Targeting the Cytosolic Branched Chain Aminotransferase (BCATc) for T Cell Driven Anti‐Lymphoma Immunotherapy

Abstract

The lymphoma microenvironment is a dynamic network between lymphoma cells and non‐malignant components that may promote tumor growth and consequently drug resistance. Progress in T cell metabolism has demonstrated that T cells experience metabolic disadvantage in the tumor microenvironment, which commonly manifests in T cell exhaustion and jeopardizes their potential to destroy cancer cells. We identified the cytosolic branched chain aminotransferase (BCATc) as a novel metabolic checkpoint of T cell activation. BCATc catalyzes the first step in the degradation of the essential amino acid leucine, which is a well‐known activator of complex 1 of the mammalian target of rapamycin (mTORC1). In T cells, BCATc is induced upon T cell activation and is a part of a negative feedback loop regulation of mTORC1. Based on extensive earlier research, we proposed that BCATc exerts immunosuppressive properties in the tumor microenvironment and we hypothesized that deleting BCATc from T cells renders them effective in eradicating lymphoma tumors.By using our newly designed mouse model with BCATc deleted from CD4+ and CD8+ T cells (T‐BCATcKO mice), we performed tumor studies where male and female mice were subcutaneously injected with 2.5x105 murine EL‐4 lymphoma cells [n=8] or phosphate‐buffered saline [n=4‐6]. As controls, mice expressing the floxed BCATc gene (T‐BCATcfl/flmice, n=9) were used. Tumor growth, body weight, and food intake were monitored for up to 15 days followed by collection of tumor tissues and organs. In addition, CD4+ T cells were isolated from spleens of T‐BCATcfl/fl and T‐BCATcKO mice, activated with a plate‐bound anti‐CD3 and soluble anti‐CD28 and left untreated or treated with 5 mM gabapentin (a competitive inhibitor of BCATc) or 10 mM N‐acetyl‐leucine amide (NALA, a structural analog of leucine) for 24‐72 hours to measure changes in the secretion of interferon gamma (IFNγ) from T helper 1 (Th1) cells or the expression of Foxp3, a master activator of regulatory T cells (CD4+CD25+ T reg cells).Deletion of BCATc from T cells or inhibition of the BCATc activity by gabapentin caused a significant increase in the secretion of IFNγ from Th1 cells after 24‐72 hours of activation, while CD4+CD25+ T reg cells expressed reduced levels of Foxp3 in response to gabapentin. In contrast, NALA blocked the IFNγ secretion and caused an increase in the expression of Foxp3. Following tumor inoculation, the T‐BCATcKO mice demonstrated a significant delay in tumor appearance and growth (~50 % reduction in tumor volumes) and the final tumor masses were reduced by 25% compared to control mice. None of the animals differed in body, organ weights or food intake and deletion of BCATc from T cells did not lead to systemic overactivation of the immune system. This study revealed that deleting BCATc from T cells may offer an advantage to better fight lymphoma and identified BCATc as a new therapeutic target to improve the efficiency of checkpoint inhibitors and help increase the low rates of remission seen in lymphoma patients.