Aberrant epigenetic reprogramming together with dysregulated mTOR signaling are hallmarks of cancer, where altered chromatin methylation and nutrient-sensing pathways cooperate to drive tumor progression. S-adenosylmethionine (SAM), the universal methyl donor, is essential for these processes, yet how tumors sustain elevated SAM availability to support oncogenic transmethylation reactions remains poorly defined. Here, using prostate cancer (PCa) as a model system, we identify nicotinamide N-methyltransferase (NNMT) as a critical metabolic-epigenetic regulator and tumor suppressor. Using a prostate-specific Nnmt knockout mouse model, we demonstrate that NNMT loss accelerates PCa progression, particularly in the context of Pten deletion, resulting in infiltrating carcinoma and reduced survival. Mechanistically, NNMT functions as a "SAM-sink," and its loss increases intracellular SAM abundance, thereby activating mTORC1 signaling through SAMTOR-dependent sensing and broadly enhancing chromatin methylation. In human PCa, recurrent genomic deletions of NNMT occur in up to 7% of cases, and NNMT protein expression is largely absent in primary tumors and metastases. NNMT-deficient PCa cells exhibit elevated SAM:SAH ratios, increased histone methylation, and heightened mTORC1 activity, enabling sustained tumor growth even under dietary methionine-restriction (MR). Notably, combined MR and pharmacologic mTORC1 inhibition synergistically suppresses the growth of NNMT-deficient tumors, revealing a previously unrecognized therapeutic vulnerability. Collectively, these findings establish NNMT as a key tumor suppressor that constrains SAM-driven epigenetic and signaling programs in PCa and suggest a rational, diet-based therapeutic strategy for advanced cancers with NNMT loss.

Cigarette smoke promotes bladder tumor growth by enhancing cancer cell survival and proliferation through smoke mediated carcinogens. FASN, a key enzyme in fatty acid synthesis, is dysregulated in many cancers and correlates with aggressive phenotypes. In this study, we demonstrate elevated fatty acid levels and FASN specifically in smokers with bladder cancer. Elevated FASN under smoke exposure imparted epigenetic alterations, particularly histone acetylation, impacts DNA repair and DNA-binding transcription factors which regulate metabolic pathways. Under cigarette smoke, bladder cancer cells undergo a metabolic shift, utilizing glutamine as a major carbon source through reductive carboxylation to fuel fatty acid biosynthesis via FASN. Genetic and pharmacological inhibition of FASN significantly reduced tumor growth in a Chicken embryo Chorio-allantoic Membrane model exposed to smoke. FASN inhibitors such as TVB-2640, currently in clinical trials, may represent an effective therapeutic strategy for smokers with bladder cancer exhibiting high FASN levels.

Changes in the methylation of proteins, nucleic acids, and metabolites are fundamental in cancer development. Specifically, when these changes affect the methylation of histones and nucleic acids, they can alter the epigenetic status of cells, impacting the expression of thousands of genes and driving cancer initiation and progression. S-adenosylmethionine (SAM) is the universal methyl donor for SAM-dependent reactions, while its byproduct, S-adenosylhomocysteine (SAH), inhibits methyltransferases, including HMTase and DNMTs. Dysregulation of SAM, SAH, and their ratio can reshape the histone and DNA methylation landscape and alter gene expression. Nicotinamide (NAM) N-methyltransferase (NNMT), a one-carbon group methyltransferase that catalyzes NAM methylation to produce SAH and 1-methylnicotinamide (1-MNAM), is a well-characterized methyl sink that reduces the overall methylation potential of cells. NNMT has been implicated in regulating multiple metabolic pathways in cancer cells through its high consumption of SAM and generation of the chemically inert metabolite 1-MNAM. High expression of NNMT in certain cancers, particularly in cancer-associated fibroblasts, results in excessive consumption of the SAM pool (a “SAM-sink”), thereby diverting SAM from DNA and histone methylation processes and ultimately leading to metastasis and cancer progression. Here, we hypothesized that NNMT could act as a tumor suppressor in prostate cancer (PCa), which relies on high SAM availability for polyamine synthesis and methylation. To test this hypothesis, we generated prostate-specific NNMT knockout mice combined with PTEN deletion developed infiltrating carcinoma associated with increased histone methylation marks (H3K36me3/H3K27me3) and activation of oncogenic pathways. Importantly, we identified a recurrent, focal genomic deletion of NNMT in up to 8% of human PCa. Additionally, a tissue microarray containing hundreds of PCa samples demonstrated tumor-specific loss of NNMT expression. CRISPR-mediated NNMT deletion or overexpression in PCa cells caused reciprocal changes in SAM/SAH ratios, global methylation patterns, methylated chromatin marks, and tumor growth in vivo. Importantly, reducing methionine levels, a precursor of SAM, through a methionine-restricted diet resulted in tumor growth inhibition in the context of NNMT activity in multiple cancer models. Together, our data suggest that tumor-specific genetic and epigenetic loss of NNMT results in sufficient SAM availability for DNA and histone methylation, even in nutrient-deprived environments, promoting epigenetic homeostasis and cancer progression. Erick L. Mitchell-Velasquez, Qu Deng, Mohammed Alhusayan, Ramakrishnan Natesan, Sharan Venkatesh, Gabriel Raytsis, Priti Lal, Rahul Mannan, Benjamin Garcia, Irfan Asangani. NNMT loss drives prostate cancer progression through epigenetic and metabolic reprogramming [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 5379.

