The Associations Between Gender Minority Stressors and PTSD Symptom Severity Among Trauma-Exposed Transgender and Gender Diverse Adults.

This study investigates associations between minority stressors, traumatic stressors, and post-traumatic stress disorder (PTSD) symptom severity in a sample of transgender and gender diverse (TGD) adults. We utilized surveys and clinical interview assessments to assess gender minority stress exposures and responses, and PTSD. Our sample (N = 43) includes adults who identified as a minoritized gender identity (i.e., 39.5% trans woman or woman, 25.6% trans man or man, 23.3% genderqueer or nonbinary, 11.6% other identity). All participants reported at least one traumatic event (i.e., life threat, serious injury, or sexual harm). The most common trauma events reported by the sample were sexual (39.5%) and physical violence (37.2%), with 40.9% of participants anchoring their symptoms to a discrimination-based event. PTSD symptom severity was positively correlated with both distal (r = 0.36, p = .017) and proximal minority stressors (r = 0.40, p < .01). Distal minority stress was a unique predictor of current PTSD symptom severity (b = 0.94, p = .017), however, this association was no longer significant when adjusting for proximal minority stress (b = 0.18, p = 0.046). This study suggests that minority stress, especially proximal minority stress, is associated with higher PTSD symptom severity among TGD adults.

Pancreatic cancer acquires resistance to MAPK pathway inhibition by clonal expansion and adaptive DNA hypermethylation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. RESULTS: We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. CONCLUSION: Overall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.

MLL-AF4 cooperates with PAF1 and FACT to drive high-density enhancer interactions in leukemia.

Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT, and the loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.

Considerations for the provision of PTSD treatment among pregnant women with substance use histories: A clinical conceptual model based on case consultation field notes.

OBJECTIVE: Increasing prevalence of substance use in pregnancy presents a public health crisis that is compounded by posttraumatic stress disorder (PTSD) comorbidity. We aimed to detail the clinical complexities of PTSD treatment provision among pregnant women with substance use histories. METHODS: We conducted a qualitative study using clinical case consultation field notes (N = 47 meetings) which were gathered during a hybrid effectiveness-implementation pilot study of Written Exposure Therapy (WET) for PTSD among pregnant women seen in an obstetrics-SUD clinic [2019-2021]. Patient baseline survey data (N = 25) were used to characterize the sample and contextualize engagement. RESULTS: Participants were exposed to a high number of trauma/adversity event types. There was no association between number of trauma/adversity event types and treatment response or dropout. Qualitative findings revealed clinical features relevant to PTSD treatment, including multi-system involvement; parental trauma and substance use; relevance of substance use to trauma context and posttraumatic cognitions, emotions, and behaviors; impact of trauma on experiences of pregnancy, attachment, and child rearing; limited social networks placing women at risk of ongoing violence; and experiences of substance use discrimination. CONCLUSION: PTSD treatment among pregnant women with substance use histories is highly important to maternal-child health.

Formative evaluation prior to implementation of a brief treatment for posttraumatic stress disorder in primary care.

