Citations

Abstract RATIONALE: The alveolar epithelium is primarily comprised of two cell types – the flat alveolar type I (AT1) cell responsible for gas exchange, and the cuboidal AT2 cell responsible for surfactant secretion, alveolar defense and acts as facultative stem cells in injury repair. Both cell types emerge from a population of bipotent distal progenitors (DPs). While the FGF signaling involved in directing AT2 fate has been identified, the mechanism by which AT2 differentiation is precisely timed is not well understood. In embryonic stem cells, it has been demonstrated that differentiation is regulated by a biophysical parameter, cell membrane tension (CMT), that acts upstream of FGF signaling by suppressing receptor endocytosis. Given this, we sought to investigate whether and how CMT plays a role in timing the alveolar epithelial differentiation, particularly focusing on how CMT might regulate FGF signaling. METHODS: DPs were isolated from E15.5 mice and plated on top of or embedded in Matrigel. DPs were treated with small molecules, to rise CMT (CHIR22901 and Methyl Beta cyclodextrin; MBCD), inhibiting either endocytosis (Dynasore) or pathways downstream of FGF signaling (FR180204, Akt VIII, KRpep-2d, NSC 74859). CMT is measured both with optical trap technology and FlipTR dye, which uses fluorescence lifetime imaging microscopy. Tissues and cells were immunostained and imaged using markers for AT1 and AT2 cells, Fgfr2, and the endocytosis marker EEA1. The transgenic mice for this project are in a C57B6 strain background and include a pan-lung epithelial Cre (TgNkx2.1-Cre) bred to Cre-dependent ERK reporter (Rosa26SAE). RESULTS: We found that CMT reduces immediately prior to alveolar differentiation, and that increasing CMT with CHIR and MBCD blocks differentiation of DPs in culture. As CMT reduces, we observe an increase in endocytosis and internalization of Fgfr2 in both cultured cells and in vivo at E17.5. Further, scRNAseq analysis showed an upregulation of ERK target genes and we have validated higher ERK activity in nAT2s in the reporter line. Further, we validated that CMT regulates FGF signaling, as decrease in CMT results in more Fgfr2 endocytosis that likely activates ERK and Akt signaling. Our small molecule inhibition data suggests that ERK and Akt signaling are required for AT2 fate selection and we propose that they are the downstream targets of FGF signaling that are activated following lowered CMT and increased Fgfr2 endocytosis. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and fate selection during lung development.

Abstract RATIONALE: Alveoli form late in development and, for first breath, alveolar type 1 (AT1) as well as AT2 epithelial cell types must be functional. While prior studies have mapped the cellular and transcriptional programs underlying this process and identified AT fate inductive cues, questions remain as to the timing and regulation of their emergence. Thus, we sought to identify when, where, and how AT2 cells first arise and motivated by recent work in embryonic stem cells, we investigated whether the biophysical parameter cell membrane tension (CMT) plays a role. METHODS: Single-cell RNA-sequencing (scRNAseq) datasets sampling mouse lung development were processed using cluster correlation, velocity, and gene ontology analysis. Marker expression and AT2 morphology was assessed by immunostaining, and their migration confirmed by time-lapse confocal microscopy on embryonic precision-cut lung slices (PCLS). CMT was measured via optical tweezers (OT) on isolated cells as well as fluorescence lifetime (FLIM) on organoids and PCLS. Progenitors (E15.5) and AT2s (E16.5- >PN50) were isolated from distal region of the mouse lung, as well as human AT2s recently isolated, expanded, and banked in Mayo Clinic's recently established patient lung cell biorepository. CMT was raised using methyl-β-cyclodextran (MBCD) or polyethylene glycol (PEG). Small molecules CHIR and ICG-001 were used in culture to raise and lower β-catenin levels, respectively. Fluorescently labeled mesenchymal cells were isolated, expanded, and added to organoids in suspension for co-association experiments. RESULTS: No evidence of distal progenitors selecting either AT fate was found until E16.5, at which time we identified a distinct nascent AT2 (nAT2) state that ontology analysis detects upregulation of single-cell migration genes. Resistin-like molecule alpha (RELM-α), a type 2 inflammation regulator, labels nAT2s – which first individually emerged around E16.5 (although instances exist at E15.5) at intermediate zones between the branch tips and stalks of the distal epithelium. Following molecular emergence, we observed nAT2s extruded basally and can subsequently interact with nearby but anatomically distinct lumens, a behavior we provisionally name interlumenal junctioning and whose dynamics we validated by timelapse microscopy. Regarding CMT, we found its reduction in distal progenitors occurs prior to, and is required for, differentiation. We observed CMT reduction is sensitive to the manner of organoid culture, with on top culture resulting in more reduction and differentiation. Finally, we observed CMT is regulated cell-intrinsically via the adaptor protein β-catenin, as well as -extrinsically through osmotic (via PEG) or mesenchymal cell interactions, both of which reduce membrane fluidity.

