Wilder-Romans, Kari

Abstract The brain avidly consumes glucose to fuel neurophysiology. Cancers of the brain, such as glioblastoma (GBM), relinquish physiological integrity and gain the ability to proliferate and invade healthy tissue. How brain cancers rewire glucose utilization to drive aggressive growth remains elusive. Here, we infused 13C-labeled glucose into patients and mice with brain cancer, coupled with quantitative metabolic flux analysis, to map the fates of glucose-derived carbon in tumor vs. cortex. Through the first direct and comprehensive measurements of carbon and nitrogen labeling in both cortex and glioma tissues, we uncover profound metabolic transformations. In human cortex, glucose carbons fuel essential physiologic processes including TCA cycle oxidation and neurotransmitter synthesis. Conversely, gliomas downregulate these processes and scavenge alternative carbon sources such as amino acids from the environment, repurposing glucose-derived carbons to generate molecules needed for proliferation and invasion. Targeting this metabolic rewiring in mice through dietary amino acid modulation selectively alters GBM metabolism, slows tumor growth, and augments the efficacy of standard-of-care treatments. These findings illuminate how aggressive brain tumors exploit glucose to suppress normal physiological activity in favor of malignant expansion and offer potential therapeutic strategies to enhance treatment outcomes.

Abstract OBJECTIVE: We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS: Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION: The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. Citation Format: Weihua Zhou, Zitong Zhao, Angelica Lin, John Yang, Jie Xu, Kari Wilder-Romans, Annabel Yang, Andrew J. Scott, Jing Li, Sumeet Solanki, Jennifer Speth, Natalie Walker, Ayesha U. Kothari, Yangyang Yao, Erik R. Peterson, Navyateja Korimerla, Christian K. Werner, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra M Obrien, Ameer L Elaimy, Sean P. Ferris, Shuang G. Zhao, Jann N. Sarkaria, Balázs Győrffy, Shuqun Zhang, Wajd N. Al-Holou, Yoshie Umemura, Meredith A Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Marc Peters-Golden, Yatrik M. Shah, Daniel R. Wahl. GTP signaling links metabolism, DNA repair, and responses to genotoxic stress [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A004.

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.

ABSTRACT How cell metabolism regulates DNA repair is incompletely understood. Here, we define a GTP-mediated signaling cascade that links metabolism to DNA repair and has significant therapeutic implications. GTP, but not other nucleotides, regulates the activity of Rac1, a guanine nucleotide-binding protein, which promotes the dephosphorylation of serine 323 on Abl-interactor 1 (Abi-1) by protein phosphatase 5 (PP5). Dephosphorylated Abi-1, a protein previously not known to activate DNA repair, promotes nonhomologous end joining. In patients and mouse models of glioblastoma, Rac1 and dephosphorylated Abi-1 mediate DNA repair and resistance to standard-of-care genotoxic treatments. The GTP–Rac1–PP5–Abi-1 signaling axis is not limited to brain cancer, as GTP supplementation promotes DNA repair and Abi-1-S323 dephosphorylation in nonmalignant cells and protects mouse tissues from genotoxic insult. This unexpected ability of GTP to regulate DNA repair independently of deoxynucleotide pools has important implications for normal physiology and cancer treatment. SIGNIFICANCE: A newly described GTP-dependent signaling axis is an unexpected link between nucleotide metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. This article is featured in Selected Articles from This Issue, p. 5