BACKGROUND: Successful implementation of evidence-based treatments (EBT) for posttraumatic stress disorder (PTSD) in primary care may address treatment access and quality gaps by providing care in novel and less stigmatized settings. Yet, PTSD treatments are largely unavailable in safety net primary care. We aimed to collect clinician stakeholder data on organizational, attitudinal, and contextual factors relevant to EBT implementation. METHODS: Our developmental formative evaluation was guided by the Consolidated Framework for Implementation Research (CFIR), including (a) surveys assessing implementation climate and attitudes towards EBTs and behavioral health integration and (b) semi-structured interviews to identify barriers and facilitators to implementation and need for augmentation. Participants were hospital employees (N = 22), including primary care physicians (n = 6), integrated behavioral health clinicians (n = 8), community wellness advocates (n = 3), and clinic leadership (n = 5). We report frequency and descriptives of survey data and findings from directed content analysis of interviews. We used a concurrent mixed-methods approach, integrating survey and interview data collected simultaneously using a joint display approach. A primary care community advisory board (CAB) helped to refine interview guides and interpret findings. RESULTS: Stakeholders described implementation determinants of the EBT related to the CFIR domains of intervention characteristics (relative advantage, adaptability), outer setting (patient needs and resources), inner setting (networks and communication, relative priority, leadership engagement, available resources), and individuals involved (knowledge and beliefs, cultural considerations). Stakeholders described strong attitudinal support (relative advantage), yet therapist time and capacity restraints are major PTSD treatment implementation barriers (available resources). Changes in hospital management were perceived as potentially allowing for greater access to behavioral health services, including EBTs. Patient engagement barriers such as stigma, mistrust, and care preferences were also noted (patient needs and resources). Recommendations included tailoring the intervention to meet existing workflows (adaptability), system alignment efforts focused on improving detection, referral, and care coordination processes (networks and communication), protecting clinician time for training and consultation (leadership engagement), and embedding a researcher in the practice (available resources). CONCLUSIONS: Our evaluation identified key CFIR determinants of implementation of PTSD treatments in safety net integrated primary care settings. Our project also demonstrates that successful implementation necessitates strong stakeholder engagement.

Study protocol for a hybrid 1 effectiveness-implementation trial of Brief Skills Training in Affective and Interpersonal Regulation (Brief STAIR) and web-administered STAIR (webSTAIR) for posttraumatic stress disorder in integrated primary care.

BACKGROUND: Posttraumatic stress disorder (PTSD) disproportionally affects low-income, racial and ethnic minoritized communities, where prevalence is high, yet access to evidence-based treatments (EBTs) is low. As such, there is a need to identify effective, feasible, and scalable interventions for PTSD. Stepped care approaches that include brief, low-intensity treatments are one approach to improving access yet have not been developed for adults with PTSD. Our study aims to test the effectiveness of a step one PTSD treatment in primary care while gathering information on implementation to maximize sustainability in the setting. METHODS: This study will be conducted in integrated primary care in the largest safety net hospital in New England using a hybrid type 1 effectiveness-implementation design. Eligible trial participants are adult primary care patients who meet full or subthreshold criteria for PTSD. Interventions include Brief clinician-administered Skills Training in Affective and Interpersonal Regulation (Brief STAIR) versus web-administered STAIR (webSTAIR) during a 15-week active treatment period. Participants complete assessments at baseline (pre-treatment), 15 weeks (post-treatment), and 9 months (follow-up) post-randomization. We will assess feasibility and acceptability post-trial using surveys and interviews with patients, study therapists, and other key informants, and will assess the preliminary effectiveness of interventions in terms of PTSD symptom change and functioning. CONCLUSION: This study will provide evidence for the feasibility, acceptability, and preliminary effectiveness of brief, low-intensity interventions in safety net integrated primary care, with the aim of including these interventions in a future stepped care approach to PTSD treatment. CLINICAL TRIAL NUMBER: NCT04937504.

Written exposure therapy for treatment of perinatal PTSD among women with comorbid PTSD and SUD: A pilot study examining feasibility, acceptability, and preliminary effectiveness.

OBJECTIVE: This pilot open trial examined the feasibility, acceptability, and preliminary effectiveness of Written Exposure Therapy (WET), a 5-session evidence-based intervention for posttraumatic stress disorder (PTSD) during pregnancy. Participants were pregnant women with comorbid PTSD and substance use disorder (SUD) receiving prenatal care in a high risk obstetrics-addictions clinic. METHODS: A total of 18 participants with probable PTSD engaged in the intervention, and 10 completed the intervention and were included in outcome analyses. Wilcoxon's Signed-Rank analyses were used to evaluate PTSD and depression symptoms and craving at pre-intervention to post-intervention and pre-intervention to the 6-month postpartum follow-up. Engagement and retention in WET and therapist fidelity to the intervention manual were used to assess feasibility. Quantitative and qualitative measures of patient satisfaction were used to assess acceptability. RESULTS: PTSD symptoms significantly decreased from pre-intervention to post-intervention (S = 26.6, p = 0.006), which sustained at the 6-month postpartum follow-up (S = 10.5, p = 0.031). Participant satisfaction at post-intervention was high. Therapists demonstrated high adherence to the intervention and excellent competence. CONCLUSIONS: WET was a feasible and acceptable treatment for PTSD in this sample. Randomized clinical trial studies with a general group of pregnant women are needed to expand upon these findings and perform a full-scale test of effectiveness of this intervention.