Abstract RATIONALE: The alveolar epithelium consists of two cell types (AT2 and AT1) derived from a common Distal Progenitor (DP). Understanding how the epithelium of alveoli functions and becomes differentiated is essential for treatment of lung diseases. One possible regulator of cell fate is cell membrane tension (CMT), a biophysical property measured by the energy required to deform the membrane and known to be an important regulator of cell movement and endocytic processes. AT1 fate selection is largely driven and reinforced by YAP (Yes-associated protein), a mechano-sensitive transcription factor gated from entering the nucleus by the size of nuclear pores, which increases upon stretch. Here, we investigated whether and how CMT regulates the transduction of stretch-dependent YAP signaling and the remodeling into AT1 cells. METHODS: DPs were isolated from E15.5 mice and cultured on Matrigel for use in differentiation experiments. Immunostaining for AT1 and AT2 markers, Phalloidin, DAPI and YAP were used to assess YAP localization and architectural changes. We generated transgenic mice containing a pan-epithelial Cre (TgNkx2.1-Cre), a Cre-dependent driver of ltta (TigreAi140), and a TetOff regulated construct expressing YAP1 (Rosa26teto-Yap1), resulting in constitutive Yap1 expression in Cre-expressing DPs. CMT is measured both with optical trap technology and with the use of FlipTR dye, which uses Fluorescence Lifetime Imaging Microscopy (FLIM) to determine CMT. RESULTS: To demonstrate the role of CMT in timing AT1 fate, we found that CMT lowers in DPs concomitantly with differentiation, and that flattening cells (likely AT1s) have even lower CMT than round cells (likely AT2s). Using organotypic culture, we found that raising CMT with MBCD, PEG, or Matrigel embedding largely stops differentiation of DPs. Surprisingly, even when YAP is overdriven in mice in E15.5 distal progenitors, early precocious AT1 differentiation is not observed, suggesting YAP signaling is not alone sufficient to drive AT1 fate selection in DPs with higher CMT. We reproduced this finding when culturing DPs under high CMT conditions-promoting high YAP/TAZ levels via LATS inhibition was also not sufficient to select for AT1 fate. Finally, we found that high CMT disrupts nuclear YAP localization, nuclear-actin attachment, and cytoskeletal remodeling that is typically when DPs remodel towards AT1 fate. We propose that high baseline CMT in DPs prevents cellular remodeling, specifically that it suppresses the formation of actin-nuclear associations that imbue the nucleus with stretch sensitivity, thereby preventing an increased sensitivity to stretch and thus higher YAP signaling.

