Mitchell, Duane A.

Abstract Current advances in the treatment of melanoma brain metastases through immune checkpoint blockade and targeted therapy has significantly improved outcomes and patient survival. Yet, half of all patients continue to die from brain metastases despite use of these therapies and the addition of localized therapies like surgery and stereotactic radiosurgery. Novel treatments are needed to improve the morbidity and mortality associated with melanoma brain metastases. We have identified the inflammatory myddosomal pathway as a novel potential target of therapy in melanoma brain metastases, with gross upregulation in both tumor tissue and tumor-infiltrating immune cells. These cells display upregulation in MYD88, IRAK-1, IRAK-4, and downstream mediators of inflammatory activation at both the gene and protein level. We have previously shown the ability of the oral IRAK-4 inhibitor emavusertib (CA-4948) to penetrate the blood brain barrier and inhibit the myddosomal pathway in metastatic melanoma and primary CNS lymphoma. In this study we further show the capacity for IRAK-4 inhibition with emavusertib to improve the therapeutic efficacy of anti-PD-1 immune checkpoint blockade in melanoma brain metastases. Emavusertib in combination with anti-PD-1 therapy results in improved tumor-infiltrating lymphocyte recruitment, decreased myeloid derived suppressor cell function, and upregulation of interferon γ signaling resulting in improved survival in aggressive mouse models of melanoma brain metastases. This work supports the development of combination strategies of emavusertib with immune checkpoint blockade in melanoma brain metastases.

Malignant glioma is considered invariably recalcitrant, thus necessitating development of new therapeutic approaches. Spontaneously arising canine glioma exhibits important molecular and pathological similarities to malignant pediatric glioma, making it an invaluable model for testing novel therapies. We evaluated safety and activity of RNA lipid particle aggregates (LPAs) designed to provoke cancer immunogenicity in 10 dogs with histologically confirmed glioma. Vaccine manufacturing was feasible in all subjects and generally well tolerated. Median survival time was 123 and 155 days in dogs receiving three (Group A) or four (Group B) vaccines, respectively; this was increased from historical reporting of subjects receiving palliative care. Responding animals had a robust cytokine response following infusions. In animals receiving non-specific RNA-LPAs, there was localization of messenger RNA (mRNA) payloads to the brain tumor microenvironment and a rapid shift in immunologic gene signatures consistent with a viral response. These data demonstrate the ability to induce rapid viremic response in the tumor microenvironment following a single peripherally administered mRNA vaccine. Since RNA-LPAs are amenable to repeat boosts, these formulations may overcome challenges of tumor immunosuppression and therapeutic access and are currently under evaluation in first-in-human trials ( NCT04573140 , NCT06389591 ).

Abstract INTRODUCTION Glioblastoma (GBM) contains metabolically distinct cell lineages: treatment-sensitive cells (TSCs) dependent on glycolysis and treatment-resistant persister cells (TRPCs) reliant on lipid metabolism. Our integrative systems biology approach, combining spatial multi-omics and targeted analyses, uncovers lipid acquisition in TRPCs as a pivotal driver of GBM progression which is supported by a retrospective study of statin-treated patients, providing clinical evidence for the therapeutic potential of targeting lipid metabolism in GBM patients stratified based on high TRPC signatures. METHODS We applied spatial transcriptomics and proteomics combined with 3D reconstruction of the tumor microenvironment to map TRPC- and TSC-enriched regions. Immunohistochemically stained tissues were analyzed using CellProfiler and our custom SNAQ algorithm for single-cell neighborhood analysis. Lipid transfer from Tumor-Associated Myeloid Cells (TAMCs) to TRPCs was tracked via flow cytometry, holotomography, and time-lapse imaging. Genetic and pharmacological targeting of CCR2, CSF1R, and FABP3 was performed in vitro and in vivo. Finally, a retrospective analysis of 4,085 GBM patients assessed the impact of statin treatment in relation to TRPC signatures. RESULTS TRPCs exploit recruited TAMCs to acquire lipids, supporting their survival and immune evasion. These interactions are mediated by CCL2/7–CCR2 and CSF1–CSF1R signaling. Disruption of TRPC-TAM lipid homeostasis significantly remodeled the immune microenvironment, reduced tumor growth, and extended survival in preclinical models. Patients with high TRPC signatures typically had poor outcomes; however, retrospective analysis revealed that statin treatment was associated with improved survival in this subgroup. These findings position lipid trafficking as a metabolic vulnerability in GBM and highlight the therapeutic relevance of targeting TRPC–TAMC interactions. CONCLUSION Our study reveals lipid trafficking to TRPCs as a key mechanism driving GBM progression. Targeting TAMC-mediated lipid transfer disrupts immune suppression and tumor growth. Transcriptomic profiling may guide patient stratification, identifying those likely to benefit from lipid metabolism-targeted therapies such as statins.