Molecular heterogeneity and commonalities in pancreatic cancer precursors with gastric and intestinal phenotype.

OBJECTIVE: Due to the limited number of modifiable risk factors, secondary prevention strategies based on early diagnosis represent the preferred route to improve the prognosis of pancreatic ductal adenocarcinoma (PDAC). Here, we provide a comparative morphogenetic analysis of PDAC precursors aiming at dissecting the process of carcinogenesis and tackling the heterogeneity of preinvasive lesions. DESIGN: Targeted and whole-genome low-coverage sequencing, genome-wide methylation and transcriptome analyses were applied on a final collective of 122 morphologically well-characterised low-grade and high-grade PDAC precursors, including intestinal and gastric intraductal papillary mucinous neoplasms (IPMN) and pancreatic intraepithelial neoplasias (PanIN). RESULTS: Epigenetic regulation of mucin genes determines the phenotype of PDAC precursors. PanIN and gastric IPMN display a ductal molecular profile and numerous similarly regulated pathways, including the Notch pathway, but can be distinguished by recurrent deletions and differential methylation and, in part, by the expression of mucin-like 3. Intestinal IPMN are clearly distinct lesions at the molecular level with a more instable genotype and are possibly related to a different ductal cell compartment. CONCLUSIONS: PDAC precursors with gastric and intestinal phenotype are heterogeneous in terms of morphology, genetic and epigenetic profile. This heterogeneity is related to a different cell identity and, possibly, to a different aetiology.

Supporting the implementation of written exposure therapy for posttraumatic stress disorder in an obstetrics-substance use disorder clinic in the Northeastern United States.

Pregnant people with comorbid posttraumatic stress disorder (PTSD) and substance use disorder (SUD) constitute a highly vulnerable population. PTSD and SUD confer risks to both the pregnant person and the fetus, including a host of physical and mental health consequences. When PTSD and SUD co-occur, potential negative impacts are amplified, and the symptoms of each may exacerbate and maintain the other. Pregnancy often increases engagement in the healthcare system, presenting a unique and critical opportunity to provide PTSD and SUD treatment to birthing people motivated to mitigate risks of losing custody of their children. This paper presents implementation process outcomes of Written Exposure Therapy (WET), a brief, scalable, and sustainable evidence-based PTSD treatment delivered to pregnant persons receiving care in an integrated obstetrical-addiction recovery program at Boston Medical Center. Trial participants (N = 18) were mostly White, non-Hispanic (61.1%), not currently working (77.8%), had a high school or lower level of education (55.5%), had an annual household income less than $35,000 (94.4%), and were living in a substance use residential program (55.6%). We examined intervention feasibility, acceptability, appropriateness, adoption; barriers and facilitators to implementation; and feedback on supporting uptake and sustainability of the intervention using coded qualitative sources (consultation field notes [N = 47] and semi-structured interviews [N = 5]) from providers involved in trial planning and treatment delivery. Results reflected high acceptability, appropriateness, and adoption of WET. Participants described system-, provider-, and patient-level barriers to implementation, offered suggestions to enhance uptake, but did not raise concerns about core components of the intervention. Findings suggest that WET is an appropriate and acceptable PTSD treatment for this difficult-to-reach, complex population, and has the potential to positively impact pregnant persons and their children.

