FAM120A couples SREBP-dependent transcription and splicing of lipogenesis enzymes downstream of mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.

Integrated mutational landscape analysis of poorly differentiated high-grade neuroendocrine carcinoma of the uterine cervix.

High-grade neuroendocrine cervical cancers (NETc) are exceedingly rare, highly aggressive tumors. We analyzed 64 NETc tumor samples by whole-exome sequencing (WES). Human papillomavirus DNA was detected in 65.6% (42/64) of the tumors. Recurrent mutations were identified in PIK3CA, KMT2D/MLL2, K-RAS, ARID1A, NOTCH2, and RPL10. The top mutated genes included RB1, ARID1A, PTEN, KMT2D/MLL2, and WDFY3, a gene not yet implicated in NETc. Somatic CNV analysis identified two copy number gains (3q27.1 and 19q13.12) and five copy number losses (1p36.21/5q31.3/6p22.2/9q21.11/11p15.5). Also, gene fusions affecting the ACLY-CRHR1 and PVT1-MYC genes were identified in one of the eight samples subjected to RNA sequencing. To resolve evolutionary history, multiregion WES in NETc admixed with adenocarcinoma cells was performed (i.e., mixed-NETc). Phylogenetic analysis of mixed-NETc demonstrated that adenocarcinoma and neuroendocrine elements derive from a common precursor with mutations typical of adenocarcinomas. Over one-third (22/64) of NETc demonstrated a mutator phenotype of C > T at CpG consistent with deficiencies in MBD4, a member of the base excision repair (BER) pathway. Mutations in the PI3K/AMPK pathways were identified in 49/64 samples. We used two patient-derived-xenografts (PDX) (i.e., NET19 and NET21) to evaluate the activity of pan-HER (afatinib), PIK3CA (copanlisib), and ATR (elimusertib) inhibitors, alone and in combination. PDXs harboring alterations in the ERBB2/PI3K/AKT/mTOR/ATR pathway were sensitive to afatinib, copanlisib, and elimusertib (P < 0.001 vs. controls). However, combinations of copanlisib/afatinib and copanlisib/elimusertib were significantly more effective in controlling NETc tumor growth. These findings define the genetic landscape of NETc and suggest that a large subset of these highly lethal malignancies might benefit from existing targeted therapies.

Intracellular calcium links milk stasis to lysosome-dependent cell death during early mammary gland involution.

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 h of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6, and TGFß3, all of which appear to be upregulated by increased intracellular calcium. We further demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis through a process involving inhibition of CDK4/6 and cell cycle progression. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.

Effect of physical activity on incident atrial fibrillation in individuals with varying duration of diabetes: a nationwide population study.

BACKGROUND: Diabetes mellitus (DM) duration affects incident atrial fibrillation (AF) risk; the effect of physical activity on mitigating AF risk related to varying DM duration remains unknown. We assessed the effect of physical activity on incident AF in patients with DM with respect to known DM duration. METHODS: Patients with type 2 DM who underwent the Korean National Health Insurance Service health examination in 2015-2016 were grouped by DM duration: new onset and < 5, 5-9, and ≥ 10 years. Physical activity was classified into four levels: 0, < 500, 500-999, 1,000-1,499, and ≥ 1,500 metabolic equivalent task (MET)-min/week, with the primary outcome being new-onset AF. RESULTS: The study enrolled 2,392,486 patients (aged 59.3 ± 12.0 years, 39.8% female) with an average follow-up of 3.9 ± 0.8 years and mean DM duration of 5.3 ± 5.1 years. Greater physical activity was associated with a lower AF risk. Lowering of incident AF risk varied with different amounts of physical activity in relation to known DM duration. Among patients with new-onset DM, DM duration < 5 years and 5-9 years and 1,000-1,499 MET-min/week exhibited the lowest AF risk. Physical activity ≥ 1,500 MET-min/week was associated with the lowest incident AF risk in patients with DM duration ≥ 10 years (by 15%), followed DM duration of 5-9 years (12%) and < 5 years (9%) (p-for-interaction = 0.002). CONCLUSIONS: Longer DM duration was associated with a high risk of incident AF, while increased physical activity generally reduced AF risk. Engaging in > 1,500 MET-min/week was associated with the greatest AF risk reduction in patients with longer DM duration, highlighting the potential benefits of higher activity levels for AF prevention.

