Abstract 1192: Branched-chain keto acids exert an immune-suppressive and neurodegenerative microenvironment in CNS leptomeningeal lymphoma
Authors: Khaled Mariam Lotfy , Wallace Gerald , Evernden Brittany , Chen Zhihua , Alhaddad Hasan , Ren Yuan , Ospina Oscar , Hall Maclean , Chen Ann , Robinson Timothy J. , Koomen John , Pilon-thomas Shari , Forsyth Peter , Smalley Inna 
Published: April 2023

Abstract
Leptomeningeal disease is a devastating complication characterized by the rapid onset of debilitating neurological symptoms and a markedly poor prognosis. It is diagnosed in 5-15% of hematological cancers. Here, we aim to identify the metabolic and immune remodeling of the tumor leptomeningeal microenvironment in Non-Hodgkin lymphoma through comprehensive multi-omic analysis of patient specimens, in vivo models, and in vitro functional studies. Cerebrospinal fluid (CSF) from leptomeningeal lymphoma (LML) patients and tumor-free controls were collected and analyzed using single-cell RNA sequencing (scRNA-Seq), metabolomics, proteomics, and lipidomics. Patients’ CSF cellular components were highly enriched with heterogeneous B-cell populations. Few T-lymphocytes in LML patients showed expression of activation or proliferation markers. Poor prognosis was associated with significant infiltration of macrophages and a lack of dendritic cells. These findings were confirmed to be unique to LML using immune-competent animal models where tumor cells were injected intrathecally, and into lymph nodes. Moreover, LML patients’ CSF showed an accumulation of branched-chain keto-acids (BKAs), which are well-known neurotoxins, metabotoxins, and acidogens. Absolute quantification of individual BKAs was confirmed by high-performance liquid chromatography and showed a 30-fold increase in LML patients’ CSF. To investigate the effect of BKAs accumulation on T-lymphocytes, we examined their viability, proliferation, and activation with BKAs treatment in the context of physiological CSF. Cell trace violet-labeled T lymphocytes showed a significant reduction in proliferation and viability with increasing doses of BKAs. Additionally, a significant decline in the secretion of TNF-α, interferon-γ, granzyme B, and IL-2 was found using ELISA assays, indicating a reduction in T-lymphocyte activation. The proteomic and lipidomic analysis of patients’ CSF also revealed a significant downregulation in proteins and lipids vital for neuronal development, synaptic organization, and myelin sheath integrity. Similarly, scRNA-Seq of the leptomeningeal layer collected from the LML mouse model revealed compromised leptomeningeal integrity. Neurological scoring of the LML mouse model showed disease progression to be associated with rapid neurological decline. To investigate the direct effect of BKAs on the leptomeningeal and neuronal integrity, we tested different concentrations of BKAs on murine primary neuronal cells and human leptomeningeal cells (HMC). MTT assays showed a significant reduction in HMC metabolic activity after 72 hours and in neuronal metabolic activity after 7 days. In conclusion, our data unveil the immunosuppressive and neurodegenerative role of BKAs in the leptomeningeal tumor microenvironment.
Citation Format: Mariam Lotfy Khaled, Gerald Wallace, Brittany Evernden, Zhihua Chen, Hasan Alhaddad, Yuan Ren, Oscar Ospina, Maclean Hall, Ann Chen, Timothy J. Robinson, John Koomen, Shari Pilon-thomas, Peter Forsyth, Inna Smalley. Branched-chain keto acids exert an immune-suppressive and neurodegenerative microenvironment in CNS leptomeningeal lymphoma [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 1192.