Abstract Background: Despite recent advances in the biology of IDH-wildtype glioblastoma, it remains a devastating disease with median survival of less than two years. However, the molecular underpinnings of the heterogeneous response to the current standard-of-care treatment regimen consisting of maximal safe resection, adjuvant radiation, and chemotherapy with temozolomide remain unknown. Methods: Comprehensive histopathologic, genomic, and epigenomic evaluation of paired initial and recurrent glioblastoma specimens from 106 patients was performed to investigate the molecular evolution and cellular phenotypes underlying differential treatment responses. Results: While TERT promoter mutation and CDKN2A homozygous deletion were early events during gliomagenesis shared by initial and recurrent tumors, most other recurrent genetic alterations (e.g. EGFR, PTEN, NF1) were commonly private to initial or recurrent tumors indicating acquisition later during clonal evolution. Further, glioblastomas exhibited heterogeneous epigenomic evolution with subsets becoming more globally hypermethylated, hypomethylated, or remaining stable. Glioblastoma that underwent sarcomatous transformation had shorter interval to recurrence and were significantly enriched in NF1, TP53, and RB1 alterations and the Mesenchymal epigenetic class. Patients who developed somatic hypermutation following temozolomide treatment had significantly longer interval to disease recurrence and prolonged overall survival, and increased methylation at four specific CpG sites in the promoter region of MGMT was significantly associated with this development of hypermutation. Finally, an epigenomic evolution signature incorporating change in DNA methylation levels across 347 critical CpG sites was developed that significantly correlated with clinical outcomes. Conclusions: Glioblastoma undergoes heterogeneous genetic, epigenetic, and cellular evolution that underlies prognostically different treatment responses. Citation Format: Calixto-Hope Lucas, Nadeem Al-Adli, Jacob Young, Rohit Gupta, Ramin Morshed, Jasper Wu, Ajay Ravindranathan, Anny Shai, Nancy Ann Oberheim Bush, Jennie Taylor, John de Groot, Javier Villanueva-Meyer, Melike Pekmezci, Philip Theodosopoulos, Manish Aghi, Edward Chang, Shawn Hervey-Jumper, David Raleigh, Annette Molinaro, Joseph Costello, Aaron Diaz, Jennifer Clarke, Nicholas Butowski, Joanna Phillips, Susan Chang, Mitchel Berger, David A. Solomon. Longitudinal multimodal profiling of IDH-wildtype glioblastoma reveals the molecular evolution and cellular phenotypes underlying prognostically different treatment responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr A061.

Abstract Inside the cell nucleus, nuclear speckles are a predominant type of nuclear body present in the cells throughout our bodies. While first observed by Ramon y Cajal over 100 years ago, speckles have only recently been recognized as a major layer of gene regulation that boosts gene expression by assisting multiple steps of RNA production. Large genomic regions spanning 10s of megabases are consistently organized around speckles, with regulation of chromatin position at speckles occurring at finer scales of ∼10kb-1Mb. Hence, speckles may help direct functional programs within a cell by coordinating expression of large, but specific, groups of genes. In cancer, we found that speckles exist in two major states that correlate strongly with patient outcomes in neuroblastoma and clear cell renal cell carcinoma (ccRCC). These speckle states showed dramatic differences in expression of the speckle-associated gene neighborhoods. Because certain types of genes reside within speckle-associated chromatin, speckle states correlate with broad functional programs such as protein translation and oxidative phosphorylation as well as ccRCC-specific functional programs such as HIF-2alpha-induced angiogenesis and metabolism pathways. Consistent with distinct gene regulatory programs, ccRCC tumors display tumor microenvironment differences, particularly in tumor vasculature, between the two speckle states. While these data indicate that speckles can be used as a multifunctional readout for ccRCC tumor phenotypes, what drives speckle states in ccRCC is unclear. In other efforts in the lab, we combined analysis of public data with experimental approaches to uncover that transcription factors, hormone signaling, and immune signaling globally regulate expression of genes within speckle-associated chromatin. These efforts provide clues as to what may regulate nuclear speckle functions. Ultimately, by understanding the upstream drivers and downstream consequences of speckle states in ccRCC, we hope to advance nuclear speckles as potential therapeutic targets as well as biomarkers capable of stratifying patient risk and informing treatment decisions. Citation Format: Sana Mir, Hiroe Namba, Milena Stepic, Kate Alexander. Nuclear speckles 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 IA025.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract 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.