Association between antithrombotic therapy after stroke in patients with atrial fibrillation and the risk of net clinical outcome: an observational cohort study.

AIMS: Data on the optimal use of antithrombotic drugs and associated clinical outcomes in patients with atrial fibrillation (AF) and acute ischaemic stroke (IS) are limited. We investigated the prescription patterns of antithrombotics in community practice and long-term clinical prognosis according to early post-stroke antithrombotic therapy in patients with AF and acute IS. METHODS AND RESULTS: Patients with AF who were admitted for acute IS at a single tertiary hospital in 2010-2020 were retrospectively reviewed. Clinical profiles including the aetiology of stroke and prescription patterns of antithrombotics were identified. The net clinical outcome (NCO)-the composite of recurrent stroke, any bleeding, hospitalization or emergency department visits for cardiovascular (CV) events, and death-was compared according to the antithrombotic therapy at the first outpatient clinic visit [oral anticoagulation (OAC) alone vs. antiplatelet (APT) alone vs. OAC/APT(s)] following discharge. A total of 918 patients with AF and acute IS (mean age, 72.6 years; male, 59.3%; mean CHA2DS2-VASc score 3.3) were analysed. One-third (33.9%, n = 310) of patients were simultaneously diagnosed with AF and IS. The most common aetiology of IS was cardioembolism (71.2%), followed by undetermined aetiology (19.8%) and large artery atherosclerosis (6.0%). OAC, APT(s), and concomitant OAC and APT(s) were prescribed in 33.4%, 11.1%, and 53.4% of patients during admission that changed to 67.0%, 9.1%, and 21.7% at the first outpatient clinic, and were mostly continued up to one year after IS. Non-prescription of OAC was observed in 11.3% of post-stroke patients with AF. During a median follow-up of 2.1 years, the overall incidence rate of NCO per 100 patient-year (PY) was 20.14. APT(s) monotherapy presented the highest cumulative risk of NCO (adjusted hazard ratio 1.47, 95% confidence interval 1.08-2.00, P = 0.015; with reference to OAC monotherapy) mainly driven by the highest rates of recurrent stroke and any bleeding. OAC/APT(s) combination therapy was associated with a 1.62-fold significantly higher risk of recurrent stroke (P = 0.040) and marginally higher risk of any bleeding than OAC monotherapy. CONCLUSION: Approximately one-third of acute IS in AF have a distinctive mechanism from cardioembolism. Although APT was frequently prescribed in post-stroke patients with AF, no additive clinical benefit was observed. Adherence to OAC treatment is essential to prevent further CV adverse events in patients with AF and IS.

Association between body mass index and results of cryoballoon ablation in Korean patients with atrial fibrillation: an analysis from the Korean Heart Rhythm Society Cryoablation registry.

AIMS: Pulmonary vein isolation using cryoablation is effective and safe in patients with atrial fibrillation (AF). Although both obesity and underweight are associated with a higher risk for incident AF, there is limited data on the efficacy and safety following cryoablation according to body mass index (BMI) especially in Asians. METHODS AND RESULTS: Using the Korean Heart Rhythm Society Cryoablation registry, a multicentre registry of 12 tertiary hospitals, we analysed AF recurrence and procedure-related complications after cryoablation by BMI (kg/m2) groups (BMI < 18.5, underweight, UW; 18.5-23, normal, NW; 23-25, overweight, OW; 25-30, obese Ⅰ, OⅠ; ≥30, obese Ⅱ, OⅡ). A total of 2648 patients were included (median age 62.0 years; 76.7% men; 55.6% non-paroxysmal AF). Patients were categorized by BMI groups: 0.9% UW, 18.7% NW, 24.8% OW, 46.1% OI, and 9.4% OII. Underweight patients were the oldest and had least percentage of non-paroxysmal AF (33.3%). During a median follow-up of 1.7 years, atrial arrhythmia recurred in 874 (33.0%) patients (incidence rate, 18.9 per 100 person-years). After multivariable adjustment, the risk of AF recurrence was higher in UW group compared with NW group (adjusted hazard ratio, 95% confidence interval; 2.55, 1.18-5.50, P = 0.02). Procedure-related complications occurred in 123 (4.7%) patients, and the risk was higher for UW patients (odds ratio, 95% confidence interval; 2.90, 0.94-8.99, P = 0.07), mainly due to transient phrenic nerve palsy. CONCLUSION: Underweight patients showed a higher risk of AF recurrence after cryoablation compared with NW patients. Also, careful attention is needed on the occurrence of phrenic nerve palsy in UW patients.