Mutations in histone 3 at or near lysine 36 (H3K36) have dominantly acting oncogenic effects in multiple tumor types by limiting H3K36-directed methyltransferases. Paradoxically, we find that expression of the H3 K36M oncohistone unexpectedly inhibits tumor formation in KRAS-driven lung adenocarcinoma by inducing a potent immune-mediated tumor clearance. Mechanistically, oncohistone expression derepresses endogenous retroviral element transcription, results in the accumulation of double-stranded RNA (dsRNA), and activates an innate antiviral-like immune response that eradicates tumor growth. Surprisingly, while inactivation of the H3K36 di-methyltransferase NSD2 replicated all effects of oncohistone expression, inactivation of the H3K36 tri-methyltransferase SETD2 abolished element derepression and all associated downstream anti-cancer effects that are induced by oncohistone expression. These observations restructure our understanding of the roles of H3K36 methylation, the consequences of its deregulation in cancer, and shape our expectations for therapeutic interventions targeting H3K36 methyltransferases.

Following prolonged liver injury, a small fraction of hepatocytes undergoes reprogramming to become cholangiocytes or biliary epithelial cells (BECs). This physiological process involves chromatin and transcriptional remodeling, but the epigenetic mediators are largely unknown. Here, we exploited a lineage-traced model of liver injury to investigate the role of histone post-translational modification in biliary reprogramming. Using mass spectrometry, we defined the repertoire of histone marks that are globally altered in quantity during reprogramming. Next, applying an in vivo CRISPR screening approach, we identified seven histone-modifying enzymes that alter the efficiency of hepatobiliary reprogramming. Among these, the histone methyltransferase and demethylase Nsd1 and Kdm2a were found to have reciprocal effects on H3K36 methylation that regulated the early and late stages of reprogramming, respectively. Although loss of Nsd1 and Kdm2a affected reprogramming efficiency, cells ultimately acquired the same transcriptomic states. These findings reveal that multiple chromatin regulators exert dynamic and complementary activities to achieve robust cell fate switching, serving as a model for the cell identity changes that occur in various forms of physiological metaplasia or reprogramming.

Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer for which development of effective therapies is urgently needed. Metabolic programs are vastly remodeled in pancreatic cancer to favor the production of certain metabolites that support tumor progression. Previous studies reported that catabolism of branched-chain amino acids (BCAAs) is the major carbon source for energy production in healthy pancreatic acinar cells. However, in PDA cells, BCAA metabolism is reprogrammed and its contribution to the TCA cycle is suppressed. Our labs recently showed that BCAAs, primarily isoleucine, are a primary supplier of propionyl-CoA (pr-CoA), which accumulates in the nuclei of cancer cells and is utilized for site-specific histone lysine propionylation (Kpr). Yet, the metabolic route through which BCAA catabolism supplies pr-CoA to the nucleus, as well as the distinct function of BCAA-derived Kpr in promoting tumor growth, are currently unknown. Here, we find that disrupting BCAA catabolism in PDA cells by either nutrient deprivation or genetic manipulation significantly decreases pr-CoA pools and the global levels of selective Kpr marks. Loss of BCAA-derived Kpr downregulates the expression of specific genes associated with lipid metabolism and immunosuppression. Accordingly, defective BCAA oxidation inhibits PDA tumor growth in vitro and in vivo, signifying the potential of targeting BCAA metabolism to attenuate PDA progression. Remarkably, we show that, in addition to their expected location in the mitochondria, BCAA metabolic enzymes also are detectable in the nuclear compartment of PDA cells and primary patient tissues, suggesting on-site synthesis of pr-CoA for Kpr. Overall, our findings support a novel link between metabolism and epigenetics in which segregation of a nuclear specific BCAA metabolic sub-network is harnessed by PDA cells to boost histone propionylation and shape malignant gene expression signatures. Christina Demetriadou, Michael Noji, Erick Mitchell-Velasquez, Sharan Venkatesh, Austin Good, Taku Harada, Daniel Kantner, Zoltan Arany, Irfan Asangani, Ben Stanger, Nathaniel Snyder, Kathryn Wellen. Branched-chain amino acid metabolism and histone propionylation in pancreatic cancer [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 3798.

Metastatic prostate cancer (PCa) is incurable and the second leading cause of cancer death among men in the Western world. Although patients initially respond to androgen deprivation therapy (ADT), most eventually develop castration-resistant prostate cancer (CRPC) and metastasize to bone. Second-generation AR signaling inhibitors, such as abiraterone acetate and enzalutamide, have been shown to provide a median survival benefit of 6 to 24 months for metastatic CRPC patients. Therefore, more effective therapies are needed, including novel and innovative therapeutic combination approaches. PARP inhibitors are used primarily in treating patients with DNA damage response (DDR) pathway (e.g. BRCA1/2) mutated cancers because these mutations cause a deficit in homology-directed DNA repair (HDR) that confers sensitivity to these agents. Though BRCA1/2 mutations are relatively rare, genomic defects in other DNA repair genes account for approximately 20% of advanced CRPC. Therefore, PARP inhibitor was recently FDA-approved for metastatic PCa carrying these mutations. However, the rest of the 80% HR-proficient CRPC patients do not benefit from PARPi. Apart from that, prostate tumors are immunologically cold tumors and only 5% of PCa patients respond to immune checkpoint blockers. Using next generation genomics approaches, we have found that inhibiting transcriptional kinase CDK7 in PCa cells creates a BRCA-deficient state (BRCAness) that sensitizes these HR-proficient tumor cells to PARP inhibitors in vitro and in vivo. Mechanistically, knockdown or inhibition of CDK7 led to transcriptional downregulation of DDR genes through loss of Mediator activity along with accumulation of R-loops resulting in the induction of DNA damage, micronuclei formation with consequent activation of cGAS-STING mediated inflammatory signaling, and synergy with anti-PD1 therapy in a syngeneic PCa model in vivo without toxicity. These findings suggest that CDK7 inhibition synergizes with PARP inhibitors and immune checkpoint blockers, offering a promising therapeutic strategy for both HR-proficient and HR-deficient metastatic PCa. This approach has the potential to overcome current limitations of immune checkpoint inhibitors and provides new avenues for targeted combination therapies in advanced PCa treatment. Chandan Kanta Das, Hesham Mohei, Brijesh Kumar Verma, Muzaffer Kassab, Phillip Wulfridge, Sharan Venkatesh, Mohammed Alhusayan, Reyaz ur Rasool, Kavitha Sarma, Roger A. Greenberg, Eric J. Brown, Malay Haldar, Irfan A. Asangani. Transcriptional regulation of DNA damage response and associated inflammatory signaling by CDK7 in prostate cancer [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 6159.

