MYC antagonizes AR-stimulated expression of ODC1 and AMD1

DFMO decreases putrecine induced by SPA and does not increase efficacy of AR inhibition

Supraphysiologic androgen (SPA) treatment can paradoxically restrict the growth of castration-resistant prostate cancer (CRPC) with high androgen receptor (AR) activity, which is the basis for the use of bipolar androgen therapy (BAT) for patients with this disease. Although androgens are widely appreciated for enhancing anabolic metabolism, how SPA-mediated metabolic changes alter prostate cancer progression and therapy response is unknown. In this study, we report that SPA markedly increased intracellular and secreted polyamines in prostate cancer models. AR binding at enhancer sites upstream of the ornithine decarboxylase 1 (ODC1) promoter increased the abundance of ODC, a rate-limiting enzyme of polyamine synthesis, and de novo synthesis of polyamines from arginine. SPA-stimulated polyamines enhanced prostate cancer fitness, as dCas9-KRAB-mediated inhibition of AR regulation of ODC1 or direct ODC inhibition by difluoromethylornithine (DFMO) increased the efficacy of SPA. Mechanistically, AR activation combined with the loss of negative feedback by polyamines increased the activity of S-adenosylmethionine decarboxylase 1, leading to the depletion of its substrate, S-adenosylmethionine, and global protein methylation. These data provided the rationale for a clinical trial testing the safety and efficacy of BAT in combination with DFMO for patients with metastatic CRPC. Pharmacodynamic studies of this therapeutic combination in the first five patients in the trial indicated that this approach effectively depleted polyamines in plasma. Thus, the AR potently stimulates polyamine synthesis, which constitutes a vulnerability in prostate cancer treated with SPA that can be targeted therapeutically. SIGNIFICANCE:Increased polyamine synthesis is a prominent metabolic change induced by the androgen receptor that drives tumor progression and confers a targetable vulnerability in advanced prostate cancer. See related commentary by Alizadeh-Ghodsi and Goldstein, p. 1095.

Characteristics of subjects in the Hamalainen autopsy study.

Background Although immune checkpoint inhibitors (ICIs) have long half-lives, preclinical and retrospective clinical studies across multiple tumor types suggest that the time-of-day of ICI infusion may influence therapeutic efficacy by aligning initial drug exposure with circadian peaks in T-cell responsiveness. The immunological basis of this phenomenon and its clinical relevance in hepatocellular carcinoma (HCC) remains unknown. Methods We followed patients with advanced HCC receiving ICI therapy at Johns Hopkins from 2021 to 2025, classifying them into a morning (first treatment before 12:00 hours) or afternoon (first treatment after 12:00 hours) group. We assessed clinical outcomes and compared immunological responses from baseline to early-on-treatment by profiling peripheral blood mononuclear cells using cytometry by time-of-flight and plasma cytokines using a 39-plex Luminex assay. Results Our cohort included 84 patients, 39 of whom received their first infusion in the morning. There were no statistically significant differences in baseline demographic or clinical characteristics between patients initiating therapy in the morning versus afternoon. The morning group had superior progression-free survival (multivariable HR 0.50, 95% CI 0.30 to 0.84, p<0.01) and higher odds of treatment response (multivariable OR 3.26, 95% CI 1.08 to 10.90, p<0.05), with no significant increase in immune-related adverse events. The timing of subsequent infusions after the first dose had no impact on outcomes. Immunological responses diverged after the initial dose, with morning-treated patients showing reduced interleukin (IL)-6 levels (p<0.01) and greater expansion of cytotoxic central memory CD8+ T cells (p=0.01) as well as cytotoxic effector and effector memory CD8+ T cells (p=0.06). Conclusions Morning first-dose infusion of ICIs in HCC was associated with improved clinical outcomes and distinct immune responses, including reduced IL-6 signaling and expansion of cytotoxic central memory CD8+ T cells. These findings suggest that the timing of the initial infusion can imprint an immunological program that shapes subsequent antitumor immunity, providing a mechanistic rationale for strategically scheduling ICI administration.