Abstract Clear cell renal cell carcinoma (ccRCC) is the most aggressive subtype of renal cell carcinoma. TCGA analysis shows that high expression of the inhibitor of apoptosis protein survivin (BIRC5) correlates with advanced stage and poor prognosis. Recent work in our lab demonstrated that survivin promotes proliferation, migration, and mitochondrial remodeling in the RENCA RCC cell model, suggesting that survivin lies at the intersection of cell cycle and metabolic control. Preliminary RNA-seq analysis of survivin siRNA-treated RENCA cells indicated that immune-recruiting cytokines are upregulated upon survivin loss, which we validated in vitro. Because RENCA does not fully recapitulate ccRCC biology, we investigated how survivin loss influences mitochondrial homeostasis and the tumor–immune interface across VHL-deficient ccRCC models. We utilized a novel murine VHL-null ccRCC line (LVRCC), the VHL-proficient RENCA line, and the human VHL-deficient 786-O ccRCC line. Survivin was depleted by siRNA, followed by assessment of mitochondrial mass and immune cell recruitment using transwell co-culture assays. Notably, survivin knockdown in LVRCC cells produced a change in mitochondrial mass opposite to that observed in RENCA, a finding potentially related to VHL status. The VHL-deficient 786-O line exhibited phenotypes similar to LVRCC cells, including survivin-dependent growth. Survivin loss increased immune cell migration toward ccRCC cells, indicating that survivin restrains immune cell infiltration into the tumor microenvironment. Collectively, these data support survivin as an oncogenic driver and putative therapeutic target in ccRCC. Citation Format: Shivani S. Tuli, Yamato Murakami, Cais Vo, Yongho Bae, David Gau. Survivin regulates tumorigenesis and immunogenicity of 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 B020.

Abstract Purpose: Antiangiogenic tyrosine kinase inhibitors (TKIs) have become a cornerstone in the management of clear cell renal cell carcinoma (ccRCC) as monotherapy or in combination with immune checkpoint blockades (ICB). However, TKIs produce limited long-term durability even in combination with ICBs, indicating that prolonged TKI treatment fosters an immunosuppressive microenvironment contributing to therapy resistance. Method: In this study, we performed Xenium in situ transcriptomic profiling with a 5,000-gene panel on tissue microarrays comprising treatment-naïve or sunitinib-treated tumors from RCC patients. Renca murine RCC model was used to evaluate therapeutic responses in vivo. Results: From over 220,000 analyzed cells, we identified two major tumor cell populations. The predominant population exhibited pronounced hypoxia and glycolytic activity, whereas a smaller subset of RCC cells showed reduced metabolic activity but increased metastatic potential and stem-like features. In sunitinib-treated tumors, both tumor and immune cells displayed a highly inflammatory activity, marked by upregulation of type I and type II interferon response pathways. Sunitinib treatment reduced the abundance of endothelial cells and cancer-associated fibroblasts and increased the infiltration of T cells and B cells. Although sunitinib did not alter the overall abundance of tumor-associated macrophages (TAMs), it markedly shifted their spatial distribution: in treatment-naïve tumors, TAMs were sequestered by endothelial cells, whereas in sunitinib-treated tumors, they were released and engaged to interaction with T cells. We further identified two TAM subsets—interferon-primed TAMs (IFN-TAMs) and proliferating TAMs (Prolif-TAMs)—that were expanded in sunitinib-treated tumors and associated with poorer progression-free survival. IFN-TAMs exhibited strong IFN-γ signaling activity and high expression of the immune checkpoint ligands PD-L1 and PD-L2. Prolif-TAMs were defined by elevated proliferation markers and by increased expression of tumor-promoting proteins, such as CSF1R, MSR1, VSIG4, and TREM2. In sunitinib-treated tumors, IFN-TAMs also showed enhanced interactions with multiple immune cell subsets, including proliferating T cells and cytotoxic T lymphocytes, both of which express PD-1. These findings suggest that IFN-TAMs facilitate tumor immune evasion through the PD-1/PD-L1/2 axis, consistent with our observation that PD-1 blockade synergizes with axitinib to enhance tumor suppression. Notably, both IFN- and Prolif-TAMs expressed the A2A adenosine receptor (A2AR), and we found that the A2AR antagonist CPI-444 further potentiated the therapeutic response to axitinib treatment. Conclusion: These results show that sunitinib treatment induces an immunosuppressive tumor microenvironment driven by macrophage reprogramming and altered spatial organization. Targeting these macrophage subsets markedly enhances the therapeutic efficacy of TKIs. Disclosure: AI assistance (ChatGPT) was used to polish the wording of this abstract. Citation Format: Xiande Liu, Hongchao He, Jingjing Liu, Xuesong Zhang, Anh Hoang, Tatiana Karpinets, Kanishka Sircar, Eric Jonasch. Spatial single-cell transcriptomics reveal targetable macrophage subsets associated with antiangiogenic therapy resistance in 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 PR009.

Abstract 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.