Purpose This study investigates medical students’ opinions on the optimal timing for taking the emergency medicine (EM) clerkship and its effects on preparedness for subsequent clerkships and the USMLE (United States Medical Licensing Examination) Step 2 examination. Methods A cross-sectional survey was conducted among third- and fourth-year students at the Mayo Clinic Alix School of Medicine. The survey collected opinions on the optimal timing for the EM clerkship and its impact on self-efficacy as well as perceived preparedness for future clerkships and the USMLE Step 2 examination. Results Only 5% of third-year EM clerkship students felt they would have been more prepared in their fourth year. Conversely, 70% of fourth-year students believed they were more prepared than if they had taken it in their third year. Students were evenly split on if an earlier EM clerkship would have been helpful for subsequent clerkships. 88% of pre-USMLE Step 2 students found it helpful for Step 2, while 37% of post-USMLE Step 2 students felt it would have helped their preparedness. Additionally, 96% of students stated the EM clerkship increased their confidence in seeing patients. 43% of those who took the clerkship in their fourth year reported they would have considered EM as a specialty had they taken it earlier. Most students recommended taking the EM clerkship during the second 6 months of clerkships. Conclusion There are mixed opinions among medical students regarding the helpfulness of taking the EM clerkship earlier for subsequent clerkships and USMLE Step 2 preparedness. However, the majority recommend taking the EM clerkship earlier. A significant proportion of participants would have considered EM as a specialty if they had taken the clerkship earlier. Furthermore, 96% reported increased self-efficacy. Therefore, encouraging students to take the EM clerkship earlier may increase self-confidence and allow for earlier career exploration.

Abstract O.A. Shariq: None. K.E. Lines: None. K.A. English: None. I. Rioja: Employee; Self; GlaxoSmithKline. A. Selberherr: None. H. Ferry: None. A.L. Oberg: None. R. Prinjha: Employee; Self; GlaxoSmithKline. D. Richards: None. R.V. Thakker: None. Background: Effective treatments for metastatic pancreatic neuroendocrine tumors (PNETs) are scarce. We previously showed that inhibiting bromodomain and extra-terminal (BET) epigenetic reader proteins via non-selective targeting of their two bromodomains (BD1 and BD2) reduces proliferation in preclinical PNET models. However, the clinical relevance of BET proteins in PNETs and functional differences between BD1/BD2 are unclear. Here, we characterised BET expression in human PNETs and performed efficacy and mechanistic studies using next-generation BD1- (BD1i) and BD2-selective (BD2i) BET inhibitors. Methods: BET expression was evaluated by immunohistochemistry in functioning and non-functioning human PNETs (n=52) vs normal adjacent islets (n=18). RNA sequencing (RNA-seq) and gene set enrichment analyses (GSEA) were performed in an independent PNET cohort (n=46) to identify biological pathways upregulated in metastatic vs non-metastatic tumors. Effects of BD1i and BD2i on viability, apoptosis, and cell cycle progression were assessed in three PNET cell lines (BON1, QGP1, MIN6). Early (6h) and late (48h) transcriptional responses to BET inhibition were examined in vitro using nascent RNA-seq (SLAM-seq) and standard RNA-seq, respectively. Epigenomic responses were explored using chromatin immunoprecipitation sequencing (ChIP-seq) for the histone mark H3K27ac. Effects of BD1i and BD2i on viability were evaluated in ex vivo neoplastic pancreatic islets derived from Men1L/L/RIP2-Cre mice. Results: BET proteins (BRD2, BRD3 and BRD4) were overexpressed by 1.5-2.5-fold in human PNETs vs islets (p<0.01). GSEA showed enrichment of BET-regulated gene sets in metastatic tumors (normalized enrichment score 1.31, adjusted