Ronald M. Evans

SMRT regulation of nuclear receptors orchestrates bile acid homeostasis

HDAC inhibition sensitizes pancreatic tumors to DNA damage by global redistribution of the transcriptional machinery

A phase II clinical trial of albumin-bound paclitaxel, cisplatin, gemcitabine (NABPLAGEM) and paricalcitol as neoadjuvant therapy in pancreatic cancer

Patient-derived organoids reveal ductal dysfunction and CFTR-modulator responses in chronic pancreatitis

Chronic pancreatitis (CP) affects ∼3 million people worldwide, yet altering the course of the disease is challenging. We developed a patient-derived organoid (PDO) platform to investigate the molecular pathogenesis of this disease and identify therapeutic strategies. We generated 37 PDOs from patients with idiopathic, hereditary, and alcohol-related CP with a high genetic concordance. PDOs retained inflammation-associated transcriptional and proteomic features. Transcriptomic profiling revealed three molecular subtypes of CP independent of etiology. We discovered widespread dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in half of the CP PDOs, including those with wild-type CFTR. Clinically available CFTR modulators stabilized mutant or wild-type CFTR, restored CFTR function, and decreased mitogenic and inflammatory signaling. This work provides a comprehensive PDO platform for modeling CP. We demonstrate the utility of this platform for precision therapeutic investigations. Our findings reveal CFTR modulators as a broadly applicable and effective therapeutic strategy.

HDAC Inhibition Sensitizes Pancreatic Tumors to DNA Damage by Global Redistribution of the Transcriptional Machinery

The DNA damage response (DDR) is critical for pancreatic ductal adenocarcinoma (PDAC) development and therapeutic responses, including to genotoxic agents. While epigenetic modulators have been shown to contribute to the DDR, how chromatin regulation dictates responses to DNA damage in PDAC remains incompletely understood. Here, we identify Class I histone deacetylases (HDACs) as critical regulators of the DDR. HDAC1/2 directs the genomic distribution of H3K27ac, ensuring sufficient BRD4 and RNA polymerase II (Pol II) occupancy at DDR gene promoters. HDAC inhibition by entinostat shifts the balance of H3K27 acetylation preferentially towards intergenic regions, diverting BRD4 and Pol II from promoters, thereby suppressing DDR gene expression. In line with this, HDAC inhibition heightens DNA damage and sensitizes PDAC to diverse DNA-damaging and DDR-targeting agents. Since the clinical development of HDAC inhibitors has been limited by systemic toxicity, we developed bottlebrush prodrug (BPD) nanoparticles for tumor-selective entinostat delivery. Entinostat-BPD achieved tumor-specific HDAC inhibition while displaying potent efficacy and reduced systemic toxicity. These findings reveal an HDAC-dependent DDR vulnerability and offer combinational and precision targeting strategies to facilitate clinical translation and improve PDAC patient outcomes. SIGNIFICANCE STATEMENT The ability of tumor cells to tolerate DNA damage limits the efficacy of many anticancer therapies. Our study reveals that pancreatic cancer cells enforce this resistance by sustaining expression of DNA damage response (DDR) genes through Class I histone deacetylases (HDACs). HDACs maintain genome-wide acetylation patterns required for efficient recruitment of the transcriptional machinery to DDR genes. Pharmacological HDAC inhibition disrupts this process and sensitizes pancreatic cancer cells to diverse DNA-damaging agents. To overcome systemic toxicity that limits translational potential, we further establish a bottlebrush prodrug nanoparticle platform that enables tumor-selective HDAC inhibition. Given the central role of the DDR in cancer, targeting HDAC-mediated DDR regulation through drug combinations and precision delivery may have broad therapeutic relevance across cancer types.

CA19-9 promotes liver metastasis of pancreatic cancer through E-selectin mediated extravasation

Multi-layered transcriptional control of glycogen metabolism coordinates thermogenic remodeling of white adipocytes in male mice

Thermogenic activation of subcutaneous white adipocytes requires glycogen synthesis and turnover. Here we show that β-adrenergic stimulation induces a distinct glycogen metabolism gene program in inguinal white adipose tissue in a cell-autonomous and adipocyte-specific manner. Among these, Gys2 and Ppp1r3c are rapidly induced following acute β3-adrenergic receptor activation. We identify Gys2 as a direct transcriptional target of PKA-CREB signaling. In contrast, sustained expression of glycogen metabolism genes under chronic β3-adrenergic activation requires the coactivator PGC1α, whose loss blunts glycogen accumulation and thermogenic capacity. Mechanistically, PGC1α cooperates with estrogen-related receptors (ERRs) to regulate chromatin accessibility and gene transcription. Although deletion of ERRα is compensated by ERRγ, combined deletion of ERRα/β/γ abolishes expression of glycogen metabolism and thermogenic genes. Chromatin profiling confirm that ERRs directly control the glycogen metabolic program in beige adipocytes. Together, our results identify a multilayered transcriptional axis that sustains glycogen metabolism during β-adrenergic activation in male mice.

