G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules.

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles.

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

PD-1-Expressing SARS-CoV-2-Specific CD8+ T Cells Are Not Exhausted, but Functional in Patients with COVID-19.

Memory T cell responses have been demonstrated in COVID-19 convalescents, but ex vivo phenotypes of SARS-CoV-2-specific T cells have been unclear. We detected SARS-CoV-2-specific CD8+ T cells by MHC class I multimer staining and examined their phenotypes and functions in acute and convalescent COVID-19. Multimer+ cells exhibited early differentiated effector-memory phenotypes in the early convalescent phase. The frequency of stem-like memory cells was increased among multimer+ cells in the late convalescent phase. Cytokine secretion assays combined with MHC class I multimer staining revealed that the proportion of interferon-γ (IFN-γ)-producing cells was significantly lower among SARS-CoV-2-specific CD8+ T cells than those specific to influenza A virus. Importantly, the proportion of IFN-γ-producing cells was higher in PD-1+ cells than PD-1- cells among multimer+ cells, indicating that PD-1-expressing, SARS-CoV-2-specific CD8+ T cells are not exhausted, but functional. Our current findings provide information for understanding of SARS-CoV-2-specific CD8+ T cells elicited by infection or vaccination.

Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.

Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy-diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. Here, we demonstrate that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

ULK1 and ULK2 Regulate Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97.

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.

Brain decoding of spontaneous thought: Predictive modeling of self-relevance and valence using personal narratives.

The contents and dynamics of spontaneous thought are important factors for personality traits and mental health. However, assessing spontaneous thoughts is challenging due to their unconstrained nature, and directing participants' attention to report their thoughts may fundamentally alter them. Here, we aimed to decode two key content dimensions of spontaneous thought-self-relevance and valence-directly from brain activity. To train functional MRI-based predictive models, we used individually generated personal stories as stimuli in a story-reading task to mimic narrative-like spontaneous thoughts (n = 49). We then tested these models on multiple test datasets (total n = 199). The default mode, ventral attention, and frontoparietal networks played key roles in the predictions, with the anterior insula and midcingulate cortex contributing to self-relevance prediction and the left temporoparietal junction and dorsomedial prefrontal cortex contributing to valence prediction. Overall, this study presents brain models of internal thoughts and emotions, highlighting the potential for the brain decoding of spontaneous thought.

Heterogeneous osteoimmune profiles via single-cell transcriptomics in osteoporotic patients who fail bisphosphonate treatment.

Postmenopausal osteoporosis arises from imbalanced osteoclast and osteoblast activity, and mounting evidence suggests a role for the osteoimmune system in bone homeostasis. Bisphosphonate (BP) is an antiresorptive agent, but its treatment failure rate can be as high as 40%. Here, we performed single-cell RNA sequencing on peripheral immune cells from carefully selected postmenopausal women: non-osteoporotic, osteoporosis improved after BP treatment, and BP-failed cases. We found an increase in myeloid cells in patients with osteoporosis (specifically, T cell receptor+ macrophages). Furthermore, lymphoid lineage cells varied significantly, notably elevated natural killer cells (NKs) in the BP-failed group. Moreover, we provide fruitful lists of biomarkers within the immune cells that exhibit condition-dependent differences. The existence of osteoporotic- and BP-failure-specific cellular information flows was revealed by cell-cell interaction analysis. These findings deepen our insight of the osteoporosis pathology enhancing comprehension of the role of immune heterogeneity in postmenopausal osteoporosis and BP treatment failure.

The TOX-RAGE axis mediates inflammatory activation and lung injury in severe pulmonary infectious diseases.

Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.

Targeting ATP2B1 impairs PI3K/Akt/FOXO signaling and reduces SARS-COV-2 infection and replication.

ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of intracellular Ca2+ content have been suggested to impair SARS-CoV-2 replication. Here, we demonstrate that a nontoxic caloxin-derivative compound (PI-7) reduces intracellular Ca2+ levels and impairs SARS-CoV-2 infection. Furthermore, a rare homozygous intronic variant of ATP2B1 is shown to be associated with the severity of COVID-19. The mechanism of action during SARS-CoV-2 infection involves the PI3K/Akt signaling pathway activation, inactivation of FOXO3 transcription factor function, and subsequent transcriptional inhibition of the membrane and reticulum Ca2+ pumps ATP2B1 and ATP2A1, respectively. The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 lacks toxicity in vitro, its prophylactic use as a therapeutic agent against COVID-19 is envisioned here.

Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions.

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Psychiatric adverse events following COVID-19 vaccination: a population-based cohort study in Seoul, South Korea.

Evidence has suggested an increased risk of psychiatric manifestations following viral infections including coronavirus disease-2019 (COVID-19). However, psychiatric adverse events (AEs) after COVID-19 vaccination, which were documented in case reports and case series, remain unclear. This study is aimed to investigate the psychiatric AEs after COVID-19 vaccination from a large population-based cohort in Seoul, South Korea. We recruited 50% of the Seoul-resident population randomly selected from the Korean National Health Insurance Service (KNHIS) claims database on 1, January, 2021. The included participants (n = 2,027,353) from the Korean National Health Insurance Service claims database were divided into two groups according to COVID-19 vaccination. The cumulative incidences per 10,000 of psychiatric AEs were assessed on one week, two weeks, one month, and three months after COVID-19 vaccination. Hazard ratios (HRs) and 95% Confidence interval (CIs) of psychiatric AEs were measured for the vaccinated population. The cumulative incidence of depression, anxiety, dissociative, stress-related, and somatoform disorders, sleep disorders, and sexual disorders at three months following COVID-19 vaccination were higher in the vaccination group than no vaccination group. However, schizophrenia and bipolar disorders showed lower cumulative incidence in the vaccination group than in the non-vaccinated group. Depression (HR [95% CI] = 1.683 [1.520-1.863]), anxiety, dissociative, stress-related, and somatoform disorders (HR [95% CI] = 1.439 [1.322-1.568]), and sleep disorders (HR [95% CI] = 1.934 [1.738-2.152]) showed increased risks after COVID-19 vaccination, whereas the risks of schizophrenia (HR [95% CI] = 0.231 [0.164-0.326]) and bipolar disorder (HR [95% CI] = 0.672 [0.470-0.962]). COVID-19 vaccination increased the risks of depression, anxiety, dissociative, stress-related, and somatoform disorders, and sleep disorders while reducing the risk of schizophrenia and bipolar disorder. Therefore, special cautions are necessary for administering additional COVID-19 vaccinations to populations vulnerable to psychiatric AEs.

Autoimmune adverse event following COVID-19 vaccination in Seoul, South Korea.

BACKGROUND: There is growing evidence that the coronavirus disease 2019 (COVID-19) vaccination can affect the regulation of the immune system, leading to the development of autoimmune diseases. However, the autoimmune adverse events (AEs) after COVID-19 vaccination remain largely unclear. OBJECTIVE: We sought to investigate the autoimmune AEs after COVID-19 vaccination from a population-based cohort in South Korea. METHODS: A total of 4,203,887 participants, representing 50% of the population residing in Seoul, were recruited from the National Health Insurance Service database and then divided into 2 groups on the basis of COVID-19 vaccination. The cumulative incidence, hazard ratios (HRs), and 95% CIs of autoimmune AEs were assessed following COVID-19 vaccination. RESULTS: The incidence of vitiligo has been observed to be significantly higher in the vaccination group compared with the no vaccination group. The cumulative incidence of vitiligo began to show a significant difference starting 2 weeks after vaccination, and it reached 2.2% in the vaccination group and 0.6% in the no vaccination group by 3 months after COVID-19 vaccination. Vitiligo (HR, 2.714; 95% CI, 1.777-4.146) was an increased risk among autoimmune AEs. Furthermore, the risk of vitiligo was the highest for heterologous vaccination (HR, 3.890; 95% CI, 2.303-6.573) compared with using cDNA vaccine (HR, 2.861; 95% CI, 1.838-4.453) or mRNA vaccine (HR, 2.475; 95% CI, 1.607-3.813). CONCLUSIONS: Vitiligo as an autoimmune AE was noted to be substantially higher in the COVID-19-vaccinated group compared with the controls. Therefore, the occurrence of vitiligo could be considered as one of the significant AEs post-COVID-19 vaccination.