Androgens increase ODC in a dose-dependent manner

Ferroptos is is an iron-dependent form of cell death converging on lipid peroxidation first identified by examining compounds with enhanced lethality to KRAS mutant cells. Despite over 90% of pancreatic ductal adenocarcinoma (PDAC) tumors harboring KRAS mutations, PDAC exhibits relative resistance to ferroptosis compared with other tumor types, and the mechanisms behind this resistance remain unclear. Here, we report that exposure to pancreatic tumor interstitial fluid in synergy with hypoxia induced robust protection against ferroptosis in a manner dependent on the hypoxia-inducible transcription factor 2 (HIF-2). HIF-2 upregulates the expression of both components of the system Xc-cystine transporter and transsulfuration pathway enzymes CBS and CTH to increase intracellular cysteine levels, enabling anti-ferroptotic glutathione production. HIF-2 also induces the Parkin mitophagy factor and suppresses mitochondrial function and reactive oxygen species (ROS) generation. Altogether, our findings uncover an unforeseen role of the HIF-2 transcription factor as a coordinator of anti-ferroptotic mechanisms in pancreatic cancer.

dCas9-KRAB blocks AR binding upstream of ODC1

Abstract Background: Tumor heterogeneity allows cancer cells to adapt to microenvironmental stress and resist therapies. MYC is commonly amplified in human cancers and its expression can be heterogeneous due to variations in extrachromosomal DNA (ecDNA) and the pulsatile nature of MYC expression. However, the heterogeneity of the MYC responsive transcriptome among single cells is unknown. Here, we evaluate the heterogeneity of MYC transcriptional response in vitro in an inducible MYC cancer cell line. Methods: We used the P493-6 human B cell line model of Epstein-Barr Virus associated Burkitt’s lymphoma (BL) with tetracycline-repressible (Tet-OFF) ectopic MYC expression. We modulated MYC expression by addition of tetracycline and performed paired bulk and single-cell RNA-sequencing (scRNA-seq). To further characterize the transcriptional response of these cells with different MYC levels, we turned off MYC in these cells and then turned it back on to capture cells with increasing levels of MYC over time and performed paired bulk and single-cell RNA sequencing. Results: We observed heterogeneous expression of MYC and strikingly diverse expression of MYC responsive genes at the single cell level. After MYC induction with the removal of tetracycline, we observe a time-dependent decrease in STAT1 and other immune-related gene expression and movement of single cell transcriptomic clusters in high dimensional space visualized with tSNE plotting. Intriguingly, we observed a small cluster of CD58+ cells whose position in the tSNE space is unaltered by MYC status. We surmise that this cluster behaves like ‘stem’ cells capable of repopulating the entire population of P493 cells. Interestingly, we also see this small cluster of cells in some patient-derived BL cell lines. Conclusions: Overall, we provide detailed evidence of MYC driven transcriptional heterogeneity at the single cell level. Our preliminary evidence of this unique ‘stem-cell’ like population of cells in BL warrants further study. Citation Format: Saravana Gowtham Baskaran, Min-Zhi Jiang, Ada Tam, Hongkai Ji, Chi Van Dang. MYC associated transcriptional heterogeneity at the single cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4773.

Why cancer arises, progresses, or proves fatal in some people but not others remains largely unresolved. Antibody repertoires, including autoantibodies targeting immune pathways, may shape cancer immunosurveillance. Mapping antibody landscapes across cancer-free, at-risk, and cancer-affected individuals could clarify their roles in cancer susceptibility and disease outcome.

ODC inhibition enhances PCa growth suppression of LN95 tumors by SPA in vivo

SPA does not alter abundance of hypusinated eIF5A

Glutamine metabolism is upregulated in many cancers. While multiple glutamine imaging agents have been developed and translated to clinical use, the short half-lives of their signal and instability in vivo limit the aspects of glutamine metabolism they capture. In phantoms at physiological pH, chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) contrast was observed at 11.7 T from glutamine, downstream metabolic products (glutamate and ammonia) and their co-substrates (alanine, aspartate, and cystine/cysteine). This contrast increased at lower pH. These results suggest that both uptake and metabolism of glutamine would increase CEST signal enhancement. We then investigated the feasibility of imaging the uptake (delivery, transport and metabolism) of naturally-occuring glutamine using CEST MRI in preclinical prostate cancer models, wherein key metabolic proteins are the glutamine transporter ASCT2 and as well as enzymes GLS1, ALT2 (GPT2), AST1 (GOT1), and GDH1 (GLUD1). The LNCaP prostate cancer line exhibited higher expression of ASCT2, GDH1, ALT2, and AST1 compared to DU-145 cells. CEST MRI enhancement upon administration of glutamine was consistently higher in LNCaP 3D spheres (phantoms) and tumors (in vivo) than their DU-145 counterparts. Mass spectrometry imaging confirmed higher uptake and metabolism of glutamine in LNCaP tumors. These findings demonstrate that CEST MRI of glutamine is capable of distinguishing preclinical prostate tumor models that differ in glutamine uptake and has potential for translation to clinical use.