The genetic structure of the Turkish population reveals high levels of variation and admixture.

The construction of population-based variomes has contributed substantially to our understanding of the genetic basis of human inherited disease. Here, we investigated the genetic structure of Turkey from 3,362 unrelated subjects whose whole exomes (n = 2,589) or whole genomes (n = 773) were sequenced to generate a Turkish (TR) Variome that should serve to facilitate disease gene discovery in Turkey. Consistent with the history of present-day Turkey as a crossroads between Europe and Asia, we found extensive admixture between Balkan, Caucasus, Middle Eastern, and European populations with a closer genetic relationship of the TR population to Europeans than hitherto appreciated. We determined that 50% of TR individuals had high inbreeding coefficients (≥0.0156) with runs of homozygosity longer than 4 Mb being found exclusively in the TR population when compared to 1000 Genomes Project populations. We also found that 28% of exome and 49% of genome variants in the very rare range (allele frequency < 0.005) are unique to the modern TR population. We annotated these variants based on their functional consequences to establish a TR Variome containing alleles of potential medical relevance, a repository of homozygous loss-of-function variants and a TR reference panel for genotype imputation using high-quality haplotypes, to facilitate genome-wide association studies. In addition to providing information on the genetic structure of the modern TR population, these data provide an invaluable resource for future studies to identify variants that are associated with specific phenotypes as well as establishing the phenotypic consequences of mutations in specific genes.

Integrated mutational landscape analysis of uterine leiomyosarcomas.

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs.

Accelerometer-derived physical activity and the risk of death, heart failure, and stroke in patients with atrial fibrillation: a prospective study from UK Biobank.

OBJECTIVE: Data on cardiovascular outcomes according to objectively measured physical activity (PA) in patients with atrial fibrillation (AF) are scarce. This study explored the associations between PA derived from wrist-worn accelerometers and the risk of death, incident heart failure (HF), and incident stroke in patients with AF. METHODS: From 37 990 patients with AF in UK Biobank, 2324 patients with accelerometer data were included. Weekly moderate-to-vigorous PA (MVPA) duration was computed from accelerometer data. The primary outcome was all-cause mortality. Secondary outcomes were cardiovascular mortality, incident HF, and incident stroke. Restricted cubic splines estimated the dose-response associations between MVPA duration and the outcomes. The adjusted HRs (aHRs) of the outcomes according to adherence to PA standard guidelines (performing MVPA≥150 min/week) were also evaluated. RESULTS: The mean age was 66.9±6.2 years and 64.9% were male. During a median follow-up of 6.7 years, there were 181 all-cause deaths, 62 cardiovascular deaths, 225 cases of incident HF, and 91 cases of incident stroke; the overall incidence rate per 1000 patient-years was 11.76, 4.03, 15.16 and 5.99, respectively. There was a linear inverse dose-response relationship between MVPA (≥108 min/week) and all-cause mortality. Performing MVPA for 105-590 min/week was associated with a lower risk of HF than those with no measurable MVPA. The risk of stroke and cardiovascular mortality was not associated with MVPA. Performing guideline-adherent MVPA was related to a 30% lower risk of all-cause mortality (aHR: 0.70 (0.50-0.98), p=0.04) and 33% lower risk of HF (aHR 0.67 (0.49-0.93), p=0.02). CONCLUSION: In patients with AF, accelerometer-derived PA data supports lower risks of all-cause mortality and HF according to a greater level of MVPA and adherence to PA guidelines. Regular MVPA should be encouraged in patients with AF as a part of integrated management.

Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activity and sperm motility in mice.