Circadian regulator REV-ERBα is a master regulator of tumor lineage plasticity and an effective therapeutic target

FGF1 ameliorates hepatic steatosis through acute activation of the unfolded protein response and VLDL production

Adipocyte heparan sulfate determines type 2 diabetes susceptibility in mice via FGF1-Mediated glucose regulation

Thalamic CGRP neurons define a spinothalamic pathway for affective pain

Pain is both a sensory and emotional experience caused by various harmful stimuli. While numerous studies have explored peripheral and central pain mechanisms, the specific neural circuits linking the spinal cord to the brain remain poorly defined. In this study, we demonstrate the involvement of calcitonin gene-related peptide (CGRP)-positive neurons in the parvicellular part of the subparafascicular nucleus (SPFp) in pain. Tracing revealed that CGRP neurons in the SPFp (CGRP SPFp ) receive projections from the dorsal horn. Increased calcium activity was observed in CGRP SPFp neurons during mechanical, thermal, and inflammatory stimuli. Genetic silencing of these neurons resulted in reduced pain responses in animals. Furthermore, optogenetic activation of CGRP SPFp neurons induced aversive memory but did not alter mechanical or thermal pain thresholds. This study reveals a distinct neural circuit involving CGRP SPFp neurons that mediates pain, which differs from CGRP neurons in the parabrachial nucleus. Understanding these circuits could lead to better pain treatments with fewer side effects.

Estrogen-related receptors regulate innate and adaptive muscle mitochondrial energetics through cooperative and distinct actions

Mitochondrial energy metabolism is vital for muscle function and is tightly controlled at the transcriptional level, both in the basal state and during adaptive muscle remodeling. The importance of the transcription factors estrogen-related receptors (ERRs) in controlling innate mitochondrial energetics has been recently demonstrated. However, whether different ERR isoforms display distinct functions in glycolytic versus oxidative myofibers is largely unknown. Moreover, their roles in regulating exercise-induced adaptive mitochondrial biogenesis remain unclear. Using muscle-specific single and combinatorial knockout mouse models, we have identified both cooperative and distinct roles of the ERR isoforms ERRα and ERRγ in regulating mitochondrial energy metabolism in different muscles. We demonstrate the essential roles of both these ERRs in mediating adaptive mitochondrial biogenesis in response to exercise training. We further show that PGC1α-induced mitochondrial biogenesis is completely abolished in primary myotubes with ERRα deletion but not ERRγ, highlighting distinct roles of these two isoforms in adaptive mitochondrial remodeling. Mechanistically, we find that both ERRs directly bind to the majority of mitochondrial energetic genes and control their expression, largely through collaborative binding to the same genomic loci. Collectively, our findings reveal critical and direct regulatory roles of ERRα and ERRγ in governing both innate and adaptive mitochondrial energetics in skeletal muscle.

BCL6 coordinates muscle mass homeostasis with nutritional states

Nutritional status is a determining factor for growth during development and homeostatic maintenance in adulthood. In the context of muscle, growth hormone (GH) coordinates growth with nutritional status; however, the detailed mechanisms remain to be fully elucidated. Here, we show that the transcriptional repressor B cell lymphoma 6 (BCL6) maintains muscle mass by sustaining GH action. Muscle-specific genetic deletion of BCL6 at either perinatal or adult stages profoundly reduces muscle mass and compromises muscle strength. Conversely, muscle-directed viral overexpression of BCL6 significantly reverses the loss of muscle mass and strength. Mechanistically, we show that BCL6 transcriptionally represses the suppressor of cytokine signaling 2 to sustain the anabolic actions of GH in muscle. Additionally, we find that GH itself transcriptionally inhibits BCL6 through the Janus kinase and signal transducer and activator of transcription 5 (JAK/STAT5) pathway. Supporting the physiologic relevance of this feedback regulation, we show the coordinated suppression of muscle Bcl6 expression with the induction of GH in the fasted state. These findings reveal the complexity of the feedback controls modulating GH signaling and identify BCL6 as a key homeostatic regulator coordinating muscle mass with nutrient availability. Moreover, these studies open avenues for targeted therapeutic strategies to combat muscle-wasting conditions.

