Abstract IA025: Nuclear speckles in ccRCC

Nuclear speckles are dynamic membraneless bodies located in the cell nucleus. They harbor RNAs and proteins, many of which are splicing factors, that together display complex biophysical properties dictating nuclear speckle formation and maintenance. Although these nuclear bodies were discovered decades ago, only recently has in-depth genomic analysis begun to unravel their essential functions in modulation of gene activity. Major advancements in genomic mapping techniques combined with microscopy approaches have enabled insights into the roles nuclear speckles may play in enhancing gene expression, and how gene positioning to specific nuclear landmarks can regulate gene expression and RNA processing. Some studies have drawn a link between nuclear speckles and disease. Certain maladies either involve nuclear speckles directly or dictate the localization and reorganization of many nuclear speckle factors. This is most striking during viral infection, as viruses alter the entire nuclear architecture and highjack host machinery. As discussed in this Review, nuclear speckles represent a fascinating target of study not only to reveal the links between gene positioning, genome subcompartments and gene activity, but also as a potential target for therapeutics.

Clear cell renal cell carcinoma (ccRCC) is the most predominant histology of renal cancer, representing 75% of all cases and accounting for the majority of associated deaths. Towards understanding the underlying molecular alterations that can drive renal oncogenesis, The Cancer Genome Atlas (TCGA) has performed extensive genomic and transcriptomic profiling, and comparative analyses. To gain insight into the impact of genomic alterations on the functional modules that drive ccRCC tumorigenesis, we leveraged comprehensive proteogenomic characterization of 110 ccRCC tumors and 84 corresponding normal adjacent tissue (NAT) samples. Pairing state-of-the-art mass spectrometry based proteomic and phosphoproteomic strategies with the comprehensive genomic analysis, we integrated global and phosphoproteomic measurements with genomic and transcriptomic data to elucidate the dysregulated cellular mechanism resulting from genomic alterations. Arm-level loss of 3p, involving genes VHL, SETD2, BAP1 and PBRM1 is a frequent event in ccRCC and investigation of chromosomal number variations (CNV), mutations, and methylation profiles of these genes, revealed a unique pattern of BAP1 regulated proteins relative to the other 3p loci genes. Comparative protein profiling of ccRCC tumors and NATs identified pathways associated with immune response and glycolysis to be regulated in ccRCC tumors, however correlating mRNA and protein revealed a non-linear relationship in cellular processes including Warburg Effect-related metabolism and protein translation. Employing unbiased consensus clustering identified three proteomic subtypes of ccRCC, discriminated by differential abundance of proteins involved in adaptive immune response, innate immunity, ribosome activity, and metabolic processes. Further evidence of tumor heterogeneity was observed in the phosphoproteome, with phosphopeptide co-expression modeling generating phospho-signaling modules that overlapped with global protein processes (i.e. adaptive and innate immunity), as well pathways only detected at the phosphoproteomic level (i.e. Notch signaling and DNA repair). The fact that ccRCC tumors are highly immunogenic prompted us to explore the degree of immune infiltration in our cohort. We deconvoluted immune, stromal, and microenvironment cell gene signatures, identifying 60 cell types in our cohort, and three immune-based subtypes of ccRCC: Inflamed, Immune-excluded, and Immune desert. These immune subtypes displayed unique patterns of PD-1, PD-L1, and CTLA4 expression, and could be discriminated by the up-regulation of distinct protein pathways including interferon-γ, HIF1, and EMT. Our results integrated multi-level “omics” analyses to provide greater depth and expand our understanding of ccRCC biology, while identifying novel proteomic, phosphoproteomic, and immune ccRCC subtypes for personalized, precision-based care. Citation Format: David J. Clark, The CPTAC Consortium. Integrated proteogenomic characterization of clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2458.