Functional noninvasive detection of glycolytic pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) lacks effective treatment options beyond chemotherapy. Although molecular subtypes such as classical and QM (quasi-mesenchymal)/basal-like with transcriptome-based distinct signatures have been identified, deduced therapeutic strategies and targets remain elusive. Gene expression data show enrichment of glycolytic genes in the more aggressive and therapy-resistant QM subtype. However, whether the glycolytic transcripts are translated into functional glycolysis that could further be explored for metabolic targeting in QM subtype is still not known. METHODS: We used different patient-derived PDAC model systems (conventional and primary patient-derived cells, patient-derived xenografts (PDX), and patient samples) and performed transcriptional and functional metabolic analysis. These included RNAseq and Illumina HT12 bead array, in vitro Seahorse metabolic flux assays and metabolic drug targeting, and in vivo hyperpolarized [1-13C]pyruvate and [1-13C]lactate magnetic resonance spectroscopy (HP-MRS) in PDAC xenografts. RESULTS: We found that glycolytic metabolic dependencies are not unambiguously functionally exposed in all QM PDACs. Metabolic analysis demonstrated functional metabolic heterogeneity in patient-derived primary cells and less so in conventional cell lines independent of molecular subtype. Importantly, we observed that the glycolytic product lactate is actively imported into the PDAC cells and used in mitochondrial oxidation in both classical and QM PDAC cells, although more actively in the QM cell lines. By using HP-MRS, we were able to noninvasively identify highly glycolytic PDAC xenografts by detecting the last glycolytic enzymatic step and prominent intra-tumoral [1-13C]pyruvate and [1-13C]lactate interconversion in vivo. CONCLUSION: Our study adds functional metabolic phenotyping to transcriptome-based analysis and proposes a functional approach to identify highly glycolytic PDACs as candidates for antimetabolic therapeutic avenues.

Viewing AML through a New Lens: Technological Advances in the Study of Epigenetic Regulation.

Epigenetic modifications, such as histone modifications and DNA methylation, are essential for ensuring the dynamic control of gene regulation in every cell type. These modifications are associated with gene activation or repression, depending on the genomic context and specific type of modification. In both cases, they are deposited and removed by epigenetic modifier proteins. In acute myeloid leukemia (AML), the function of these proteins is perturbed through genetic mutations (i.e., in the DNA methylation machinery) or translocations (i.e., MLL-rearrangements) arising during leukemogenesis. This can lead to an imbalance in the epigenomic landscape, which drives aberrant gene expression patterns. New technological advances, such as CRISPR editing, are now being used to precisely model genetic mutations and chromosomal translocations. In addition, high-precision epigenomic editing using dCas9 or CRISPR base editing are being used to investigate the function of epigenetic mechanisms in gene regulation. To interrogate these mechanisms at higher resolution, advances in single-cell techniques have begun to highlight the heterogeneity of epigenomic landscapes and how these impact on gene expression within different AML populations in individual cells. Combined, these technologies provide a new lens through which to study the role of epigenetic modifications in normal hematopoiesis and how the underlying mechanisms can be hijacked in the context of malignancies such as AML.

Consideration of racism experiences in the implementation of trauma-focused therapy in primary care.