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that thioredoxin-glutathione reductase (TXNRD3), a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male fertility. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We find that mitochondria develop more defined cristae during capacitation in wildtype sperm. Furthermore, we show that absence of TXNRD3 alters thiol redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.

Risk of newly developed atrial fibrillation by alcohol consumption differs according to genetic predisposition to alcohol metabolism: a large-scale cohort study with UK Biobank.

BACKGROUND: The predictive relationship between mild-to-moderate alcohol consumption and the risk of incident atrial fibrillation (AF) remains controversial. OBJECTIVE: We investigated whether the relationship between alcohol consumption and the risk of incident AF could be associated with the genetic predisposition to alcohol metabolism. METHODS: A total of 399,329 subjects with genetic data from the UK Biobank database, enrolled between 2006 and 2010, were identified and followed for incident AF until 2021. Genetic predisposition to alcohol metabolism was stratified according to the polygenic risk score (PRS) tertiles. Alcohol consumption was categorized as non-drinkers, mild-to-moderate drinkers (< 30 g/day), and heavy drinkers (≥ 30 g/day). RESULTS: During the follow-up (median 12.2 years), 19,237 cases of AF occurred. When stratified by PRS tertiles, there was a significant relationship between genetic predisposition to alcohol metabolism and actual alcohol consumption habits (P < 0.001). Mild-to-moderate drinkers showed a decreased risk of AF (HR 0.96, 95% CI 0.92-0.99), and heavy drinkers showed an increased risk of AF (HR 1.06, 95% CI 1.02-1.10) compared to non-drinkers. When stratified according to PRS tertiles for genetic predisposition to alcohol metabolism, mild-to-moderate drinkers had equivalent AF risks, and heavy drinkers showed increased AF risk in the low PRS tertile group. However, mild-to-moderate drinkers had decreased AF risks and heavy drinkers showed similar risks of AF in the middle/high PRS tertile groups. CONCLUSIONS: Differential associations between alcohol consumption habits and incident AF across genetic predisposition to alcohol metabolism were observed; individuals with genetic predisposition to low alcohol metabolism were more susceptible to AF.

Accumulated hypertension burden on atrial fibrillation risk in diabetes mellitus: a nationwide population study.

BACKGROUND: Patients with diabetes mellitus have an increased risk of incident atrial fibrillation (AF). The effect of accumulated hypertension burden is a less well-known modifiable risk factor. We explored the relationship between accumulated hypertension burden and incident AF in these patients. METHODS: We evaluated data for 526,384 patients with diabetes who underwent three consecutive health examinations, between 2009 and 2012, from the Korean National Health Insurance Service. Hypertension burden was calculated by assigning points to each stage of hypertension in each health examination: 1 for stage 1 hypertension (systolic blood pressure [SBP] 130-139 mmHg; diastolic blood pressure [DBP] 80-89 mmHg); 2 for stage 2 (SBP 140-159 mmHg and DBP 90-99 mmHg); and 3 for stage 3 (SBP ≥ 160 mmHg or DBP ≥ 100 mmHg). Patients were categorized into 10 hypertensive burden groups (0-9). Groups 1-9 were then clustered into 1-3, 4-6, and 7-9. RESULTS: During a mean follow-up duration of 6.7 ± 1.7 years, AF was newly diagnosed in 18,561 (3.5%) patients. Compared to patients with hypertension burden 0, those with burden 1 to 9 showed a progressively increasing risk of incident AF: 6%, 11%, 16%, 24%, 28%, 41%, 46%, 57%, and 67% respectively. Clusters 1-3, 4-6, and 7-9 showed increased risks by 10%, 26%, and 45%, respectively, when compared to a hypertension burden of 0. CONCLUSIONS: Accumulated hypertension burden was associated with an increased risk of incident AF in patients with diabetes. Strict BP control should be emphasized for these patients.

Elimusertib (BAY1895344), a novel ATR inhibitor, demonstrates in vivo activity in ATRX mutated models of uterine leiomyosarcoma.