Chromosome mis-segregation during cell division occurs in diverse tumor types, promoting aneuploidy and intratumoral genetic heterogeneity. While the mechanisms that govern chromosome segregation are well-established, how changes in the activities of tumor suppressors or oncogenes drive mitotic errors remain poorly understood. Loss of the tumor suppressor SETD2, the methyltransferase responsible for tri-methylation of histone 3 lysine 36 (H3K36me3), correlates with clear cell renal cell carcinoma (ccRCC) tumors exhibiting extensive intratumoral heterogeneity. Yet, how loss of H3K36me3 or SETD2 promotes heterogeneity is unknown. Here, we show that loss of SETD2 promotes chromosome mis-segregation during mitosis and interphase DNA bridges driven by the formation of dicentric chromosomes. Cytogenetic analyses revealed that these dicentrics loss were largely comprised of mirror-imaged isodicentric chromosomes that contain two active centromeres. In addition to isodicentrics, cells lacking SETD2 or H36K36me3 generated iso- and acentric chromosomes, however loss of SETD2 protein additionally caused chromosome instability. These data directly link loss of a tumor suppressor to these mutable chromatin structures that initiate intratumor heterogeneity by promoting gross chromosomal rearrangements. Citation Format: Frank M. Mason, Anteneh T. Tebeje, Emily S. Kounlavong, Rashmi Dahiya, Logan Vlach, Tiffany Guess, Ruhee Dere, Ryoma Ohi, Peter Ly, Cheryl L. Walker, W. Kimryn Rathmell. SETD2 safeguards the genome against isochromosome formation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR012.

Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.

Introduction. Translocation Renal Cell Carcinoma (tRCC) is a distinct and aggressive molecular subtype of kidney cancer unusually prevalent in children and adolescents. tRCC has a poor prognosis with no specific therapies and is incurable in the metastatic setting. tRCC is characterized by chromosomal translocations involving three out of the four Microphthalmia (MiT) family transcription factors, TFE3, TFE3, and MITF. The most commonly involved gene is TFE3, which most often fuses with ASPSCR1. However, the molecular mechanisms by which ASPSCR1-TFE3 (and more broadly MiT/TFE fusion proteins) dictates tRCC tumorigenesis remain unclear and advances are hampered by the paucity of animal models. Herein, we sought to model tRCC and performed integrative genomic analyses to dissect molecular mechanisms underpinning tRCC tumorigenesis with the goal of translating this knowledge into therapeutic opportunities. Methods. We conditionally expressed a human ASPSCR1-TFE3 fusion in the Pax8 and Sglt2 lineages and performed phenotypic analyses as well as WES and RNAseq of emerging tRCC tumors. Similar genomic analyses of a human tRCC cohort were performed. The mutational landscape, cooperating pathways, and potential therapeutic targets were identified through comparative genomic and transcriptomic analyses. Emerging hypotheses were tested through in vitro and in vivo assays. Results. We show that human ASPSCR1-TFE3 driven by Pax8-Cre, a credentialled RCC driver, disrupted nephron development causing neonatal death, whilst Sglt2-Cre resulted in tRCC with complete penetrance and short latency. In both models, ASPSCR1-TFE3 induced characteristic morphological changes, loss of epithelial markers, and an EMT program, but only in the Sglt2-Cre model was proliferation observed. Given that Pax8-Cre, but not Sglt2-Cre¸ induce clear cell RCC (ccRCC), these data show that tRCC and ccRCC originate from different cell types. Comparative murine/human genomic analyses show that tumorigenesis by MiT/TFE fusion proteins is broadly cooperative (i.e. p53, ATM/ATR, Fanconi anemia, SWI/SNF, Hippo, and Wnt). Gene expression and functional studies implicate EPHA5 as well as Met/Ret in tRCC tumorigenesis. Antitumor/antiproliferative activity was observed with the therapeutic targeting of these pathways. Conclusion. This study provides insight into the tRCC mutational landscape, the cell of origin, and MiT/TFE-driven transcriptional programs offering several avenues for therapeutic development. Citation Format: Gopinath Prakasam, Akhilesh Mishra, Alana Christie, Jeffrey Miyata, Vanina T. Tcheuyap, Hui Ye, Quyen N. Do, Kevin B. Jones, Mai-Carmen Requena-Komuro, Ivan Pedrosa, Dinesh Rakheja, Payal Kapur, James Brugarolas. Comparative genomic analysis of novel translocation renal cell carcinoma model reveals molecular mechanisms of disease progression and therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR004.