Recent approvals of immune checkpoint inhibitor (ICI) based regimens have transformed the clinical landscape of advanced hepatocellular carcinoma (HCC), however, clinical responses are only observed in a minority of patients. Trimethylamine-N-oxide (TMAO) is an amine oxide biosynthesized from trimethylamine (TMA), generated when gut bacteria metabolize dietary choline via the enzyme CutC. While prior studies have shown that TMAO concentrations positively correlate with the risk of several gastrointestinal malignancies, newer studies have demonstrated that TMAO may enhance antitumor immunity in some tumor types. TMAO may therefore play an important role in modulating the tumor immune response, but its significance in HCC is unknown. We prospectively collected whole blood from patients with HCC prior to initiating standard-of-care ICI-based therapy at Johns Hopkins (IRB #00267960). Clinical records were reviewed for demographic characteristics and clinical outcomes. Additionally, we studied the impact of acute and chronic choline supplementation on anti-PDL1 efficacy in a syngeneic murine model of HCC (Hep53.4 in C57BL/6 mice). Baseline choline, TMA, and TMAO plasma concentrations in clinical and preclinical specimens were quantified via liquid chromatography-mass spectrometry (LC-MS) utilizing the SCIEX Triple Quad 6500+ system. The clinical cohort included 42 patients with advanced HCC. Patients were predominantly male (81.0%) with Barcelona Clinic Liver Cancer (BCLC) stage C disease (73.8%) who underwent treatment with atezolizumab + bevacizumab (52.4%) or durvalumab + tremelimumab (31.0%). In a multivariable Cox model, higher plasma TMAO concentration at baseline was associated with inferior progression-free survival (PFS; HR 1.36 per interquartile range [IQR] increase, p=0.032) and overall survival (OS; HR 1.63 per IQR increase, p<0.001) after controlling for BCLC stage, albumin-bilirubin index, alpha fetoprotein (AFP) concentration, and baseline choline and TMA concentrations. In our preclinical model, mice given chronic choline supplementation (1% choline diet for 14 days prior to treatment with anti-PDL1 therapy) had increased plasma TMAO concentrations (p<0.001) and exhibited no response to anti-PDL1 therapy, compared to mice given standard and acute choline (1% choline diet starting concurrently with anti-PDL1 treatment) diets. Our clinical and preclinical results suggest that high plasma TMAO concentration is associated with inferior outcomes on ICI therapies in HCC. These findings contrast with existing preclinical studies that suggest TMAO may potentiate responses to ICI therapy. We hypothesize that chronic TMAO exposure may lead to immune cell exhaustion via promotion of an inflammatory microenvironment and oxidative stress. Studies examining the effect of CutC inhibition are ongoing in our laboratory and may provide further rationale for targeting TMAO in HCC. Mari Nakazawa, Vivian L. Raj, NV Rajeshkumar, Noushin Rastkari, Michael Davis, Elizabeth De Oliveria, Madelena Brancati, Ervin Griffin, Kabeer Munjal, James Leatherman, Sarah Mitchell, Marina Baretti, Won Jin Ho, Mark Yarchoan, Chi V. Dang. Trimethylamine-N-oxide (TMAO) mediates immunotherapy resistance in advanced HCC [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 2222.

BACKGROUND:Neuroblastoma is a heterogeneous disease with adrenergic (ADRN)-like cells and therapy-resistant mesenchymal (MES)-like cells driven by distinct transcription factor networks. Here, we investigate the expression of immunotherapeutic targets in each neuroblastoma subtype and propose pan-neuroblastoma and cell state-specific targetable cell surface proteins. METHODS:We characterized cell lines, patient-derived xenografts, and patient samples as ADRN-dominant or MES-dominant to define subtype-specific and pan-neuroblastoma gene sets. Targets were validated with ChIP-sequencing, immunoblotting, and flow cytometry in neuroblastoma cell lines and isogenic ADRN-to-MES transition cell line models. Finally, we evaluated the activity of MES-specific agents in vivo and in vitro. RESULTS:Most immunotherapeutic targets being developed for neuroblastoma showed significantly higher expression in the ADRN subtype with limited expression in MES-like tumor cells. In contrast, CD276 (B7-H3) and L1CAM maintained expression across both ADRN and MES states. We identified several receptor tyrosine kinases (RTKs) enriched in MES-dominant samples and showed that AXL targeting with ADCT-601 was potently cytotoxic in MES-dominant cell lines and showed specific antitumor activity in a MES cell line-derived xenograft. CONCLUSIONS:Immunotherapeutic strategies for neuroblastoma must address the potential of epigenetic downregulation of antigen density as a mechanism for immune evasion. We identified several RTKs as candidate MES-specific immunotherapeutic target proteins for the elimination of therapy-resistant cells. We hypothesize that the phenomena of immune escape will be less likely when targeting pan-neuroblastoma cell surface proteins such as B7-H3 and L1CAM, and/or dual targeting strategies that consider both the ADRN and MES cell states.