Abstract INTRODUCTION Glioblastoma (GBM) is a brain cancer known for its complex tumor microenvironment and ability to reprogram glucose metabolism and evade host immune responses. CAR-T cell therapy has attracted considerable attention due to its potential for high specificity and efficacy across various cancer types. However, its success in GBM has been limited due to the unique and hostile metabolic landscape of the tumor microenvironment. GBM tumor cells exhibit enhanced aerobic glycolysis, resulting in elevated glucose consumption. This restricts glucose availability for T cells, impairing activation and anti-tumor response. In our study, we enhanced the tumoricidal activity of CD70 CAR T-cells by overexpressing the glucose transporters GLUT1 and GLUT3, thereby improving their metabolic fitness and enabling them to overcome tumor-induced glucose deprivation. METHODS The CD70 CAR T-cell glucose metabolic pathway was modulated via glucose transporter overexpression. The efficacy and safety of metabolically modified CD70 CAR T-cells was investigated in in vitro and in vivo platforms. RESULTS GLUTs overexpression does not negatively affect T-cell differentiation and function but improves glucose consumption and intra-tumoral T-cell trafficking. We then used CD70 CAR-T platform and overexpress GLUT1 and GLUT3 to engineer T-cells. GLUT overexpression provided metabolic advantage to CD70CAR T-cells, which showed enhanced activation and cell avidity when co-cultured with GBM cells in both glucose enriched and restricted conditions. The metabolic modifications regulate CAR T-cell efficacy by enhancing intra-tumoral trafficking and persistence, followed by improved anti-tumor response and survival in mice. The lack of significant alterations in body weight and organ morphology in mice receiving GLUT-overexpressing CD70 CAR T-cells suggests that the adoptive transfer of these metabolically enhanced T cells does not elicit toxicity, thereby demonstrating a favorable safety profile during treatment. CONCLUSION Our study demonstrates development of immunotherapeutic strategies by incorporating core principles of tumor and immune cell metabolism, thus providing evidence that targeting immune-metabolic pathways is a potential approach to treat brain tumor.

Abstract Introduction: Glioblastoma (GBM) tumors contain distinct cell lineages, including treatment-sensitive cells (TSCs) relying on aerobic glycolysis and treatment-resistant persister cells (TRPCs) dependent on lipid metabolism. Our systems biology-based, spatial multi-omics and multi-targeted approach decodes a critical node in GBM progression, lipid acquisition in TRPCs, and validates this through a retrospective analysis of statin-treated patients, offering clinical insights into the potential benefits of targeting lipid metabolism in high TRPC signature GBM patients. Methods: We performed spatial transcriptomics and proteomics, combined with 3D rendering of the tumor microenvironment (TME) and geospatially resolved single-cell neighborhood analysis of IHC-stained GBM tissues using our custom-developed SNAQ algorithm to investigate the immune microenvironment and lipid distribution specific to TSCs and TRPCs. To characterize lipid dynamics, we employed flow cytometry, time-lapse imaging, and holotomography to track lipid transfer from Tumor-Associated Myeloid Cells (TAMCs) to TRPCs. A critical node for GBM progression was identified through in vivo survival and tumor progression studies that assessed effects of genetic targeting of CCR2 and pharmacological targeting of CSF1R and FABP3. Retrospective analysis was conducted on 4,085 GBM patients to evaluate the effect of statin treatment on GBM outcomes based on their TRPC signature. Results: Our study establishes the skewed accumulation of TAMCs within the TRPC microenvironment through upregulation of the CCL2/7-CCR2 and CSF1-CSF1R pathways. TAMCs actively transfer lipids to TRPCs to fulfill their metabolic demands. Targeting elements responsible for lipid incorporation in TRPCs, such as CCR2, CSF1R, and FABP3 in vivo, resulted in tumor immune microenvironment remodeling, significant tumor growth reduction, and improved survival. This reinforces the critical role of metabolic communications via lipid trafficking in TRPCs as an essential node in GBM progression. Our retrospective study showed that GBM patients with elevated TRPC signatures, typically associated with shorter survival, had no significant survival differences when treated with lipid-lowering statins. This suggests statins may disrupt lipid metabolism in TRPCs, reducing TAMC lipid support and improving patient outcomes. Conclusion: Our study highlights lipid trafficking in TRPCs as a key mechanism in GBM progression, suggesting that targeting TAMC recruitment and lipid acquisition in TRPCs may offer a promising therapy. Additionally, our retrospective analysis highlights the potential of transcriptomic profiling for patient stratification based on TRPC signature expression levels, enabling the identification of patients to benefit from lipid metabolism-targeted therapies. Citation Format: Avirup Chakraborty, Changlin Yang, Diana Feier, Aryeh Silver, Nyla T. Searl, Miruna Anica, Avinash Pittu, Illena West, Olusegun O. Sobanjo, Ethan D. Hodge, Mia K. Engelbart, Srabasti Sengupta, Nagheme Thomas, Christina Von Roemeling, Maryam Rahman, Matthew Sarkisian, Jianping Huang, Jeffrey Harrison, Duane A. Mitchell, Loic P. Deleyrolle. Disrupting lipid metabolism in GBM: A systems biology approach to targeting treatment-resistant persister cells and tumor associated myeloid cell-mediated support [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB082.