[Objective] Mitochondria are the places for the energy production of the cells, while reactive oxygen species (ROS) are also produced alongside. In recent years, it has been reported that cancer stem cells metabolize predominantly through oxidative phosphorylation (OXPHOS) rather than glycolysis in certain cancer cells. Targeting OXPHOS achieved by suppression of ATP synthesis through mitochondrial ATP synthase could be a potential therapeutic option against cancer stem cells. In the current study, we have identified the mitochondria metabolism as the potential therapeutic target in osteosarcoma (OS) stem cells, presenting the synergistic effects of combination of OXPHOS inhibition by pterostilebene (PTE) with c-Myc inhibitor, which target both OXPHOS-dominant cancer stem cells and glycolysis-dominant non-cancer stem cells as a ‘two hit’ or ‘dual inhibition’ of metabolic pathways, OXPHOS and glycolysis. [Materials & Methods] Using human OS cell lines of SaOS2, U2OS and MG63 cells, cell survival and the ability of sphere formation was assessed with or without PTE, and the expression of stem cell markers mRNA such as Oct3, NS, CD44 was examined by RT-PCR. Next, the activity of mitochondrial ATP synthase, mitochondrial respiration capacity of oxygen consumption rate, and the amount of ATP as well as ROS production were measured under the treatment of PTE. Furthermore, we examined the synergistic effect of PTE with cMyc transcription inhibitors of JQ1 or Honokiol (HNK). [Results] PTE treatment on human OS cell lines reduced the viabilities of all cell lines in dose-dependent manner and expression of stem cell marker and the ability of sphere formation were also decrease in terms of sphere number and size. PTE reduced the activity of F0F1-ATP synthase; Complex V predominantly, and the mitochondrial oxygen consumption rates and synthetic amount of ATP were also decreased in spheroid condition. These results suggest that PTE possibly targets stem cell population which preferably relies on OXPHOS, suppressing ATP synthesis via F0F1-ATP synthase inhibition as well as increased ROS production in OS cells and changes metabolic flax to glycolysis dependent feature. The dual inhibition of OXPHOS by PTE and c-Myc inhibition by HNK or JQ1 showed the synergistically inhibition of OS cell growth in a dose-dependent manner. [Discussion] Prognosis of the patients with osteosarcoma has reached to plateau without any breakthroughs over the last quarter century, and nearly 30% of patients still have to face very severe poor prognosis, especially with metastatic disease. Current study suggests that modulation of metabolic flux by c-Myc and OXPHOS inhibitors showed a greater synergistic effect with ‘two metabolic hit’ or ‘dual metabolic inhibition’ of distinct metabolic features and it could be a novel therapeutic strategy against osteosarcoma, possibly targeting both stem-like cell population and general tumor cell population. Citation Format: Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Yasuhito Tanaka, Hiroki Kuniyasu. Dual inhibition of distinct metabolic features targets osteosarcoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 801.

Renal cell carcinoma (RCC) comprises a diverse group of tumors with varied molecular drivers and clinical outcomes. Despite this heterogeneity, most therapeutic approaches are based on the biology of clear cell RCC (ccRCC), the most prevalent subtype, leaving rarer subtypes such as translocation RCC (tRCC) without tailored treatments. tRCC is an aggressive and rare RCC subtype characterized by oncogenic gene fusions involving MiT/TFE transcription factors, most commonly TFE3. Standard therapies developed for ccRCC are typically less effective against tRCC, in part due to a limited understanding of its tumor-intrinsic features and tumor microenvironment (TME). To address this gap, we employed a comprehensive multi-omics approach to profile 20 tRCC tumors from 15 patients using single-nucleus RNA sequencing, single-nucleus ATAC sequencing, spatial transcriptomics, and T cell receptor sequencing. These data were compared to single-cell profiles from 32 ccRCC tumors and validated using over 1,000 bulk RNA-seq datasets. Despite a shared cell-of-origin in proximal tubule cells, tRCCs diverged markedly from ccRCC at the molecular level. tRCC tumors showed upregulation of oxidative phosphorylation, respiration, lysosomal gene programs, and a MITF-like transcriptional program, all driven by TFE3 fusions. We additionally identified six conserved oncogenic meta-programs across tRCC tumors, reflecting intratumoral heterogeneity. Among these, epithelial-mesenchymal transition (EMT) and proximal tubule identity programs were mutually exclusive and modulated by TFE3-fusion activity - higher fusion levels promoted EMT. The TME in tRCC was notably immunosuppressive, with sparse infiltration of CD8 T cells, many displaying exhausted phenotypes along with pro-tumor SPP1+ macrophages and matrix-associated fibroblasts. EMT-high tumors showed a greater association with these suppressive macrophage and stromal components, suggesting that fusion-driven EMT may contribute to shaping an immune-evasive niche. Together, our findings define tRCC as a molecularly and immunologically distinct RCC subtype, characterized by fusion-driven transcriptional programs and a highly suppressive TME. These insights help explain the poor response of tRCC to current immunotherapies and provide a foundation for rational therapeutic development, including targeting fusion-mediated pathways and reprogramming the TME to enhance immune responsiveness in tRCC. Citation Format: Prathyusha Konda, Cary Weiss, Yantong Cui, Sayed Matar, Jinyu Wang, Yasmin Nabil, Riva Deodhar, Jiao Li, Jack Horst, Sabrina Camp, Aseman Sheshdeh, Jonathan Hecht, David Einstein, Yashika Rustagi, Anwesha Nag, Aaron Thorner, Cheng-Zhong Zhang, Eliezer Van Allen, Sabina Signoretti, Toni Choueiri, Srinivas Viswanathan. Fusion-driven oncogenic programs shape the immune landscape in translocation renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr B025.

