Abstract 3804: Accumulation of branched chain keto acids promotes immunosuppression and neurodegeneration in leptomeningeal disease
Authors: Mariam Lotfy Khaled, Ethan Vallebuona, Min Liu, M.Baraa Boozo, Zhihua Chen, Gerald C. Wallace, Yuan Ren, Ronak Kundalia, Hasan Alhaddad, Oscar Ospina, Brittany Evernden, Victoria Izumi, Lancia NF Darville, Ann Chen, MacLean Hall, Michael Jain, Shari Pilon-Thomas, Paul Stewart, Fredrick L. Locke, Timothy J. Robinson, John M. Koomen, Peter A. Forsyth, Inna Smalley
Journal: Cancer Research (2025)

Leptomeningeal disease (LMD) occurs when malignant cells seed into the leptomeningeal space and cerebrospinal fluid (CSF), leading to severe neurological symptoms and very short survivals. LMD occurs in 5to15% of cancer patients and is most common in breast, melanoma, lung, and lymphomas. LMD tumors are generally resistant to all standard of care therapies. To better understand LMD biology we used comprehensive multiomic analyses of CSF specimens from LMD patients with different primary tumors to understand its immune and metabolic tumor microenvironment. Single cell RNA sequencing on patient CSF showed a distinct immunosuppressive landscape regardless of the primary histology. We identified significant enrichment of CD4Tregs and exhausted T cells in LMD patients. Lack of active, proliferating T cells and protumorigenic macrophage infiltration were associated with LMD poor survival (&amp;lt;10months, p&amp;lt;0.01). Additionally, proteomic and lipidomic analysis of patients’ CSF showed downregulation in proteins and lipids vital for neuronal development and myelin sheath integrity. Strikingly, the metabolomic analysis demonstrated an accumulation of branched-chain keto acids (BCKA), well known neurotoxins, in CSF of LMD versus controls (p&amp;lt;0.05). Next, we performed correlation analysis between single cell RNA seq data and levels of BCKA in LMD samples (n=8). Results revealed positive correlations between high BCKA concentrations and CD4Tregs (p&amp;lt;0.01), and two exhausted T cell populations (p&amp;lt;0.05), suggesting an immunosuppressive impact of BCKA in LMD patients. Our in-vitro functional data demonstrated that BCKA drastically disrupts the T-lymphocyte viability, proliferation, and effector cytokine secretion. Moreover, BCKA reduced the viability of chimeric antigen receptor CART cells, neurons, and meningeal cells but not tumor cells. These results were validated in immunocompetent LMD animal models. Consistent with BCKA-induced neurotoxicity, we found that LMD mice had a rapid neurological decline (p&amp;lt;0.0001). Furthermore, we found the leptomeningeal layer in LMD mice was compromised and had significantly high levels of BCKA (p&amp;lt;0.01). Highlighting the translatability of this work, we found that phenylbutyrate, a BCKA lowering agent, improved neurological function (p&amp;lt;0.01), survival outcomes (p&amp;lt;0.01), and efficacy of CART therapy in lymphoma LMD model (p&amp;lt;0.05). Phenylbutyrate also improved responses to chemotherapy in breast cancer LMD model. This is the first report describing BCKA accumulation in LMD as a cause of neurotoxicity and immunosuppression and provides a unique strategy to treat these deadly tumors. Repurposing of FDA approved phenylbutyrate improves the quality of life, survival, and efficacy of immune therapies in LMD. Citation Format: Mariam Lotfy Khaled, Ethan Vallebuona, Min Liu, M.Baraa Boozo, Zhihua Chen, Gerald C. Wallace, Yuan Ren, Ronak Kundalia, Hasan Alhaddad, Oscar Ospina, Brittany Evernden, Victoria Izumi, Lancia NF Darville, Ann chen, MacLean Hall, Michael Jain, Shari Pilon-Thomas, Paul Stewart, Fredrick L. Locke, Timothy J. Robinson, John M. Koomen, Peter A. Forsyth, Inna Smalley. Accumulation of branched chain keto acids promotes immunosuppression and neurodegeneration in leptomeningeal disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3804.