2)), is critical for the development of a migratory DC phenotype. PGE(2) is important for inducing efficient immune responses, but, if deregulated, contributes to chronic inflammation, autoimmune diseases through Th17-cell development and tumorigenesis. In contrast, activation of liver X receptor (LXR)α has recently been shown to interfere with CCR7 expression and migration of DCs resulting in a reduced immune response. Here, we demonstrate that PGE(2) downregulates LXRα expression in human monocyte derived as well as ex vivo DCs. Moreover, PGE(2) stimulation dampens LXR activation, auto-regulation and LXR-mediated gene transcription. Consequently, we show that PGE(2) enhances CCR7 expression and migration of LXR-activated DCs. Furthermore, we provide evidence that PGE(2) signaling and LXR activation specifically elicit converse effects on CCR7 expression and DC migration. In contrast, production of MMP9, CCL4, COX-2, and IL-23 is solely regulated by PGE(2) , but not by LXR activation, offering new perspectives for therapeutic interventions. Show less

Preliminary studies have shown that systemic beta-human chorionic gonadotrophin (betaHCG) therapy alleviates endometriosis-related chronic pelvic pain. The underlying mechanism, however, is completely unknown. This study has investigated the dose-dependent alterations in the overall gene expression profile of endometriosis-derived stromal cells under increasing concentrations of betaHCG by using the Affymetrix GeneChip U133 Set. It has been previously shown that betaHCG concentrations of 0.1U/ml and higher lead to a significant and dose-dependent increase in the expression of 68 genes. This study reports on a cluster analysis which identified three clusters of genes with a comparable expression pattern in response to increasing concentrations of betaHCG. Most of the up-regulated genes encoded proteins that are involved in cell adhesion, intercellular communication, extracellular matrix remodelling, apoptosis and inflammation. Stromal monocultures from eight patients, treated with and without 50U/ml of betaHCG, were then incubated and real-time polymerase chain reaction for the highly up-regulated genes PAI2, DUSP6, PLAU and MMP1 performed in order to validate the cDNA array findings in patients with endometriosis. Taken together, this study shows that betaHCG induces dose-dependent characteristic response clusters in the gene expression profile of stromal cells obtained from endometriotic lesions which could explain the differential biological responses of betaHCG in endometriosis. Show less

Retinal Müller glial cells, in addition to providing homeostatic support to retinal neurons, have been shown to engage in modulation of neuronal activity and regulate vasomotor responses in the retina, among other functions. Calcium-mediated signaling in Müller cells has been implicated to play a significant role in the intracellular and intercellular interactions necessary to carry out these functions. Although the basic molecular mechanisms of calcium signaling in Müller cells have been described, the dynamics of calcium responses in Müller cells have not been fully explored. Here, we provide a quantitative characterization of calcium signaling in an in vitro model of Müller cell signaling using the rMC-1 cell line, a well-established line developed from rat Müller cells. rMC-1 cells displayed robust intracellular calcium transients and the capacity to support calcium transient-mediated intercellular calcium waves with signaling dynamics similar to that reported for Müller cells in in situ retinal preparations. Furthermore, pharmacological perturbations of intracellular calcium transients with thapsigargin and intercellular calcium waves with purinergic receptor antagonists and gap junction blockers (PPADS and FFA, respectively) suggest that the molecular mechanisms that underlie calcium signaling in rMC-1 cells has been conserved with those of Müller cells. This model provides a robust in vitro system for investigating specific mechanistic hypotheses of intra- and intercellular calcium signaling in Müller cells. Show less

For cells of the yeast Saccharomyces cerevisiae, heat shock causes a transient inhibition of the cell cycle-regulatory step START. We have determined that this heat-induced START inhibition is accompanied by decreased CLN1 and CLN2 transcript abundance and by possible posttranscriptional changes to CLN3 (WHI1/DAF1) cyclin activity. Persistent CLN2 expression from a heterologous promoter or the CLN2-1 or CLN3-1 alleles that are thought to encode cyclin proteins with increased stability eliminated heat-induced START inhibition but did not affect other aspects of the heat shock response. Heat-induced START inhibition was shown to be independent of functions that regulate cyclin activity under other conditions and of transcriptional regulation of SWI4, an activator of cyclin transcription. Cells lacking Bcy1 function and thus without cyclic AMP control of A kinase activity were inhibited for START by heat shock as long as A kinase activity was attenuated by mutation. We suggest that heat shock mediates START blockage through effects on the G1 cyclins. Show less

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression. Show less

For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step "start". The threshold amount of cell mass (reflected as a "critical size") necessary for "start" is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at "start", termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for "start", causing the smaller cells, produced in the previous cell cycle, to be delayed at "start" while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and "start", nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product. Show less