Introduction ccRCC exhibits diverse tumor microenvironments, and associations with ICI response have been demonstrated using bulk RNA sequencing and molecular subtype analyses. However, the factors that drive this microenvironmental heterogeneity remain unclear. This study aimed to analyze large-scale single-cell data to identify multicellular co-occurrence patterns within the tumor microenvironment and the tumor-intrinsic programs that shape these ecosystems. Methods We analyzed single-cell RNA-seq data from 58 ccRCC cases (224,018 cells), including 6 untreated, 31 pre-ICI, 14 post-ICI, 3 pre-TKI, and 4 post-TKI (Kashima, ASCO 2024). We performed high-resolution clustering for each non-tumor cell lineage (CD8+ T, CD4+ T, regulatory T cells, myeloid cells, endothelial cells, and fibroblasts). Pairwise correlations of cluster frequencies across samples were computed to detect “modules” representing co-existing immune and stromal populations. These modules were validated using an independent single-cell RNA-seq dataset (HCRN GU16-260, N = 16; Hugaboom, Cancer Discov. 2025). Tumor cells (50,346 cells) were separately analyzed using consensus NMF to identify shared gene programs. We compared the prevalence of modules in responders (R) (n = 10; complete or partial response) with non-responders (NR) (n = 8; progressive disease) to ICI therapies. Results We identified 78 fine clusters and 21 co-existence modules. These clusters and modules captured features not fully explained by bulk RNA-seq or small-scale single-cell data. Most modules consisted of multiple cell types rather than a single lineage, i.e., a CXCL13+ CD4+ T cell–memory B cell module. Module structure was supported in the HCRN cohort (Mantel r = 0.3, P < 0.001). Patients exhibited highly heterogeneous combinations of these modules. Several modules were associated with favorable ICI responses, including exhausted CD8+ T cells–MACRO+ macrophages (Module R1) and cDC–iCAF (Module R2). Conversely, modules such as Th17 CD4+ T cells–GNLY+ ILCs–pDCs (Module NR1) and GZMA+ CD4+ T cells–Trm CD8+ T cells–IFNγ+ ILCs (Module NR2) were associated with poor ICI response. Consensus NMF of tumor cells revealed six interpretable gene programs shared across samples. Notably, the tumor-intrinsic IL6 and VEGF programs were correlated with Module NR1, while Module NR2 was mainly associated with the MYC program. Modules R1 and R2 also demonstrated distinct associations with specific tumor gene programs. These findings suggest that patient-specific, multiple biological pathways may contribute to both resistance and responsiveness to ICI therapy. Conclusion Our large-scale single-cell analysis identifies reproducible multicellular modules within the ccRCC tumor microenvironment, several of which are positively or negatively associated with ICI response. The observed relationships between non-tumor cell modules and tumor-intrinsic gene programs indicate that tumor cell states and microenvironmental composition are interdependent, highlighting potential cellular crosstalk that may influence immunotherapy efficacy. Citation Format: Katsuhiro Ito, Soki Kashima, Rishabh Rout, Zhaochen Ye, Nicholas R. Schindler, David A. Braun. Multicellular modules and tumor programs shape the tumor microenvironment and predict immune checkpoint inhibitor (ICI) response in clear cell renal cell carcinoma (ccRCC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr A009.