Abstract Introduction/Objectives: Purines regulate DNA repair in brain tumors through uncertain mechanisms. We sought to define the mechanisms by which purines regulate DNA repair and therapy response. Methods: Crispr/Cas9, siRNA, and CDNA overexpression and immunoblot were used to modulate and confirm protein levels. γ-H2AX and Rad51 foci were enumerated using immunofluorescence. Fluorescent reporter assays were performed to detect DNA repair activity. Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after RT and antibodies generated against novel sites. Celltiter-glo or clonogenic assay were performed to evaluate drug/RT responses. IHC was used to detect protein expression. Animal models of glioblastoma and normal tissue (irradiation of GI system) were used to assess DNA repair and treatment responses in vivo. Results: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT, slowed the repair of RT-induced DSBs and inhibited the activity of non-homologous end joining (NHEJ), but not homologous recombination (HR). These effects could be rescued by GTP (but not ATP) supplementation. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced DSB repair and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to the small GTP-activated protein Rac1. Expression of constitutively active Rac1 promoted the dephosphorylation of Abi-1 (S323) and DSB repair while these effects were blocked by dominant negative Rac1. Pharmacogenomic inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. These findings have therapeutic relevance. In GBM PDX samples, levels of p-Abi-1 (S323) negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibition of Rac1 enhanced RT responses and suppressed Abi-1 S323 dephosphorylation. Abi-1 knockout enhanced efficacy of numerous genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in normal astrocytes and enteroids and protected mice from RT-mediated gastrointestinal injury. Conclusions: GTP promotes DNA repair by activating Rac1 and PP5 to dephosphorylate Abi-1 and stimulate NHEJ. Disrupting this regulation could improve brain tumor treatment responses while augmenting it could help protect normal tissues from genotoxic injury. Citation Format: Weihua Zhou, Angelica Lin, Zitong Zhao, John Yang, Kari Wilder-Romans, Jie Xu, Sumeet Solanki, Jing Li, Annabel Yang, Andrew Scott, Ayesha Kothari, Erik Peterson, Navyateja Korimerla, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra Obrien, Sean Ferris, Shuang Zhao, Jann N. Sarkaria, Meredith Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Yatrik Shah, Daniel R. Wahl. GTP-dependent signaling links metabolism, non-homologous end joining and treatment responses in cancer and normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3498.

Abstract Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming radiation (RT) resistance. To discover genotype-independent mediators of RT resistance, we correlated RT resistance with the concentration of approximately 700 metabolites across 23 GBM cell lines. Purine metabolites, especially those containing the base guanine, were most correlated with RT resistance. Similarly, increased abundance of tumor purines was associated with decreased survival in GBM patients treated with RT. This relationship is causal. Purine supplementation protected RT-sensitive GBMs from RT and promoted the repair of RT-induced double strand DNA breaks (DSBs). In vitro and in vivo stable isotope tracing confirmed that GBM cell lines and orthotopic patient-derived xenografts primarily generated purines through the de novo synthetic pathway. RT treatment further increased de novo purine synthesis in GBM through signaling via the DNA damage response. Inhibition of de novo GTP synthesis with mycophenolic acid (MPA) sensitized multiple GBM cell lines and neurospheres to RT by slowing the repair of RT-induced DSBs. MPA-induced radiosensitization was GTP-dependent as it was rescued by nucleoside supplementation. Modulating pyrimidine metabolism affected neither RT resistance nor DSB repair, suggesting these GTP-specific effects are due to active signaling rather than its ability to act as a physical substrate for DNA repair and candidate signaling molecules have been identified. These results were recapitulated in vivo with mycophenolate mofetil (MMF), the orally bioavailable FDA-approved prodrug of MPA. MMF potentiated RT efficacy, reduced tumor guanylates and slowed the repair of RT-induced DSBs across multiple models. Because de novo purine synthesis is activated by many of the oncogenic alterations that drive GBM, its inhibition is a promising genotype-independent strategy to overcome GBM RT resistance. We have now begun a clinical trial to determine whether combining MMF and RT is safe and potentially efficacious in patients with GBM.