Multi-label classification with XGBoost for metabolic pathway prediction.

BACKGROUND: Metabolic pathway prediction is one possible approach to address the problem in system biology of reconstructing an organism's metabolic network from its genome sequence. Recently there have been developments in machine learning-based pathway prediction methods that conclude that machine learning-based approaches are similar in performance to the most used method, PathoLogic which is a rule-based method. One issue is that previous studies evaluated PathoLogic without taxonomic pruning which decreases its performance. RESULTS: In this study, we update the evaluation results from previous studies to demonstrate that PathoLogic with taxonomic pruning outperforms previous machine learning-based approaches and that further improvements in performance need to be made for them to be competitive. Furthermore, we introduce mlXGPR, a XGBoost-based metabolic pathway prediction method based on the multi-label classification pathway prediction framework introduced from mlLGPR. We also improve on this multi-label framework by utilizing correlations between labels using classifier chains. We propose a ranking method that determines the order of the chain so that lower performing classifiers are placed later in the chain to utilize the correlations between labels more. We evaluate mlXGPR with and without classifier chains on single-organism and multi-organism benchmarks. Our results indicate that mlXGPR outperform other previous pathway prediction methods including PathoLogic with taxonomic pruning in terms of hamming loss, precision and F1 score on single organism benchmarks. CONCLUSIONS: The results from our study indicate that the performance of machine learning-based pathway prediction methods can be substantially improved and can even outperform PathoLogic with taxonomic pruning.

Barocaloric Effects in Dialkylammonium Halide Salts.

Barocaloric effects─solid-state thermal changes induced by the application and removal of hydrostatic pressure─offer the potential for energy-efficient heating and cooling without relying on volatile refrigerants. Here, we report that dialkylammonium halides─organic salts featuring bilayers of alkyl chains templated through hydrogen bonds to halide anions─display large, reversible, and tunable barocaloric effects near ambient temperature. The conformational flexibility and soft nature of the weakly confined hydrocarbons give rise to order-disorder phase transitions in the solid state that are associated with substantial entropy changes (>200 J kg-1 K-1) and high sensitivity to pressure (>24 K kbar-1), the combination of which drives strong barocaloric effects at relatively low pressures. Through high-pressure calorimetry, X-ray diffraction, and Raman spectroscopy, we investigate the structural factors that influence pressure-induced phase transitions of select dialkylammonium halides and evaluate the magnitude and reversibility of their barocaloric effects. Furthermore, we characterize the cyclability of thin-film samples under aggressive conditions (heating rate of 3500 K s-1 and over 11,000 cycles) using nanocalorimetry. Taken together, these results establish dialkylammonium halides as a promising class of pressure-responsive thermal materials.

Increased risk of contralateral breast cancer for BRCA1/2 wild-type, high-risk Korean breast cancer patients: a retrospective cohort study.

BACKGROUND: This study aimed to investigate the contralateral breast cancer (CBC) recurrence rate in Korean breast cancer patients according to their BRCA1/2 germline mutation status, focusing particularly on the CBC recurrence risk in BRCA1/2 negative (BRCAx) patients. METHODS: We conducted a retrospective study on 13,107 primary breast cancer patients. The patients were divided into high-risk and low-risk groups for hereditary breast cancer based on the Korean National Health Insurance Service's eligibility criteria for BRCA1/2 germline mutation testing. The high-risk group was further categorized into the BRCA mutation group, the BRCAx group, and the not tested group. We evaluated the overall survival and cumulative risk of developing CBC in these patients. RESULTS: Among 4494 high-risk patients, 973 (21.7%) underwent genetic testing for BRCA1/2 germline mutation, revealing mutations in 158 patients (16.2%). We observed significant overall survival differences across all four groups, with the high-risk, not-tested group demonstrating notably worse overall survival (p < 0.001). However, when adjusted for other prognostic factors, there was no significant differences in hazard ratio of death between the four groups. The cumulative risk of CBC also varied among the groups. Patients with BRCA1/2 mutations showed a 7.3-fold increased risk of CBC compared to the low-risk group (95% CI 4.11-13.0, p < 0.001). Interestingly, BRCAx patients also demonstrated a significantly higher risk of CBC (HR 2.77, 95% CI 1.76-4.35, p < 0.001). The prognostic importance of the BRCAx for CBC recurrence persisted after adjusting for the age and subtype, but became insignificant when the family history of breast cancer was adjusted. CONCLUSION: Breast cancer patients who are at high risk of hereditary breast cancer but with wild-type BRCA 1/2 genes (BRCAx) have increased risk of developing contralateral breast cancer when compared to the low-risk patients. More careful surveillance and follow-up can be offered to these patients especially when they have family history of breast cancer.