OBJECTIVE: To understand providers' perceptions of how a patient's experience of racism may impact the successful implementation of a brief posttraumatic stress disorder (PTSD) treatment in the safety net integrated primary care setting. To conduct a developmental formative evaluation prior to a hybrid type I effectiveness-implementation trial. DATA SOURCES AND STUDY SETTING: From October 2020 to January 2021, in-depth qualitative interviews were conducted with integrated primary care stakeholders (N = 27) at the largest safety net hospital in New England, where 82% of patients identify as racial or ethnic minorities. STUDY DESIGN: Interviews with clinical stakeholders were used to (a) contextualize current patient and provider experiences and responses to racism, (b) consider how racism may impact PTSD treatment implementation, (c) gather recommendations for potential augmentation to the proposed PTSD treatment (e.g., culturally responsive delivery, cultural adaptation), and (d) gather recommendations for how to shift the integrated primary care practice to an antiracist framework. DATA COLLECTION/EXTRACTION METHODS: Interview data were gathered using remote data collection methods (video conferencing). Participants were hospital employees, including psychologists, social workers, primary care physicians, community health workers, administrators, and operations managers. We used conventional content analysis. PRINCIPAL FINDINGS: Clinical stakeholders acknowledged the impact of racism, including racial stress and trauma, on patient engagement and noted the potential need to adapt PTSD treatments to enhance engagement. Clinical stakeholders also characterized the harms of racism on patients and providers and provided recommendations such as changes to staff training and hiring practices, examination of racist policies, and increases in support for providers of color. CONCLUSIONS: This study contextualizes providers' perceptions of racism in the integrated primary care practice and provides some suggestions for shifting to an antiracist framework. Our findings also highlight how racism in health care may be a PTSD treatment implementation barrier.

MLL::AF9 degradation induces rapid changes in transcriptional elongation and subsequent loss of an active chromatin landscape.

MLL rearrangements produce fusion oncoproteins that drive leukemia development, but the direct effects of MLL-fusion inactivation remain poorly defined. We designed models with degradable MLL::AF9 where treatment with small molecules induces rapid degradation. We leveraged the kinetics of this system to identify a core subset of MLL::AF9 target genes where MLL::AF9 degradation induces changes in transcriptional elongation within 15 minutes. MLL::AF9 degradation subsequently causes loss of a transcriptionally active chromatin landscape. We used this insight to assess the effectiveness of small molecules that target members of the MLL::AF9 multiprotein complex, specifically DOT1L and MENIN. Combined DOT1L/MENIN inhibition resembles MLL::AF9 degradation, whereas single-agent treatment has more modest effects on MLL::AF9 occupancy and gene expression. Our data show that MLL::AF9 degradation leads to decreases in transcriptional elongation prior to changes in chromatin landscape at select loci and that combined inhibition of chromatin complexes releases the MLL::AF9 oncoprotein from chromatin globally.

Redesign of a brief PTSD treatment in safety net integrated primary care: Supporting implementation in the context of the COVID-19 pandemic.

OBJECTIVE: We conducted a formative evaluation to understand the impact of the COVID-19 pandemic on the safety net integrated primary care setting and to identify (and respond to) new implementation barriers prior to a hybrid type I effectiveness-implementation trial of a posttraumatic stress disorder (PTSD) treatment. METHOD: We used surveys and qualitative interviews with employee stakeholders (N = 27) to (1) understand pandemic-related factors that may influence implementation, including changes in patient needs, provider experiences, and the practice, and (2) assess the need for augmentation to study design, implementation plan, or intervention. RESULTS: Conventional content analysis and survey findings suggest that patient acuity and volume increased provider burden, leading to high burnout. Although the shift to telehealth improved behavioral health access, issues with technology access and literacy were common. Changes to the study design and implementation plan, based on findings, included the provision of multi-modality treatments (in person, telehealth, web-administered), technology and administrative support, and other strategies for reducing provider burnout. CONCLUSIONS: This study describes how an ongoing research study adapted to major changes to the implementation setting during the pandemic. Changes to study design and implementation plan were responsive to the shift to telehealth and therapist burden (and burnout) concerns.

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients.

Secondary resistance to anti-EGFR therapy by transcriptional reprogramming in patient-derived colorectal cancer models.