p=0.04). BD2i marginally affected cell viability (IC50 55-90 µM), apoptosis, and cell cycle profile, while BD1i had a moderate impact on all phenotypes (IC50 1-1.5 µM). Synergistic effects were observed when BD1i+BD2i were combined (IC50 0.2-0.5 µM). RNA-seq and SLAM-seq revealed transcriptional changes that were minimal with BD2i (n=73 and n=10 differentially expressed genes, respectively), modest with BD1i (n=354 and n=60 genes), and greatest with BD1i+BD2i combined (n=1739 and n=375 genes). ChIP-seq showed a reduction in H3K27ac enrichment at promoters and super-enhancers of genes involved in BET protein-regulated pathways (e.g. cell cycle progression), with the greatest decrease observed with BD1i+BD2i combined. In ex vivo cultured neoplastic islets, viability was reduced by <30% with BD2i (p<0.005), ∼40% with BD1i (p<0.0001), and ∼60% with BD1i+BD2i (p<0.0001). Conclusions: Our studies confirm BET proteins as clinically-relevant targets in human PNETs and show that dual (BD1+BD2) inhibition has synergistic efficacy. These findings have important implications for the clinical application of next-generation epigenetic modulators. Sunday, June 2, 2024

AbstractINTRODUCTIONHigher male prevalence in sporadic behavioral variant frontotemporal dementia (bvFTD) has been reported. We hypothesized differences in phenotypes between genetic and sporadic bvFTD females resulting in underdiagnosis of sporadic bvFTD females.METHODSWe included genetic and sporadic bvFTD patients from two multicenter cohorts. We compared behavioral and cognitive symptoms, and gray matter volumes, between genetic and sporadic cases in each sex.RESULTSFemales with sporadic bvFTD showed worse compulsive behavior (p = 0.026) and language impairments (p = 0.024) compared to females with genetic bvFTD (n = 152). Genetic bvFTD females had smaller gray matter volumes than sporadic bvFTD females, particularly in the parietal lobe.DISCUSSIONFemales with sporadic bvFTD exhibit a distinct clinical phenotype compared to females with genetic bvFTD. This difference may explain the discrepancy in prevalence between genetic and sporadic cases, as some females without genetic mutations may be misdiagnosed due to atypical bvFTD symptom presentation.Highlights Sex ratio is equal in genetic behavioral variant of frontotemporal dementia (bvFTD), whereas more males are present in sporadic bvFTD. Distinct neuropsychiatric phenotypes exist between sporadic and genetic bvFTD in females. Phenotype might explain the sex ratio difference between sporadic and genetic cases.

Abstract Background Studies evaluating the risk of post-polypectomy bleeding (PPB) after colorectal endoscopic mucosal resection (EMR) in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) are limited. Methods This retrospective cohort study utilized the U.S. Collaborative Network to assess the risk of PPB after colorectal EMR in patients with CKD compared to controls. Using one-to-one propensity score matching (PSM), the primary outcome measured was PPB within 30 days after colorectal EMR. The PPB risk was further stratified by CKD severity: non-advanced CKD and advanced CKD. Results After PSM, each cohort included 7,460 patients. Overall, CKD was associated with increased risk of PPB following colorectal EMR (5.5% vs. 4%, odds ratio [OR] 1.38, 95% confidence interval [CI] 1.19–1.61, p < 0.001). The PPB risk was significantly higher in patients with advanced CKD (8.1% vs, 4.6%, OR 1.83, 95% CI 1.43–2.34, p < 0.001), while those with non-advanced CKD showed modest increase in risk of PPB (4.7% vs. 3.9%, OR 1.21, 95% CI 1.001–1.46, p = 0.049). Conclusion Patients with CKD had higher risk of PPB than patients without CKD, but absolute risk remained low. The PPB risk was notably increased in patients with advanced CKD. Optimizing patients with CKD, especially advanced CKD, before colorectal EMR and monitoring for post-procedure bleeding remains important.