INTRODUCTION: Uterine leiomyosarcoma (uLMS) is a rare, highly aggressive malignancy. Recent data suggest 50% of uLMS may harbor alterations in the ATRX gene and such mutations may confer sensitivity to ataxia-telangiectasia-and-Rad3-related (ATR) kinase inhibitors. We sought to investigate the in vivo activity of Elimusertib (BAY1895344), a novel ATR-inhibitor, against ATRX-mutated uLMS patient-derived xenografts (PDXs). METHODS: Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or BAY1895344 (20 mg/kg dosed twice daily 3 days on 4 days off) were given via oral gavage and tumor measurements as well as weights obtained twice weekly. Tumor volume differences were calculated with a two-way ANOVA. Mechanistic studies were performed ex vivo using BAY1895344 treated uLMS tumor samples by western blot analysis. RESULTS: Both PDX LEY-11 and PDX LEY-16 harboring ATRX gene mutations demonstrated an aggressive behavior in vivo (i.e., control mice were euthanized on average at day 12.5 for PDX LEY-11 and at day 33 for PDX LEY-16). In both tumor models BAY1895344 20 mg/kg dosed with an intermittent oral schedule was able to induce significant growth inhibition compared to vehicle control treatment (p < 0.001 for both LEY-11 and LEY-16) and prolong median overall survival [PDX LEY-11 (12.5 vs. 42 days, p < 0.001) and PDX LEY-16 (33 vs. 60 days, p < 0.001)]. There were not significant changes in weight between treatment and controls. By western blot assays BAY1895344 exposure decreased phosphorylated-ATR and increased expression of apoptotic molecules in LMS PDXs. CONCLUSIONS: BAY1895344 demonstrates promising in vivo activity against biologically aggressive PDX models of uLMS harboring ATRX mutations, with no significant toxicity. Clinical trials of BAY1895344 in uLMS patients are warranted.

Ovarian and uterine carcinosarcomas are sensitive in vitro and in vivo to elimusertib, a novel ataxia-telangiectasia and Rad3-related (ATR) kinase inhibitor.

BACKGROUND: Carcinosarcoma of the ovary (OCS) and uterus (UCS) are rare highly aggressive malignancies. Ataxia-telangiectasia-and-Rad3-related (ATR) kinase and homologous recombination play a pivotal role in DNA damage repair. Homologous recombination deficiency (HRD) has been demonstrated in >30% of OCS/UCS. We investigated the preclinical activity of elimusertib, a selective ATR kinase inhibitor, against carcinosarcoma (CS) cell lines and xenografts. METHODS: Sensitivity to elimusertib was evaluated in vitro against nine whole exome-sequenced (WES) primary CS cell lines and in vivo against HRD CS xenografts. Western blots were performed to determine baseline ATR and p-ATR protein expression in CS, and ATR pathway downstream effectors and apoptosis markers in CS HRD cell lines after Elimusertib treatment. RESULTS: Out of the 9 CS cell lines, 3 harbored HRD and 6 homologous recombination proficient (HRP) features. Most of CS (i.e., 7/9 = 85%) were found to be sensitive to Elimusertib in vitro. Among the 5 primary CS cell lines with a high-grade pure serous epithelial component, HRD cell lines were more sensitive to elimusertib than HRP tumors (mean IC50 ± SEM HRD CS = 61.3 nM ±15.2 vs HRP = 361.6 nM ±24.4 (p = 0.01)). Baseline ATR and p-ATR protein expression was higher in HRD CS cell lines. Elimusertib showed tumor growth inhibition in HRD CS xenografts (p < 0.0001) and increased overall animal survival (p < 0.0001). Western blot demonstrated dose-dependent inhibition of ATR, p-ATR and its downstream effector p-CHK1, and a dose-dependent increase in caspase-3 expression. CONCLUSIONS: Elimusertib is preclinically active in vitro and in vivo against primary CS cell lines and xenografts, respectively. CS models harboring HRD or with pure/mixed endometrioid histology demonstrated higher sensitivity to ATR inhibition. Clinical trials with elimusertib in CS patients are warranted.

Uterine leiomyosarcomas harboring MAP2K4 gene amplification are sensitive in vivo to PLX8725, a novel MAP2K4 inhibitor.