Abstract New approaches are needed to overcome intrinsic therapy resistance in glioblastoma (GBM). Because GBMs exhibit sexual dimorphism and are reported to express steroid hormone receptors, we reasoned that signaling through the androgen receptor (AR) could mediate therapy resistance in GBM, as it does in AR-positive prostate and breast cancers. Using RNAseq, immunoblot and immunohistochemistry, we found that nearly half of GBM cell lines, patient-derived xenografts and human tumors express AR transcript and protein with levels that overlap those of primary prostate cancer. AR expression in GBM did not vary by sex, age or common molecular alterations. We identified two cell line models of GBM that expressed AR protein (LN18 and T98G: termed “AR positive”) and two that did not (8MGBA and AM38: termed “AR negative”). Seviteronel, a blood-brain barrier permeable CYP17 lyase inhibitor and antiandrogen slowed growth in AR positive GBM cell lines (GI50 3-4 µM) but not AR negative lines (GI50 > 500 µM) as measured by the colony formation assay. The antiandrogen enzalutamide, which also penetrates the blood brain barrier, similarly preferentially slowed growth in AR positive GBM cell lines. Seviteronel and enzalutamide sensitized AR positive GBM cell lines to radiation with enhancement ratios of 1.3-1.6 as measured by the clonogenic survival assay. Antiandrogens had no effect on the radiosensitivity of AR negative GBM cell lines. Seviteronel treatment did not affect the growth of AR positive T98G xenografts grown in vivo, but did sensitize these tumors to radiation (median time to tripling: 15 d with radiation alone and not reached with radiation combined with seviteronel). Enzalutamide similarly had modest single agent effects on an AR positive GBM patient-derived xenograft (GBM26 from the Mayo Clinic GBM PDX national resource) grown in vivo but sensitized these tumors to radiation (median time to tripling: 25.5 d with radiation alone and 39 d with radiation combined with enzalutamide). RNAseq performed on GBM26 tumors grown in vivo revealed that enzalutamide treatment caused minimal transcriptional changes when given as monotherapy but, when given in combination with radiation, blocked the ability of AR-positive GBMs to engage adaptive transcriptional programs related to multiple DNA repair pathways. We confirmed these mechanistic findings in vitro, as antiandrogens selectively impaired the repair of radiation-induced double strand DNA breaks in AR positive GBM cell lines. These results suggest that AR signaling may mediate therapy resistance in AR positive GBMs, and patients with these tumors could derive clinical benefit from combination therapies involving radiation and blood-brain-barrier permeable antiandrogens. Citation Format: Christian K. Werner, Uchechi Nna, Hanshi Sun, Kari Wilder-Romans, Joseph Dresser, Ayesha Kothari, Weihua Zhou, Yangyang Yao, Arvind Rao, Stefanie Stallard, Carl Koschmann, Tarik Bor, Waldemar Debinski, Alexander Hegedus, Meredith Morgan, Sriram Venneti, Edwina Baskin-Bey, Daniel Spratt, Howard Colman, Jann Sarkaria, Arul Chinnayain, Joel Eisner, Corey Speers, Theodore S. Lawrence, Roy Strowd, Daniel R. Wahl. Repurposing antiandrogens to overcome therapy resistance in androgen receptor-positive glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6267.

Abstract New approaches are needed to overcome intrinsic therapy resistance in glioblastoma (GBM). Because GBMs exhibit sexual dimorphism and are reported to express steroid hormone receptors, we reasoned that signaling through the androgen receptor (AR) could mediate therapy resistance in GBM, much as it does in AR-positive prostate and breast cancers. We found that nearly half of GBM cell lines, patient-derived xenografts (PDX), and human tumors expressed AR at the transcript and protein level—with expression levels overlapping those of primary prostate cancer. Analysis of gene expression datasets also revealed that AR expression is higher in GBM patient samples than normal brain tissue. Multiple clinical-grade antiandrogens slowed the growth of and radiosensitized AR-positive GBM cell lines and PDXs in vitro and in vivo. Antiandrogens blocked the ability of AR-positive GBM PDXs to engage adaptive transcriptional programs following radiation and slowed the repair of radiation-induced DNA damage. These results suggest that combining blood–brain barrier permeable antiandrogens with radiation may have promise for patients with AR-positive GBMs.

Abstract Purpose: Protein kinases are known to play a prominent role in oncogenic progression across multiple cancer subtypes, yet their role in prostate cancer progression remains underexplored. The purpose of this study was to identify kinases that drive prostate cancer progression. Experimental Design: To discover kinases that drive prostate cancer progression, we investigated the association between gene expression of all known kinases and long-term clinical outcomes in tumor samples from 545 patients with high-risk disease. We evaluated the impact of genetic and pharmacologic inhibition of the most significant kinase associated with metastatic progression in vitro and in vivo. Results: DNA-dependent protein kinase (DNAPK) was identified as the most significant kinase associated with metastatic progression in high-risk prostate cancer. Inhibition of DNAPK suppressed the growth of both AR-dependent and AR-independent prostate cancer cells. Gene set enrichment analysis nominated Wnt as the top pathway associated with DNAPK. We found that DNAPK interacts with the Wnt transcription factor LEF1 and is critical for LEF1-mediated transcription. Conclusions: Our data show that DNAPK drives prostate cancer progression through transcriptional regulation of Wnt signaling and is an attractive therapeutic target in aggressive prostate cancer.