Ewing Sarcoma (EwS) is a rare pediatric malignancy characterized by a unique t(11:22) (q24;q12) translocation resulting in the pathognomonic EWSR1::FLI1 fusion. Recent reports indicate that the EWSR1::FLI1 oncofusion drives aberrant expression of numerous transcripts, including Lipoxygenase Homology Domains 1 (LOXHD1). Given its highly restricted protein expression pattern and role in EwS tumorigenesis and metastasis, LOXHD1 may serve as a novel immunotherapeutic target in this malignancy. LOXHD1 immunogenic epitopes restricted to HLA-A*02:01 allowed for the isolation of a high avidity αβTCR. LOXHD1-specific TCR engineered CD8+ T cells conferred cytotoxic activity against a panel of HLA-A*02:01+ EwS tumor cell lines and adoptive transfer led to tumor eradication in a mouse xenograft model of EwS. This study nominates LOXHD1 as an oncofusion regulated, non-mutated tumor associated antigen (TAA) with expression limited to inner hair cells of the cochlea, adult testis, and EwS.

Androgen receptor (AR) is a ligand-responsive transcription factor that drives terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to activate malignant phenotypes, the molecular mechanisms of which remain unknown. Here, we show that tumor-specific AR enhancers are critically reliant on H3K36 dimethyltransferase activity of NSD2. NSD2 expression is abnormally induced in prostate cancer, where its inactivation impairs AR transactivation potential by disrupting over 65% of its cistrome. NSD2-dependent AR sites distinctively harbor the chimeric FOXA1:AR half-motif, which exclusively comprise tumor-specific AR enhancer circuitries defined from patient specimens. NSD2 inactivation also engenders increased dependency on the NSD1 paralog, and a dual NSD1/2 PROTAC degrader is preferentially cytotoxic in AR-dependent prostate cancer models. Altogether, we characterize NSD2 as an essential AR neo-enhanceosome subunit that enables its oncogenic activity, and position NSD1/2 as viable co-targets in advanced prostate cancer. CRISPR screen identifies coactivators of the androgen receptor (AR) complex, including NSD2. NSD2 contributes to AR cistrome reprogramming during prostate cancer progression, and its degradation via a novel PROTAC reduces prostate cancer cell viability in vitro.

Despite recent treatment advances, non-small cell lung cancer (NSCLC) remains one of the leading causes of cancer-related deaths worldwide, and therefore it necessitates the exploration of new therapy options. One commonly shared feature of malignant cells is their ability to hijack metabolic pathways to confer survival or proliferation. In this study, we highlight the importance of the polyol pathway (PP) in NSCLC metabolism. This pathway is solely responsible for metabolizing glucose to fructose based on the enzymatic activity of aldose reductase (AKR1B1) and sorbitol dehydrogenase (SORD). Via genetic and pharmacological manipulations, we reveal that PP activity is indispensable for NSCLC growth and survival in vitro and in murine xenograft models. Mechanistically, PP deficiency provokes multifactorial deficits, ranging from energetic breakdown and DNA damage, that ultimately trigger the induction of apoptosis. At the molecular level, this process is driven by pro-apoptotic JNK signaling and concomitant upregulation of the transcription factors c-Jun and ATF3. Moreover, we show that fructose, the PP end-product, as well as other non-glycolytic hexoses confer survival to cancer cells and resistance against chemotherapy via sustained NF-kappa B activity as well as an oxidative switch in metabolism. Given the detrimental consequence of PP gene targeting on growth and survival, we propose PP pathway interference as a viable therapeutic approach against NSCLC.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate “reprogramming” by opening new chromatin sites for expression that can attract transcription factors from the starting cell’s enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver. In lineage-traced cells, we assessed the timing of SOX4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, SOX4 directly binds to and closes hepatocyte regulatory sequences via an overlapping motif with HNF4A, a hepatocyte master regulatory transcription factor. Subsequently, SOX4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

SETD2 is a tumor suppressor that is frequently inactivated in several cancer types. The mechanisms through which SETD2 inactivation promotes cancer are unclear, and whether targetable vulnerabilities exist in these tumors is unknown. Here we identify heightened mTORC1-associated gene expression programs and functionally higher levels of oxidative metabolism and protein synthesis as prominent consequences of Setd2 inactivation in KRAS-driven mouse models of lung adenocarcinoma. Blocking oxidative respiration and mTORC1 signaling abrogates the high rates of tumor cell proliferation and tumor growth specifically in SETD2-deficient tumors. Our data nominate SETD2 deficiency as a functional marker of sensitivity to clinically actionable therapeutics targeting oxidative respiration and mTORC1 signaling.

sSupplementary Legends for Figures 1-5 from Cetuximab Attenuates Metastasis and u-PAR Expression in Non–Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity

Supplementary Figure 3. Expression of PCAT29 in VCaP and LNCaP cells.