Philadelphia chromosome-like B-cell acute lymphoblastic leukemia (Ph-like ALL) is driven by genetic alterations that induce constitutive kinase signaling and is associated with chemoresistance and high relapse risk in children and adults. Preclinical studies in the most common CRLF2-rearranged/JAK pathway-activated Ph-like ALL subtype have shown variable responses to JAK inhibitor-based therapies, suggesting incomplete oncogene addiction and highlighting a need to elucidate alternative biologic dependencies and therapeutic vulnerabilities, while the ABL-class Ph-like ALL subtype appears preferentially sensitive to SRC/ABL- or PDGFRB-targeting inhibitors. Which patients may be responsive versus resistant to tyrosine kinase inhibitor (TKI)-based precision medicine approaches remains a critical knowledge gap. Using bulk and single-cell multiomics analyses, we profiled residual cells from CRLF2-rearranged or ABL1-rearranged Ph-like ALL patient-derived xenograft models treated in vivo with targeted inhibitors to identify TKI-resistant subpopulations and potential mechanisms of therapeutic escape. We detected a specific MYC dependency in Ph-like ALL cells and defined a new leukemia cell subpopulation with senescence-associated stem cell-like features regulated by AP-1 transcription factors. This dormant ALL subpopulation was effectively eradicated by dual pharmacologic inhibition of BCL-2 and JAK/STAT or SRC/ABL pathways, a clinically-relevant therapeutic strategy. Single cell-derived molecular signatures of this senescence and stem/progenitor-like subpopulation further predicted poor clinical outcomes associated with other high-risk genetic subtypes of childhood B-ALL and thus may have broader prognostic applicability beyond Ph-like ALL.

BACKGROUND & AIMS:Fibrolamellar carcinoma (FLC) is a pediatric and adolescent liver cancer that is characterized by a recurrent fusion of DNAJB1 and PRKACA, yielding a chimeric translated protein, DNAJ-PKAc. PRKACA encodes the catalytic subunit of protein kinase A (PKA), a regulator of cellular metabolism. METHODS:We generated a syngeneic murine model of FLC, TIBxDNAJ-PKAc. We utilized preclinical models of FLC and human specimens to characterize the metabolic and immune effects of DNAJ-PKAc. RESULTS:DNAJ-PKAc induced a high glycolytic and glutamine flux to support nucleotide metabolism. As compared to parental TIBx tumors, TIBxDNAJ-PKAc tumors demonstrated reduced T-cell infiltration with impaired T-cell activation. Systemic administration of a glutamine antagonist reversed the immune-inactivated phenotype of TIBxDNAJ-PKAc tumors and provided tumor control in combination with immune checkpoint inhibitors. CONCLUSION:The presence of DNAJ-PKAc creates a vulnerability to the combination of glutamine antimetabolite and immune checkpoint inhibitor therapy in FLC. IMPACT AND IMPLICATIONS:The DNAJ-PKAc fusion in fibrolamellar carcinoma induces metabolic reprogramming, including enhanced glutamine metabolism, which promotes immune evasion. Targeting this metabolic vulnerability with a glutamine antagonist, in combination with immune checkpoint inhibitors, reverses the immunosuppressive tumor microenvironment, offering a promising therapeutic strategy for fibrolamellar carcinoma treatment.

Otto Warburg sparked the field of cancer metabolism in the 1920s through his observations that human and animal cancer tissues converted significant amounts of glucose to lactate with an elusive underlying mechanism. The discovery of oncogenes led to the notion that neoplasia results from deregulated cell division control with metabolism at the margin, standing by to support cell growth. Studies over the past several decades have linked oncogenes to the direct regulation of metabolism, such as the myelocytomatosis (MYC) oncogene, driving glycolysis and other central metabolic pathways, necessary for cell growth and proliferation. Deregulated oncogenic drive of metabolism renders tumor cells addicted to glucose and other nutrients, such that nutrient deprivation can trigger cancer cell death. The revelation of this addiction stimulated pharmaceutical companies to target metabolism for cancer therapy, but due to several failed clinical studies, this exuberance fizzled commercially. However, the transformative impact of cancer immunotherapy ushered in an interest in understanding the hostile metabolic tumor microenvironment that limits the function of anti-tumor T cells and clinical responses to immunotherapy. This interest drives the convergence of immunometabolism and cancer cell metabolism research to provide a richer understanding of tumor metabolic vulnerability. Herein, I discuss the historical and current context of opportunities and challenges to targeting cancer metabolism.