Pembrolizumab ± paricalcitol in metastatic pancreatic cancer postmaximal cytoreduction

LESSONS LEARNED: Intravenous paricalcitol did not improve the efficacy of pembrolizumab, likely related to the short half-life. BACKGROUND: Immunotherapy has limited benefit in the treatment of advanced pancreatic cancer with the tumor microenvironment playing a key role in immune resistance. In preclinical studies, vitamin D receptor (VDR) agonists have been shown to sensitize pancreatic tumors to PD-1 blockade. METHODS: This was a randomized, double-blinded, placebo-controlled, phase II trial to evaluate pembrolizumab with or without paricalcitol as maintenance therapy for patients with metastatic pancreatic ductal adenocarcinoma (PDAC). Participants were ≥18 years; histologically or cytologically confirmed metastatic PDAC showing no disease progression after frontline systemic therapy, and achieving maximal cytoreduction (eg, with no further antitumor effect), Eastern Cooperative Oncology Group (ECOG) status of 0 or 1; adequate organ function. Study treatment included: pembrolizumab 200 mg IV every 3 weeks and either paricalcitol 25 mcg IV 3 times per week or placebo. The primary objective was to evaluate 6-month progression free survival (PFS). Secondary objectives include evaluating the toxicity of the combination and overall survival (OS). RESULTS: There was no significant difference in 6-month PFS, median PFS, median OS, nor treatment-related AEs between the 2 arms. CONCLUSIONS AND RELEVANCE: Paricalcitol did not improve the efficacy of pembrolizumab likely related to its short half-life of only 5-7 hours. Microbiome analysis revealed significant difference between long-term (>12 weeks) and short-term (<12 weeks) survival groups across treatment arms. Modulation of the tumor microenvironment will likely require more sustained VDR activity. TRIAL REGISTRATION: Clinicaltrials.gov, ID: NCT03331562.

Chronic pancreatitis patient-derived organoids reveal new paths to precision therapeutics

Chronic pancreatitis (CP) affects ~3 million people worldwide, yet altering the course of disease is challenging. We developed a patient-derived organoid (PDO) platform to investigate the molecular pathogenesis of this disease and identify therapeutic strategies. We generated 36 PDOs from patients with idiopathic, hereditary, and alcohol-related CP with high genetic concordance. PDOs retained inflammation-associated transcriptional and proteomic features. Transcriptomic profiling revealed three molecular subtypes of CP independent of etiology. We discovered widespread dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in half of the CP PDOs, including those with wildtype CFTR. Clinically available CFTR modulators stabilized mutant or wildtype CFTR, restored CFTR function, and decreased mitogenic and inflammatory signaling. This work provides the first comprehensive PDO platform for modeling CP. We demonstrate the utility of this platform for precision therapeutic investigations. Our findings reveal CFTR modulators as a broadly applicable and effective therapeutic strategy.

Functional inversion of circadian regulator REV-ERBα leads to tumorigenic gene reprogramming

Profound functional switch of key regulatory factors may play a major role in homeostasis and disease. Dysregulation of circadian rhythm (CR) is strongly implicated in cancer with mechanisms poorly understood. We report here that the function of REV-ERBα, a major CR regulator of the orphan nuclear receptor subfamily, is dramatically altered in tumors in both its genome binding and functional mode. Loss of CR is linked to a functional inversion of REV-ERBα from a repressor in control of CR and metabolic gene programs in normal tissues to a strong activator in different cancers. Through changing its association from NCoR/HDAC3 corepressor complex to BRD4/p300 coactivators, REV-ERBα directly activates thousands of genes including tumorigenic programs such as MAPK and PI3K-Akt signaling. Functioning as a master transcriptional activator, REV-ERBα partners with pioneer factor FOXA1 and directly stimulates a large number of signaling genes, including multiple growth factors, receptor tyrosine kinases, RASs, AKTs, and MAPKs. Moreover, elevated REV-ERBα reprograms FOXA1 to bind new targets through a BRD4-mediated increase in local chromatin accessibility. Pharmacological targeting with SR8278 diminishes the function of both REV-ERBα and FOXA1 and synergizes with BRD4 inhibitor in effective suppression of tumorigenic programs and tumor growth. Thus, our study revealed a functional inversion by a CR regulator in driving gene reprogramming as an unexpected paradigm of tumorigenesis mechanism and demonstrated a high effectiveness of therapeutic targeting such switch.