Refractory ascites often requires therapeutic paracentesis, which is associated with potential risks and diminished quality of life. Terlipressin is a vasopressin analog that is indicated for i.v. bolus injection for hepatorenal syndrome, with the potential to reduce large-volume ascites and its complications. Continuous infusion of terlipressin is associated with fewer adverse effects than bolus dosing. The efficacy and safety of continuous infusion of a novel liquid formulation of terlipressin acetate (BIV201) were evaluated in this open-label phase 2 study. Patients with cirrhosis and refractory ascites were randomly assigned (2:1) to receive two 28-day cycles of continuous infusion BIV201 plus standard of care (SOC) separated by a ≤56-day washout (n=10), or SOC alone (n=5). Data analysis was limited by the small sample size and confounded by a potential interaction with gabapentinoids in the BIV201+SOC group. Nonetheless, there were differences in favor of BIV201+SOC versus SOC in the coprimary efficacy endpoints and several quality of life assessments. The beneficial effects of BIV201 on liver complications (mean: 90% CI; BIV201-completers=2.87: 1.51; 5.46 vs. SOC=2.38: 1.20; 4.73) and the change in cumulative ascites (mean: 90% CI; BIV201-completers=−10.76: −26.51; 5.00 vs. SOC=−4.99: −21.95; 11.97) were more pronounced versus SOC in the 5 BIV201+SOC patients who completed both treatment cycles. There were also greater improvements in exploratory quality of life assessments and the percent change in therapeutic paracenteses with BIV201+SOC (−27.94±41.80) versus SOC (−16.67±45.64). Despite the high rate of hyponatremia in the BIV201+SOC group (4/10 patients), the safety profile suggested that continuous BIV201 infusion was well tolerated. These findings support further development of BIV201 in confirmatory trials.

PURPOSE Sertoli-Leydig cell tumors (SLCTs) are rare sex cord-stromal tumors, representing <0.5% of all ovarian tumors. We analyze the role of germline DICER1 status in outcomes of ovarian SLCT. METHODS Patients with SLCT were enrolled in the International Pleuropulmonary Blastoma/ DICER1 Registry and/or the International Ovarian and Testicular Stromal Tumor Registry. Medical records were systematically abstracted, and those with known germline DICER1 status were selected for analysis. RESULTS Of 162 patients with SLCT, 60% had a germline DICER1 pathogenic or likely pathogenic (P/LP) variant. The adjusted 3-year recurrence-free survival (RFS) was 87.2% (95% CI, 79.4 to 95.8) for patients with a germline DICER1 P/LP variant compared with 78.1% (95% CI, 66.4 to 91.9) for those without a germline DICER1 P/LP variant ( P = .043). The adjusted 3-year and 5-year overall survival (OS) was 93.9% (95% CI, 87.3 to 100.0) for those with a germline DICER1 P/LP variant compared with the 3-year OS of 91.3% (95% CI, 83.4 to 100.0) and the 5-year OS of 78.2% (95% CI, 63.8 to 95.9) for those without a germline DICER1 P/LP variant ( P = .021). Among patients with a germline DICER1 P/LP variant, the risk of a subsequent, nonrecurrent event was 36.2% (95% CI, 21.4 to 48.1) within 10 years. Previous/concurrent and subsequent neoplasms were rare among those without a germline DICER1 P/LP variant. CONCLUSION This cohort study of patients with SLCT demonstrated that those with germline DICER1 P/LP variants had superior RFS and OS even when adjusting for other prognostic factors. Beyond prognostic implications of a germline DICER1 P/LP variant, germline testing helps identify patients at risk of subsequent neoplasms, including metachronous SLCT.