Proposal of New ypStage Grouping System for Esophageal Squamous Cell Carcinoma Patients who Underwent Neoadjuvant Chemoradiotherapy followed by Surgery.

OBJECTIVE: To propose a new ypTNM grouping system to address these limitations and improve prognostic relevance. SUMMARY BACKGROUND DATA: The current 8th edition of the American Joint Committee on Cancer (AJCC) ypStage system shows unsatisfactory prognostic relevance in patients with esophageal squamous cell carcinoma (ESCC) treated with neoadjuvant chemoradiotherapy (nCRT) followed by esophagectomy. METHODS: The study cohort included 501 ESCC patients who received nCRT followed by esophagectomy at the Samsung Medical Center in Korea between 1994 and 2018 (development cohort) and 422 patients treated at Asan Medical Center (validation cohort). Recursive partitioning with a tree-structured regression model was used to develop and validate a new ypStage grouping system. RESULTS: In the new ypStage grouping system, ypStage I includes ypT0N0 only; ypStage II includes ypTis-T2N0 or ypT0-T2N1; ypStage III includes ypT3N0-N1; and ypStage IV includes ypT4N0-N1 or ypTanyN2-3. This system adequately addressed the limitations of the existing AJCC classification system, including overlapping and reversal of survival rates. Moreover, the discrimination ability of the new system was higher than that of the existing system [concordance-index (C-index): 61.9%] in the development (C-index: 66.6%) and validation (C-index: 66.0%) cohorts. NRIe was 0.17 [95% confidence interval (CI): 0.09-0.26, P-<0.001) and 0.18 (95% CI: 0.10-0.27, P-<0.001)] in the development and validation cohorts, respectively. CONCLUSIONS: The current study proposes a clear revised version of the 8th edition of the AJCC ypStage grouping system that exhibits superior prognostic stratification in patients with ESCC treated with nCRT followed by esophagectomy.

Tumor-intrinsic and Cancer-associated Fibroblast Subtypes Independently Predict Outcomes in Pancreatic Cancer.

OBJECTIVE: To assess the utility of tumor-intrinsic and cancer-associated fibroblast (CAF) subtypes of pancreatic ductal adenocarcinoma (PDAC) in predicting response to neoadjuvant therapy (NAT) and overall survival. BACKGROUND: PDAC remains a deadly disease with limited treatment options, and both the tumor as well as the microenvironment play an important role in pathogenesis. Gene expression-based tumor-intrinsic subtypes (classical and basal-like) have been shown to predict outcomes, but tumor microenvironment subtypes are still evolving. METHODS: RNA-sequencing was performed on 114 deidentified resected PDAC tumors. Clinical data were collected by retrospective chart review. Single sample classifiers (SSCs) were used to determine classical and basal-like subtypes as well as tumor-permissive permCAF and tumor-restraining restCAF subtypes. Survival was analyzed using log-rank test. RESULTS: Patients who received NAT had an increase in overall survival (OS), with median survival of 27.9 months compared to 20.1 months for those who did not receive NAT, but the difference did not reach statistical significance (HR 0.64, P=0.076). Either tumor-intrinsic or CAF subtypes alone were associated with OS regardless of NAT or no NAT, and patients with classical or restCAF subtype had the best outcomes. When evaluated together, patients with classical-restCAF subtype had the best OS and basal-permCAF the worst OS (P<0.0001). NAT patients with classical-restCAF subtype demonstrated the longest OS compared to the other groups (P=0.00041). CONCLUSIONS: CAF subtypes have an additive effect over tumor-intrinsic subtypes in predicting survival with or without neoadjuvant FOLFIRINOX in PDAC. Molecular subtyping of both tumor and CAF compartments of PDAC may be important steps in selecting first-line systemic therapy.