Introduction The Florida-California Cancer Research, Education, and Engagement (CaRE 2 ) Health Equity Center is a triad partnership committed to increasing institutional capacity for cancer disparity research, the diversity of the cancer workforce, and community empowerment. This article provides an overview of the structure, process innovations, and initial outcomes from the first 4 years of the CaRE 2 triad partnership. Methods CaRE 2 serves diverse populations in Florida and California using a “molecule to the community and back” model. We prioritize research on the complex intersection of biological, environmental, and social determinants health, working together with scientific and health disparities communities, sharing expertise across institutions, bidirectional training, and community outreach. Partnership progress and outcomes were assessed using mixed methods and four Program Steering Committee meetings. Results Research capacity was increased through development of a Living Repository of 81 cancer model systems from minority patients for novel cancer drug development. CaRE 2 funded 15 scientific projects resulting in 38 publications. Workforce diversity entailed supporting 94 cancer trainees (92 URM) and 34 ESIs (32 URM) who coauthored 313 CaRE 2 -related publications and received 48 grants. Community empowerment was promoted via outreaching to more than 3000 individuals, training 145 community cancer advocates (including 28 Community Scientist Advocates), and publishing 10 community reports. CaRE 2 members and trainees together have published 639 articles, received 61 grants, and 57 awards. Conclusion The CaRE 2 partnership has achieved its initial aims. Infrastructure for translational cancer research was expanded at one partner institution, and cancer disparities research was expanded at the two cancer centers.

Abstract Purpose: An ongoing challenge in cancer is the management of primary and metastatic brain malignancies. This is partly due to restrictions of the blood-brain barrier and their unique microenvironment. These challenges are most evident in cancers such as lymphoma and melanoma, which are typically responsive to treatment in systemic locations but resistant when established in the brain. We propose interleukin-1 receptor-associated kinase-4 (IRAK-4) as a potential target across these diseases and describe the activity and mechanism of oral IRAK-4 inhibitor CA-4948. Experimental Design: Human primary central nervous system lymphoma (PCNSL) and melanoma brain metastases (MBM) samples were analyzed for expression of IRAK-4 and downstream transcription pathways. We next determined the central nervous system (CNS) applicability of CA-4948 in naïve and tumor-bearing mice using models of PCNSL and MBM. The mechanistic effect on tumors and the tumor microenvironment was then analyzed. Results: Human PCNSL and MBM have high expression of IRAK-4, IRAK-1, and nuclear factor kappa B (NF-κB). This increase in inflammation results in reflexive inhibitory signaling. Similar profiles are observed in immunocompetent murine models. Treatment of tumor-bearing animals with CA-4948 results in the downregulation of mitogen-activated protein kinase (MAPK) signaling in addition to decreased NF-κB. These intracellular changes are associated with a survival advantage. Conclusions: IRAK-4 is an attractive target in PCNSL and MBM. The inhibition of IRAK-4 with CA-4948 downregulates the expression of important transcription factors involved in tumor growth and proliferation. CA-4948 is currently being investigated in clinical trials for relapsed and refractory lymphoma and warrants further translation into PCNSL and MBM.