INTRODUCTION: Uterine leiomyosarcomas (uLMS) are rare, highly aggressive tumors. Up to 30% of uLMS may harbor gain of function (GOF) in the MAP2K4 gene, important for tumor cell proliferation, differentiation and metastasis. We investigated the in vivo activity of a novel MAP2K4 inhibitor, PLX8725, against uLMS harboring MAP2K4 gene-amplification. METHODS: Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or PLX8725 (50 mg/kg) were given via oral gavage daily on weekdays for up to 60 days. Tumor volume differences were calculated with two-way ANOVA. Pharmacokinetic (PK) and mechanistic studies of PLX8725 in uLMS PDX models were also performed. RESULTS: Both uLMS tumors evaluated demonstrated GOF in MAP2K4 (i.e., 3 CNV in both LEY-11 and LEY-16). Tumor growth inhibition was significantly greater in both PDX LEY-11 and PDX LEY-16 treated with PLX8725 when compared to controls (p < 0.001). Median overall survival was also significantly longer in both PDX LEY-11 (p = 0.0047) and PDX LEY-16 (p = 0.0058) treatment cohorts when compared to controls. PLX8725 oral treatment was well tolerated, and PK studies demonstrated that oral PLX8725 gives extended exposure in mice. Ex vivo tumor samples after PLX8725 exposure decreased phosphorylated-ATR, JNK and p38, and increased expression of apoptotic molecules on western blot. CONCLUSION: PLX8725 demonstrates promising in vivo activity against PDX models of uLMS harboring GOF alterations in the MAP2K4 gene with tolerable toxicity. Phase I trials of PLX8725 in advanced, recurrent, chemotherapy-resistant uLMS patients are warranted.

Factors Associated With the Development and Severity of Polycystic Liver in Patients With Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Factors related to the development and severity of polycystic liver disease (PLD) have not been well established. We aimed to evaluate the genetic and epidemiologic risk factors of PLD in patients with autosomal dominant polycystic kidney disease (ADPKD). METHODS: Adult patients with inherited cystic kidney disease were enrolled from May 2019 to May 2021. Demographic, clinical, and laboratory data were collected at the initial study visit. The severity of PLD was graded based on the height-adjusted total liver volume: < 1,000 mL/m (Gr1), 1,000-1,800 mL/m (Gr2), and > 1,800 mL/m (Gr3). Targeted exome sequencing was done by a gene panel including 89 ciliopathy-related genes. We searched out the relative factors to the presence and the severity of PLD using logistic regression analysis. RESULTS: Of 602 patients with typical ADPKD, 461 (76.6%) patients had PLD. The patients with PLD showed female predominance and a higher frequency of other ADPKD-related complications. The genetic variants with truncating mutation of PKD1 (PKD1-protein-truncating [PT]) or PKD2 commonly affected the development and severity of PLD. An older age, female sex, and higher kidney volume with Mayo classification 1C-1E was significantly associated with the development of PLD, but not with the severity of PLD. On the other hand, higher body mass index, lower hemoglobin, and higher alkaline phosphatase (ALP) were the significant risk factors of severe PLD (≥ Gr2). CONCLUSION: Hepatic involvement in ADPKD could be related to kidney manifestations and genetic variants including PKD1-PT or PKD2. Monitoring hemoglobin and ALP and evaluating the genetic variants might help predict severe PLD. TRIAL REGISTRATION: Clinical Research Information Service Identifier: KCT0005580.

Whole-exome sequencing reveals damaging gene variants associated with hypoalphalipoproteinemia.

Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4-36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. We found 120 occurrences of probably damaging variants (116 heterozygous; four homozygous) among 45 of 104 recognized HDL candidate genes. Those with the highest prevalence of damaging variants were ABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4). Binomial analysis for damaging missense or loss-of-function variants identified the ABCA1 and LDLR genes at genome-wide significance. In conclusion, whole-exome sequencing of individuals with low HDL-C showed the burden of damaging rare variants in the ABCA1 and LDLR genes is particularly high and revealed numerous occurrences in HDL candidate genes, including many genes identified in genome-wide association study reports. Many of these genes are involved in cancer biology, which accords with epidemiologic findings of the association of HDL deficiency with increased risk of cancer, thus presenting a new area of interest in HDL genomics.