Tumor-associated Macrophage-derived Interleukin-23 Interlinks Kidney Cancer Glutamine Addiction with Immune Evasion

Clear cell renal cell carcinoma (ccRCC) harbors metabolic vulnerabilities beyond canonical HIF signaling, yet non-genetic dependencies remain largely undefined. Here, using patient-derived regulatory networks (SJARACNe→NetBID2) coupled with in vitro and in vivo functional genomics, we identify never-in-mitosis A–related kinase 5 (NEK5) as a hidden driver selectively required for VHL-deficient ccRCC. NEK5 activity—but not mRNA expression—is elevated in VHL-mutant tumors. Genetic ablation of NEK5 suppresses anchorage-independent growth, delays orthotopic tumor formation, and reduces metastasis, while sgRNA-resistant NEK5 fully restores tumorigenicity. Mechanistically, NEK5 is a mitochondrial protein that binds the translocator protein TSPO to control respiration, membrane potential, and redox homeostasis. NEK5 loss increases oxygen consumption yet decreases ΔΨm and ATP/ADP ratios, producing uncoupled oxidative phosphorylation and excessive mitochondrial ROS; ROS scavenging restores growth. A kinase-dead NEK5 mutant rescues proliferation and ATP-competitive inhibition is ineffective, demonstrating a largely kinase-independent mechanism. Epistasis positions TSPO downstream of NEK5, and VHL loss strengthens NEK5–TSPO association, linking this axis to the VHL pathway. Guided by these mechanistic insights, we developed Cereblon-based PROTACs that degrade NEK5, recapitulate the phenotypes of genetic loss, and selectively impair VHL-deficient ccRCC growth. These findings unveil NEK5 as an activity-defined, mitochondria-centered vulnerability in ccRCC and demonstrate how network-inference frameworks can expose non-enzymatic targets amenable to protein degradation therapies. Citation Format: Cheng Zhang, Zhen Xie, Shanshan Bradford. Yu, Jamie Jarusiewicz, Nina Gildor, Gisele Nishiguchi, Kaiwen Yu, Qingfei Pan, Junmin Peng, Jiyang Yu, Qing Zhang. Activity-based network analysis identifies NEK5 as a PROTAC-targetable vulnerability in ccRCC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr PR018.

The purpose of our study is to determine whether and how lymphocyte-specific protein tyrosine kinase (Lck) may exhibit novel functions in cancer cell mitochondria. Mitochondria are responsible for producing cellular energy via oxidative phosphorylation (OXPHOS) of glucose and fatty acids. Compared to normal differentiated cells, cancer cells rely more on aerobic glycosis than mitochondrial OXPHOS. This shift in energy metabolism allows more resources to be converted into biomass for cancer cell's uncontrolled growth and proliferation. Numerous mechanisms contribute to altered mitochondrial activity, including phosphorylation of mitochondrial proteins. Recent studies showed that cytoplasmic protein tyrosine kinases (PTK) also translocate to the mitochondria and regulate mitochondrial functions. It represents an important non-canonical pathway of how PTK modulates mitochondrial activity. As a Src-family PTK, Lck is essential for T cell activation by transmitting signals downstream of cell surface receptors. Overexpression and aberrant activity of Lck have been reported in both blood malignancies and solid tumors. We showed previously that Lck exhibited additional oncogenic property when translocated to the nucleus (BBRC 417:1058). To explore the possibility that Lck may also translocate to mitochondria, we examined human Jurkat T cell leukemia and its Lck-deficient derivative Jcam cell line. Subcellular fractionation and microscopy both confirmed the presence of Lck in Jurkat mitochondria. Compared to Jcam, Jurkat cells exhibit high levels of mitochondrial protein tyrosine phosphorylation, disrupted mitochondrial morphology, and reduced OXPHOS. Subsequent mass spectrometry and in situ proximity ligation assay identified CR6-interacting factor 1 (CRIF1) as a novel Lck-interacting partner in mitochondria. CRIF1 interacts with mitoribosome and Tid1 to facilitate translation and membrane insertion of mitochondrion-encoded proteins, respectively. Compared to Jcam, Jurkat cells express less mitochondrial proteins and show reduced CRIF1-Tid1 interaction. Knocking down CRIF1 in Jcam also results in decreased mitochondrial protein level and mitochondrial dysfunction. Interestingly, CRIF1 is not phosphorylated on tyrosine by Lck, suggesting the presence of other Lck substrates in mitochondria. All together, these results support the role of mitochondrial Lck as a negative regulator of CRIF1 through competitive binding and independent of its kinase activity. Disrupted mitochondrial protein translation and membrane insertion then lead to reduced OXPHOS and abnormal morphology. These findings raise the possibility of targeting mitochondrial Lck to redirect the metabolic shift in cancer cells. Citation Format: Shahrooz Vahedi, Fu-Yu Chueh, Chao-Lan Yu. Lymphocyte-specific protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in mitochondria to repress oxidative phosphorylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2015-3041

Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3' untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.

Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways Haleigh Parker1, Kayla Haberman1, Alexander Kornienko2, Antonio Evidente3, Daniel Romo4, Benny Abraham Kaipparettu5,6, Joseph Taube1,6 1 Department of Biology, Baylor University, Waco, TX, USA 2 Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA 3 Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Naples, Italy 4 Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA 5 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 6 Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA Background Metastatic progression in patients with triple negative breast cancer (TNBC) occurs in approximately half of all patients, reducing median overall survival. Metastasis may be facilitated through the epithelial to mesenchymal transition (EMT), which generates cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics, features which are partially mediated by alterations in metabolic pathways and mitochondrial function. Here, we show a drug-like small molecule possesses EMT-specific cytotoxic activity via EMT-imparted variations in metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, Ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, though its target and mechanism of action remain unknown. Objectives The objectives of this study are to characterize alterations in metabolic pathways and mitochondrial function in the context of EMT, determine OpA induced alterations in specific metabolic pathways involved in metastasis and identify metabolic targets for the treatment of chemotherapeutic-resistant, metastatic triple negative breast cancer. Methods This study identifies distinct, novel metabolic alterations associated with EMT status and specific changes in mitochondrial and metabolic function associated with OpA treatment via analysis of intermediate metabolites, rates of oxidative phosphorylation and glycolysis, metabolic pathway capacity and dependency, protein expression, iron localization, reactive oxygen species accumulation and transmission electron microscopy. Results Our analysis indicates that OpA acts in a mitochondria-specific manner to cause a loss of membrane potential in EMT-positive, but not EMT-negative, cells with specific effects on complex III of the electron transport chain and oxidative phosphorylation as a whole. Furthermore, distinct shifts in glycolysis not only support previous data, but also provides insight as to mitochondrial functionality. Cells were also found to have distinct metabolic pathway capacities/dependencies and ROS and iron accumulation, depending on EMT status and OpA treatment. Finally, specific morphological alterations were uncovered via TEM image analysis. Therefore, we conclude that EMT imparts alterations in mitochondrial function and metabolic pathways, conferring sensitivity to the cytotoxic effects of OpA. Citation Format: Haleigh Parker, Kayla Haberman, Joseph Taube, Alexander Kornienko, Antonio Evidente, Daniel Romo, Benny Kaipparettu. Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-08.