Abstract DAC combinations serve as frontline therapies for AML. DAC forms DNA-DNMT1 crosslinks and activates the ATR-CHK1-WEE1 DDR. Combinations of DNA-protein crosslinking agents with ATR, CHK1, or WEE1 inhibitors (i) have shown efficacy in solid tumors. This study evaluates the cytotoxic synergy of DAC plus ATRi, CHK1i, or WEE1i in AML. Public datasets were queried for mRNA expression and dependency. Commercial cell lines (U937, KG1a, Molm13, MV-4-11) were genomically validated. Molm13 TP53-/- clones were generated via CRISPR. Cells were cultured in complete RPMI. To treat, cells were cultured for 48 h in RPMI with drugs or diluent (0.2% v/v DMSO). Protein expression was evaluated by Western blotting (WB). Apoptosis was assayed by flow cytometry with annexin V or propidium iodide. Primary AML samples were obtained at study centers after informed consent. Isolated mononuclear cells (Ficoll) were used in colony forming unit (CFU) assays with DMSO or drug for 12-16 days. Synergy was determined by median-effect (CalcuSyn) where a combination index &lt;1 is synergistic and &gt;1 antagonistic. DNMT1 expression (TCGA) was highest in AML vs all other tumor types (p&lt;0.001). In CRISPR screens (DepMap), AML cell lines were increasingly dependent on DNMT1, ATR, CHK1, and WEE1 (ANOVA p&lt;0.001). WB of AML cell lines treated with DAC (25 - 200 nM) for 24 h showed phosphorylations of H2AX and CHK1 S296/S345 that were most pronounced in TP53MT lines. In apoptosis assays, ATRi, CHK1i, and WEE1i were synergistic with clinical concentrations of DAC (Table 1). In isogenic Molm13 lines, TP53 loss conferred DAC resistance that was overcome with combination treatment. Selective CHK1i were generally more synergistic than CHK1/2i, consistent with a CHK1 DDR. CFU assays of primary AML samples also showed synergy between DAC and ATRi, CHK1i, and especially WEE1i. Table 1. Synergy between decitabine and replication checkpoint inhibitors in preclinical models of AML. Cell line data are derived from subdiploid apoptosis assays. U937 data were replicated using annexin V. Prexasertib showed no activity in KG1a cells (suggesting drug efflux) requiring the use of the alternate CHK1 inhibitor, rabusertib. Abbreviations: ATR, ataxia telangiectasia and Rad3 related; CHK1/2, checkpoint kinase 1 and/or 2; CI, combination index; Conc, concentration; DNMT1, DNA methylatransferase 1; IQR, interquartile range; MT, mutated; ND, not determined; SD, standard deviation; WT, wild type. Drug Target Conc. (nM) Molm13 (TP53WT) Molm13 (TP53-/-) U937 (TP53G187fs*/-) KG1a (TP53V225fs*/-) Primary AML (TP53WT orTP53MT) Units Decitabine DNMT1 6.25 – 200 25 ± 11 350 ± 260 310 ± 180 6.1 ± 1.6 ND EC50, nM (mean, SD) Ceralasertib ATR 250 – 500 0.93 (0.66 – 1.24) 0.90 (0.84 – 0.96) 0.15 (0.08 – 0.21) 0.74 (0.63 – 1.02) 0.66 (0.43 – 2.43) CI with decitabine (median, IQR) Camonsertib ATR 10 – 100 0.27 (0.19 – 0.59) 0.24 (0.22 – 0.34) 0.34 (0.21 – 0.39) ND ND Prexasertib CHK1/2 1.0 – 6.0 0.89 (0.82 – 1.06) 0.78 (0.68 – 0.90) 0.95 (0.93 – 1.08) ND 0.88 (0.29 – 1.28) MK-8776 CHK1 250 – 1, 000 0.57 (0.48 – 0.80) 0.76 (0.73 – 1.19) 0.89 (0.57 – 1.39) ND ND Rabusertib CHK1 600 – 1, 000 ND ND ND 0.51 (0.48 – 0.56) ND Adavosertib WEE1 100 – 400 0.70 (0.63 – 1.11) 0.99 (0.78 – 1.14) 0.67 (0.60 – 0.74) 0.67 (0.37 – 0.93) 0.65 (0.43 – 0.88) DAC synergizes with ATRi, CHK1i, and WEE1i in AML. Future work will test these combinations in vivo and incorporate BH3 mimetics. Citation Format: Clifford M. Csizmar, Antoine N. Saliba, Olivia K. Rossman, Kevin L. Peterson, X. Wei Meng, B. Douglas Smith, Gabriel Ghiaur, Jonathan A. Webster, Mrinal M. Patnaik, Aref Al-Kali, Scott H. Kaufmann. Decitabine (DAC) induces a DNA damage response (DDR) and synergizes with replication checkpoint inhibitors in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1170.