PTHrP induces STAT5 activation, secretory differentiation and accelerates mammary tumor development.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers, and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled. METHODS: We developed a tetracycline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression. RESULTS: Overexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5) as well as a delay in post-lactation involution. In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells nor treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling. CONCLUSIONS: Our study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells.

A phase 2 evaluation of pembrolizumab for recurrent Lynch-like versus sporadic endometrial cancers with microsatellite instability.

BACKGROUND: Microsatellite instability-high (MSI-H)/mismatch repair deficiency (dMMR) is a biomarker for responses to immune checkpoint inhibitors (ICIs). Whether mechanisms underlying microsatellite instability alter responses to ICIs is unclear. This article reports data from a prospective phase 2 pilot study of pembrolizumab in patients with recurrent MSI-H endometrial cancer (EC) analyzed by whole exome sequencing (WES) and potential mechanisms of primary/secondary ICI resistance (NCT02899793). METHODS: Patients with measurable MSI-H/dMMR EC confirmed by polymerase chain reaction/immunohistochemistry were evaluated by WES and received 200 mg of pembrolizumab every 3 weeks for ≤2 years. The primary end point was the objective response rate (ORR). Secondary end points included progression-free survival (PFS) and overall survival (OS). RESULTS: Twenty-five patients (24 evaluable) were treated. Six patients (25%) harbored Lynch/Lynch-like tumors, whereas 18 (75%) had sporadic EC. The tumor mutation burden was higher in Lynch-like tumors (median, 2939 mutations/megabase [Mut/Mb]; interquartile range [IQR], 867-5108 Mut/Mb) than sporadic tumors (median, 604 Mut/Mb; IQR, 411-798 Mut/Mb; P = .0076). The ORR was 100% in Lynch/Lynch-like patients but only 44% in sporadic patients (P = .024). The 3-year PFS and OS proportions were 100% versus 30% (P = .017) and 100% versus 43% (P = .043), respectively. CONCLUSIONS: This study suggests prognostic significance of Lynch-like cancers versus sporadic MSI-H/dMMR ECs for ORR, PFS, and OS when patients are treated with pembrolizumab. Larger confirmatory studies in ECs and other MSI-H/dMMR tumors are necessary. Defective antigen processing/presentation and deranged induction in interferon responses serve as mechanisms of resistance in sporadic MSI-H ECs. Oligoprogression in MSI-H/dMMR patients appears salvageable with surgical resection and/or local treatment and the continuation of pembrolizumab off study. Clinical studies evaluating separate MSI-H/dMMR EC subtypes treated with ICIs are warranted.

The transcription factor ORA59 exhibits dual DNA binding specificity that differentially regulates ethylene- and jasmonic acid-induced genes in plant immunity.

Jasmonic acid (JA) and ethylene (ET) signaling modulate plant defense against necrotrophic pathogens in a synergistic and interdependent manner, while JA and ET also have independent roles in certain processes, e.g. in responses to wounding and flooding, respectively. These hormone pathways lead to transcriptional reprogramming, which is a major part of plant immunity and requires the roles of transcription factors. ET response factors are responsible for the transcriptional regulation of JA/ET-responsive defense genes, of which ORA59 functions as a key regulator of this process and has been implicated in the JA-ET crosstalk. We previously demonstrated that Arabidopsis (Arabidopsis thaliana) GDSL LIPASE 1 (GLIP1) depends on ET for gene expression and pathogen resistance. Here, promoter analysis of GLIP1 revealed ERELEE4 as the critical cis-element for ET-responsive GLIP1 expression. In a yeast one-hybrid screening, ORA59 was isolated as a specific transcription factor that binds to the ERELEE4 element, in addition to the well-characterized GCC box. We found that ORA59 regulates JA/ET-responsive genes through direct binding to these elements in gene promoters. Notably, ORA59 exhibited a differential preference for GCC box and ERELEE4, depending on whether ORA59 activation is achieved by JA and ET, respectively. JA and ET induced ORA59 phosphorylation, which was required for both activity and specificity of ORA59. Furthermore, RNA-seq and virus-induced gene silencing analyses led to the identification of ORA59 target genes of distinct functional categories in JA and ET pathways. Our results provide insights into how ORA59 can generate specific patterns of gene expression dynamics through JA and ET hormone pathways.