Renal cell carcinoma (RCC) metastatic to the lung and liver remains a lethal disease, with five-year survival rates below ten percent. Employing novel isogenic patient-derived primary and metastatic organoids (PDOs) we identified targetable metabolic adaptations promoting lung and liver metastasis. In vivo metabolic tracing analyses identified distinct metabolite utilization patterns between primary tumors and their matched metastatic lesions. Primary tumors predominantly use glucose derived pyruvate to generate lactate, whereas lung and liver metastatic lesions oxidize it to meet their cellular energetic demands. Reductive glutamine metabolism was predominant in primary tumors and liver metastasis, while oxidative glutamine metabolism was augmented in lung metastasis. Transcriptomic analysis of invasive RCC tumors identified increased expression of Oxphos genes in metastatic tumors relative to non-metastatic tumors. Suppressing electron transport chain (ETC) activity by depleting COX6C or NDUFA4, which are enriched in metastatic lesions significantly reduces metastatic incidence and burden. Residual metastatic cells compensate for ETC inhibition by increasing glycolysis and the reductive carboxylation of glutamine, thereby reducing NADH accumulation and maintaining redox homeostasis. Genetic depletion of key redox regulators, aspartate aminotransferase (GOT1) and malate dehydrogenase (MDH1) attenuates metastatic potential and progression while aspartate and pyruvate supplementation rescued this phenotype. Finally, pharmacological inhibitors targeting both ETC function and glutamine metabolism effectively suppresses metastasis and improve survival in preclinical metastatic PDO models. Collectively, these findings highlight distinct metabolic vulnerabilities of metastatic RCC and support the clinical development of combination metabolic therapies to address this urgent unmet need. Citation Format: Shuhei Kamada, Tuyen T. Dang, Kangsan Kim, Jessica Sudderth, Suvranil Ghosh, Paromita Mitra, Ciara Newman, Dongho Kim, Claire B . Llamas, Qionghua Shen, Chendong Yang, Vanina T. Tcheuyap, Charlotte Small, Brooklyn Jackson, Sophronia Hipsh, Lu Diao, Lauren G. Zacharias, Ashwathi Rajeevan, Tanner Reese, Thomas P. Mathews, Liraz Shmuel-Galia, Prashant Mishra, Qing Zhang, Kevin Dean, Payal Kapur, James Brugarolas, Ralph J. DeBerardinis, Srinivas Malladi. Patient derived organoids identify targetable metabolic adaptations in metastatic ccRCCs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr A028.

T cell engager (TCE) antibodies have been proven to be effective for a variety of hematological malignancies, with at least 10 marketed drugs to date; however, their efficacy in many solid tumors is still very limited due to a number of challenges. One of the leading challenges is the lack of tumor-specific targets, leading to an increased risk of on-target, off-tumor toxicity. An approach to address this challenge is to build a “prodrug” TCE that is conditionally activated in the tumor microenvironment (TME), taking advantages of various masking technology platforms that block the release of active TCE drug outside of the TME. To develop such a novel TCE molecule with improved tumor selectivity, potency, and safety profile, we first discovered a masking peptide specific to a CD3 VHH antibody that we previously developed, and demonstrated that the masked CD3 VHH can effectively reduce T cell activation several hundred-fold. Next, we fused this masking peptide to the CD3 VHH arm via a proteolytically-labile linker containing sequence motifs sensitive to proteases enriched in the TME, and showed that upon cleavage by proteases, the T cell-activating potency of such a masked CD3 VHH can be effectively recovered. Finally, with such a CD3 VHH armed with cleavable linker and masking peptide, we built a series of prodrug TCE molecules targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3), a tumor associated antigen with good tumor selectivity for renal cell carcinoma. Our in vitro validation studies demonstrated that the potency of these TCE prodrugs in killing ENPP3+ tumor cells can be restored to a similar level as the unmasked, active TCE molecule upon proteolytic cleavage. Our in vivo studies using an ENPP3+ CDX model with transplanted human PBMCs demonstrated two important points: (i) the prodrug/masked ENPP3-targeted TCE can potently inhibit tumor growth to a comparable level as the unmasked version and clinical benchmarks, and (ii) the prodrug TCE can be dosed at least 100-fold higher than the active drugs without any observed toxicity, suggesting a greatly improved safety profile. Further preclinical development of these ENPP3-targeted prodrug TCE molecules, including pharmacokinetics and toxicity studies in non-human primates, is ongoing. Citation Format: Yu Liang, Li Chen, Lindi Wang, Yingyu Li, Xin Wang, Yang Xin, Mingzhu Shao, Jiangtao Ning, Ziyu Chen, Cheng Luo, Jingjing Nie, Yue Wu, Tingting Yang, Hai Huang, Miaomiao Song. The discovery and development of a bispecific t cell engager prodrug targeting ENPP3 for renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr A010.