Abstract A029: Evaluating and interpreting scGPT: A foundation model for single-cell biology in real-world cancer clinical trial data

The success of large language models (LLMs) with attention mechanisms in natural language processing has inspired a series of LLMs for single-cell analysis, such as scGPT. Such LLMs claim to have excellent performance as foundation models across various downstream tasks. This study aims to validate scGPT and explore its potential in cancer research using real-world clinical trial data. We assessed scGPT's performance in both zero-shot and fine-tuned scenarios on various tasks using single-nucleus sequencing data from pancreatic ductal adenocarcinoma (PDAC) patients undergoing different treatments, including ones treated at the Dana-Farber Cancer Institute. Our methodology encompassed a comprehensive evaluation of scGPT's capabilities, including zero-shot clustering, gene expression prediction, and cell reconstruction. We fine-tuned the model for downstream tasks like cell type annotation, treatment perturbation prediction, and gene regulatory network inference. Additionally, we extracted and analyzed multi-head, multi-layer embeddings and attention matrices, visualizing the flow of information within the model and how they respond to different perturbations or fine-tuning objectives to investigate the model's learning process and its correlation with biological information. The study utilized two main datasets for fine-tuning and evaluating scGPT: an unpublished clinical trial dataset featuring metastatic PDAC patients under three comparative treatment arms with pre- and on-treatment single-nucleus sequencing data, and a published clinical trial dataset containing sequencing data from PDAC patients who underwent various treatment regimens. Our results validate scGPT's potential to effectively extract biological information as a foundation model for single-cell biology. For zero-shot, scGPT demonstrated strong performance in tasks like clustering, while showing areas with suboptimal performance like gene expression prediction and reconstruction. In addition, fine-tuning significantly boosts the model's capabilities across various tasks. The model responds well to multiple fine-tuning objectives, capturing information that distinguishes between individuals and treatments, and accurately predicting patient and treatment groups at the single-cell level. The analysis of model structure and information flow revealed that multi-head attention and representations could, to some extent, capture biological information. For example, the model’s attention to certain genes varies in different heads and layers, with being altered to certain patterns responding to fine-tuning objectives. Our analysis of the model's architecture and attention mechanisms offers preliminary insights into the relationship between model behavior and biological processes. It also provides a framework for interpreting complex biological information through the lens of attention mechanisms in single cell LLMs, paving the way for future studies exploring the intersection of foundation models and cancer biology, potentially leading to transformative progress in clinical cancer care. Citation Format: Runzi Tan, Haotian Cui, Bo Wang, Kimberly Perez, Andressa Dias Costa, Alexander Jordan, Thomas Karacic, Dalia Elganainy, Dan Y Gui, Suryun Kim, Chen Yuan, Morgan Truitt, Michael Downes, Ronald Evans, Tae Gyu Oh, Peter O’Dwyer, Andrew Aguirre, Jonathan A Nowak, Brian Wolpin, Simona Cristea. Evaluating and interpreting scGPT: A foundation model for single-cell biology in real-world cancer clinical trial data [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A029.

Vitamin D Receptor Regulates Liver Regeneration After Partial Hepatectomy in Male Mice

Vitamin D signals through the vitamin D receptor (VDR) to induce its end-organ effects. Hepatic stellate cells control development of liver fibrosis in response to stressors and vitamin D signaling decreases fibrogenesis. VDR expression in hepatocytes is low in healthy liver, and the role of VDR in hepatocyte proliferation is unclear. Hepatocyte-VDR null mice (hVDR) were used to assess the role of VDR and vitamin D signaling in hepatic regeneration. hVDR mice have impaired liver regeneration and impaired hepatocyte proliferation associated with significant differential changes in bile salts. Notably, mice lacking hepatocyte VDR had significant increases in expression of conjugated bile acids after partial hepatectomy, consistent with failure to normalize hepatic function by the 14-day time point tested. Real-time PCR of hVDR and control livers showed significant changes in expression of cell-cycle genes including cyclins D1 and E1 and cyclin-dependent kinase 2. Gene expression profiling of hepatocytes treated with vitamin D or control showed regulation of groups of genes involved in liver proliferation, hepatitis, liver hyperplasia/hyperproliferation, and liver necrosis/cell death. Together, these studies demonstrate an important functional role for VDR in hepatocytes during liver regeneration. Combined with the known profibrotic effects of impaired VDR signaling in stellate cells, the studies provide a mechanism whereby vitamin D deficiency would both reduce hepatocyte proliferation and permit fibrosis, leading to significant liver compromise.