Abstract BACKGROUND PCNSL is an aggressive brain tumor accounting for 3% of all CNS malignancies and is associated with poor prognosis. Standard of care treatment includes induction with high-dose methotrexate based chemoimmunotherapy followed by consolidation with autologous stem cell transplant or whole brain radiation. However, more than half of PCNSL patients cannot tolerate this intensive therapeutic intervention. Novel treatments are urgently needed. Toll-like receptor signaling pathway via MyD88/IRAK-4 signalosome is constitutively active in PCNSL secondary to MYD88 L265P mutation and represents an excellent therapeutic target. Emavusertib (CA-4948) is an oral first-in-class small molecule inhibitor of IRAK-4 that demonstrates clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space. HYPOTHESIS: Emavusertib has anti-tumor activity against preclinical PCNSL. METHODS Using multiparameter immunohistochemistry (IHC) and proteomics analysis, IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient PCNSL tissues as compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for emavusertib drug concentration following single oral high dose in a murine model. Survival responses in preclinical PCNSL models were measured in response emavusertib treatment, along with in vivo tumor proliferation and downstream biomarker expression by multi-parameter IHC. RESULTS Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We show that emavusertib can achieve therapeutically relevant concentrations in the brain and CSF. Emavusertib impairs tumor cell proliferation in vitro and in vivo, and shows dose-dependent single-agent activity in preclinical PCNSL models. We confirm decreased ERK1/2, MAPK and NF-κB activation, indicative of downstream IRAK-4 inhibition. CONCLUSIONS Our data supports that emavusertib demonstrates anti-tumor activity in the CNS space, warranting further preclinical and clinical evaluation of this agent for the treatment of PCNSL.

Abstract Background: Central nervous system (CNS) lymphoma, both primary and secondary disease, carries the worst prognosis of all Non-Hodgkin’s lymphoma with a 5 year survival of less than 30% with current therapeutic options. Recent studies have shown constitutive activation of both NF-κB and MAPK signaling pathways in the maintenance and progression of CNS lymphoma. This often coincides with mutations in MYD88. IRAK4 is a serine/threonine kinase that mediates interleukin 1 receptor (IL-1R) and Toll-like receptor (TLR) signaling downstream of MYD88, and is a novel therapeutic target for this disease. CA-4948 is an oral first-in-class small molecule inhibitor of IRAK4 that has demonstrated clinical activity in patients with systemic Non-Hodgkin’s Lymphoma. Our study goal was to evaluate CA-4948 efficacy in a syngeneic preclinical model of primary CNS B cell lymphoma (A20), supporting future clinical investigation of this agent in this difficult to treat disease. Materials and Methods: Plasma, cerebrospinal fluid (CSF), and brain tissue (both naïve and tumor bearing) were assessed for CA-4948 drug concentration following single high-dose oral administration using UPLC-MS/MS. Biomarker expression, including NF-κB and MAPK, was measured in control and CA-4948 treated brain tissue resected from tumor-bearing animals using multi-parameter immunohistochemistry and 3D modeling of cleared tissue. Dose-dependent survival responses were also measured using the A20 preclinical model of primary CNS lymphoma. Results: While brain concentrations of CA-4948 remained low with respect to plasma (~4-5%), therapeutic dose levels were attained in both CSF and brain tissue (>502 nM). No significant difference between naïve and A20 tumor bearing brain tissue was observed, indicating the blood brain barrier remained intact in this model. Biomarker analyses revealed significant downregulation of both MAPK and NF-κB signaling pathways in response to CA-4948, as evidenced by reduced protein levels of phospho-p38Thr180/Tyr182, phospho-ERK1/2Thr202/Tyr204, and phospho-p65Ser536 in both the tumor cells and the tumor microenvironment. Finally, CA-4948 treatment produced a dose-dependent survival response in syngeneic animals harboring CNS A20 tumors. Conclusions: CA-4948 is able to reach therapeutic dose levels in the CNS in a preclinical murine model of CNS lymphoma, producing significant and dose-dependent anti-tumor activity and survival advantage. These data support further evaluation of CA-4948 in clinical investigation and treatment of patients with CNS lymphoma. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Lan Hoang-Minh, Han W. Tun, Elizabeth Martinez, Raul Soikes, Reinhard von Roemeling, Duane A. Mitchell. The IRAK4 inhibitor CA-4948 demonstrates antitumor activity in a preclinical model of CNS lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P243.