Supplementary Table S2. Positive and negative correlations.

Supplementary Table S1. shRNA and primer sequence table.

Supplementary Data from EWS/ETS-Driven Ewing Sarcoma Requires BET Bromodomain Proteins

The androgen receptor (AR) is a ligand-responsive transcription factor (TF) that binds as a homodimer at FOXA1-pioneered enhancer elements containing a palindromic DNA motif. Prostate cancer (PCa) is highly dependent on the AR enhanceosome complex, and in castration-resistant disease, this dependency is reinforced through alterations in the AR pathway. This centrally involves extensive rewiring of the AR cistrome to gain de novo binding at enhancer sites (aka neo-enhancers) that activate hyperproliferative and metastatic gene programs. However, requisite subunits of the AR neo-enhanceosome and the genes it uniquely activates remain uncharacterized. Here, we CRISPR-engineered an endogenous AR reporter system by fusing the mCherry coding sequence in-frame with the KLK3/PSA gene and conducted a CRISPR knock-out screen to identify druggable chromatin/epigenetic cofactors of AR. Using a sgRNA library covering >200 druggable transcriptional coregulators, ranked alongside BRD4 and TRIM24/28, we identified NSD2 as a novel AR coactivator. NSD2 is an H3K36 di-methyltransferase that activates gene expression through antagonism of the PRC2/EZH2 complex. In PCa cells, genetic inhibition of NSD2 function significantly attenuated the expression of AR targets without affecting the AR protein level, suggesting that NSD2 regulates either AR’s chromatin assembly or trans-activational competence. Cistromic profiling of AR (ChIPseq) in NSD2-null PCa cells showed complete loss of binding at >40,000 genomic sites (~65% of the cistrome), with HOMER motif analyses of NSD2-dependent AR sites showing a higher recurrence of hexameric 5′-AGAACA-3′ AR half-site juxtaposed to the FOXA1, ETS, or HOXB13 motifs. In contrast, NSD2-independent AR sites, a large fraction of which show increased binding in NSD2-null cells, housed the canonical 15-bp palindromic AR motif with two invertedly-oriented half-sites separately recognized by each half of the AR homodimer. Notably, analyses of primary AR cistromes from human tissues revealed AR loading at half-motifs to be 20-30-fold enriched in the tumor-specific AR enhancer circuitry. In phenotypic assays, we found NSD2-deficient PCa cells to lose cancer hallmarks such as colony formation, invasion, and hyper-proliferation. We further developed a potent NSD2 PROTAC, LLC0150, which showed preferential cytotoxicity in AR/FOXA1+ PCa relative to the AR-negative disease, and other cancer and normal cell lines from 22 distinct lineages. In a pan-cancer screen comprising over 100 cell lines, treatment with LLC0150 triggered apoptotic death in AR/FOXA1+ malignancies and NSD2-mutant acute lymphocytic leukemia, and multiple myeloma. Altogether, we identify NSD2 as a novel AR neo-cofactor that assists oncogenic TFs in loading the AR enhanceosome at degenerate, low-affinity AR elements, thereby wiring its cancer-specific gene programs. We develop a novel NSD2 PROTAC that selectively kills AR/FOXA1-driven prostate and NSD2-altered tumors in preclinical models, positioning NSD2 therapeutics for safety and efficacy evaluation in human clinical trials. Citation Format: Abhijit Parolia, Brijesh K. Verma, Sanjana Eyunni, Sweta Aras, Sandra E. Carson, Eleanor Young, Chandan K. Das, James M. George, Reyaz ur Rasool, Prathibha Gajjala, Mohammed Alhusayan, Erick Mitchell-Velasquez, Lanbo Xiao, Jie Luo, Mustapha Jaber, Xuhong Cao, Fengyun Su, Rui Wang, Lianchao Liu, Zhen Wang, Ke Ding, Arul M. Chinnaiyan, Irfan Asangani. NSD2 is a requisite and targetable subunit of the AR/FOXA1 neo-enhanceosome complex in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A055.

Supplementary Methods and Data (posted 7/5/2011) from Characterization of KRAS Rearrangements in Metastatic Prostate Cancer