Abstract CT218: Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer

Background: Triptolide and its water-soluble prodrug, Minnelide™, have shown promising activity against pancreatic ductal adenocarcinoma (PDAC) in both in vitro and in vivo experiments. Triptolide induces apoptosis in pancreatic cancer cells by different pathways, including caspase-dependent apoptotic death and caspase -independent autophagic death. It inhibits the activity of the TFIIH general transcription factor complex and modulates the transcriptional landscape of both the stroma and cancer cell compartments. In the epithelial tumour compartment, triptolide down-regulates key transcription factors such as c-MYC. Preclinical investigations indicate significant activity against super enhancer targets. In the phase I clinical trial of Minnelide™ activity in highly refractory metastatic pancreatic carcinoma was observed. Hence, we undertook this study in patients with chemotherapy refractory metastatic PDAC. Our aims were to determine clinical anticancer effects, and to define effects of the drug on super enhancer expression and downstream effects. Methods: Eligibility criteria included stage IV PDAC progressing on standard chemotherapy, Karnofsky performance status ≥ 70%, and measurable disease by RECIST 1.1. Planned sample size was up to 35 patients with interim analysis after the first 18 evaluable patients, aiming for at least a 20% difference in DCR (10% v 30%). Minnelide™ 0.67 mg/m2 was administered intravenously over 30 minutes on days 1-21, followed by a 7-day rest period in a 28-day cycle. Exploratory objectives included: analyses of paired tumour biopsies for changes in immune cell infiltration, stromal activation, chromatin accessibility, proteomics and metabolism. Results: 17 patients were treated. There were no RECIST responses or stable disease. However, evidence of activity was observed in 4 of 15 evaluable patients. This activity included progression after 117 days treatment in 1 patient and reductions in CT tumour size or FDG-PET uptake and/or significant decreases in circulating CA19.9 levels in 3 others. Minnelide was generally well tolerated. Treatment related toxicities > 3 were: neutropenia 4 patients and cerebellar ataxia 1 patient.Exploratory analyses included ATAC-Seq on paired biopsies where gene ontology analyses showed downregulation of genes negatively regulating cell cycle and upregulation of genes controlling cell cycle checkpoints. Other exploratory analyses will be presented. Conclusions: The study identified a tolerable drug dose and evidence of activity in a highly chemotherapy-refractory population which provides encouraging proof for the principle of targeting TFIIH in human PDAC. Supported by SU2C, Cancer Research UK, Lustgarten Foundation Citation Format: David Propper, Erkut Borazanci, Peter J. O'Dwyer, Daniel Von Hoff, Joseph Hartlebury, Corina E. Antal, Gerard I. Evan, HaiYong Han, Michael Downes, Ronald Evans, Joshua Rabinowitz, Emmanuel Petricoin, Ashok Saluja, Mohana Velagapudi, Hitendra Patel. Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT218.

CAnceR IN PreGnancy (CARING) – a retrospective study of cancer diagnosed during pregnancy in the United Kingdom

Background The incidence of cancer diagnosed during pregnancy is increasing. Data relating to investigation and management, as well as maternal and foetal outcomes is lacking in a United Kingdom (UK) population. Methods In this retrospective study we report data from 119 patients diagnosed with cancer during pregnancy from 14 cancer centres in the UK across a five-year period (2016-2020). Results Median age at diagnosis was 33 years, with breast, skin and haematological the most common primary sites. The majority of cases were new diagnoses (109 patients, 91.6%). Most patients were treated with radical intent (96 patients, 80.7%), however, gastrointestinal cancers were associated with a high rate of palliative intent treatment (63.6%). Intervention was commenced during pregnancy in 68 (57.1%) patients; 44 (37%) had surgery and 31 (26.1%) received chemotherapy. Live births occurred in 98 (81.7%) of the cases, with 54 (55.1%) of these delivered by caesarean section. Maternal mortality during the study period was 20.2%. Conclusions This is the first pan-tumour report of diagnosis, management and outcomes of cancer diagnosed during pregnancy in the UK. Our findings demonstrate proof of concept that data collection is feasible and highlight the need for further research in this cohort of patients.