BACKGROUND: The development of secondary resistance (SR) in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (anti-EGFR) antibodies is not fully understood at the molecular level. Here we tested in vivo selection of anti-EGFR SR tumors in CRC patient-derived xenograft (PDX) models as a strategy for a molecular dissection of SR mechanisms. METHODS: We analyzed 21 KRAS, NRAS, BRAF, and PI3K wildtype CRC patient-derived xenograft (PDX) models for their anti-EGFR sensitivity. Furthermore, 31 anti-EGFR SR tumors were generated via chronic in vivo treatment with cetuximab. A multi-omics approach was employed to address molecular primary and secondary resistance mechanisms. Gene set enrichment analyses were used to uncover SR pathways. Targeted therapy of SR PDX models was applied to validate selected SR pathways. RESULTS: In vivo anti-EGFR SR could be established with high efficiency. Chronic anti-EGFR treatment of CRC PDX tumors induced parallel evolution of multiple resistant lesions with independent molecular SR mechanisms. Mutations in driver genes explained SR development in a subgroup of CRC PDX models, only. Transcriptional reprogramming inducing anti-EGFR SR was discovered as a common mechanism in CRC PDX models frequently leading to RAS signaling pathway activation. We identified cAMP and STAT3 signaling activation, as well as paracrine and autocrine signaling via growth factors as novel anti-EGFR secondary resistance mechanisms. Secondary resistant xenograft tumors could successfully be treated by addressing identified transcriptional changes by tailored targeted therapies. CONCLUSIONS: Our study demonstrates that SR PDX tumors provide a unique platform to study molecular SR mechanisms and allow testing of multiple treatments for efficient targeting of SR mechanisms, not possible in the patient. Importantly, it suggests that the development of anti-EGFR tolerant cells via transcriptional reprogramming as a cause of anti-EGFR SR in CRC is likely more prevalent than previously anticipated. It emphasizes the need for analyses of SR tumor tissues at a multi-omics level for a comprehensive molecular understanding of anti-EGFR SR in CRC.

Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPß binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPß, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPß binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.

BET inhibition disrupts transcription but retains enhancer-promoter contact.

Enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes in higher eukaryotes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. Bromodomain and Extra-Terminal domain (BET) and Mediator proteins have been shown capable of forming phase condensates and are thought to be essential for super-enhancer function. Here, we show that targeting of cells with inhibitors of BET proteins or pharmacological degradation of BET protein Bromodomain-containing protein 4 (BRD4) has a strong impact on transcription but very little impact on enhancer-promoter interactions. Dissolving phase condensates reduces BRD4 and Mediator binding at enhancers and can also strongly affect gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further indicate that enhancer-promoter interactions are not dependent on high levels of BRD4 and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and, at some sites, CTCF and cohesin.

H3K79me2/3 controls enhancer-promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells.

MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer-promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.

ITIH5 and ECRG4 DNA Methylation Biomarker Test (EI-BLA) for Urine-Based Non-Invasive Detection of Bladder Cancer.

Bladder cancer is one of the more common malignancies in humans and the most expensive tumor for treating in the Unites States (US) and Europe due to the need for lifelong surveillance. Non-invasive tests approved by the FDA have not been widely adopted in routine diagnosis so far. Therefore, we aimed to characterize the two putative tumor suppressor genes ECRG4 and ITIH5 as novel urinary DNA methylation biomarkers that are suitable for non-invasive detection of bladder cancer. While assessing the analytical performance, a spiking experiment was performed by determining the limit of RT112 tumor cell detection (range: 100-10,000 cells) in the urine of healthy donors in dependency of the processing protocols of the RWTH cBMB. Clinically, urine sediments of 474 patients were analyzed by using quantitative methylation-specific PCR (qMSP) and Methylation Sensitive Restriction Enzyme (MSRE) qPCR techniques. Overall, ECRG4-ITIH5 showed a sensitivity of 64% to 70% with a specificity ranging between 80% and 92%, i.e., discriminating healthy, benign lesions, and/or inflammatory diseases from bladder tumors. When comparing single biomarkers, ECRG4 achieved a sensitivity of 73%, which was increased by combination with the known biomarker candidate NID2 up to 76% at a specificity of 97%. Hence, ITIH5 and, in particular, ECRG4 might be promising candidates for further optimizing current bladder cancer biomarker panels and platforms.