Arginine, glutamine, and the branched chain amino acids (BCAAs) are a focus of increased interest in the field of oncology due to their importance in the metabolic reprogramming of cancer cells. In the tumor microenvironment (TME), these amino acids serve to support the elevated biosynthetic and energy demands of cancer cells, while simultaneously maintaining the growth, homeostasis, and effector function of tumor-infiltrating immune cells. To escape immune destruction, cancer cells utilize a variety of mechanisms to suppress the cytotoxic activity of effector T cells, facilitating T cell exhaustion. One such mechanism is the ability of cancer cells to overexpress metabolic enzymes specializing in the catabolism of arginine, glutamine, and the BCAAs in the TME. The action of such enzymes supplies cancer cells with metabolic intermediates that feed into the TCA cycle, supporting energy generation, or providing precursors for purine, pyrimidine, and polyamine biosynthesis. Armed with substantial metabolic flexibility, cancer cells redirect amino acids from the TME for their own advantage and growth, while leaving the local infiltrating effector T cells deprived of essential nutrients. This review addresses the metabolic pressure that cancer cells exert over immune cells in the TME by up-regulating amino acid metabolism, while discussing opportunities for targeting amino acid metabolism for therapeutic intervention. Special emphasis is given to the crosstalk between arginine, glutamine, and BCAA metabolism in affording cancer cells with metabolic dominance in the TME.

Rapidly growing lymphoma cells deplete the tumor microenvironment (TME) of nutrients needed to maintain anti-tumor T cell immunity. This metabolic disadvantage leads to diminished T cell function and reduced clearance of tumor cells. The branched chain amino acids (BCAAs) are required for growth of tumor and immune cells. In the TME, BCAAs are preferentially taken up by tumor cells for protein synthesis or energy production. The first step in BCAA degradation is reversible transamination catalyzed by the cytosolic branched chain aminotransferase, BCATc. Recent investigations suggest that BCATc is immunosuppressive in the TME. We found BCATc is induced upon T cell activation, during which time, BCATc negatively regulates T cell metabolism. We hypothesized that a loss of BCATc from T cells restores effector T cell function and limits the immunosuppressive Treg cells in the TME. Tumor studies were conducted using mice with BCATc deleted from CD4+ and CD8+ T cells (T-BCATcKO mice) and corresponding controls expressing the floxed transgene of BCATc in T cells (T-BCATcfl/fl mice). Mice were subcutaneously injected with 2.5 × 105 murine EL-4 lymphoma cells [tumor-injected mice, n=8-9/group] or phosphate-buffered saline [vehicle-injected mice, n=4-6/group]. Tumor growth, body weight, and food intake were monitored for 15 days followed by collection of tumors and organs. Splenocytes were purified and stained for expression of Foxp3 or used to isolate CD4+ and CD8+ T cells that were activated with anti-CD3 and anti-CD28, and, after initial expansion with cytokine IL-2, reactivated to hypotolerant or fully activated CD4+ Th1 cells, or expanded to effector CD8+T cells for 96h, in the presence or the absence of the leucine antagonist, N-acetyl-leucine amide (NALA). Supernates were tested for secretion of IFNγ (CD4+T cells) or perforin and granzyme B (CD8+T cells). T-BCATcKO mice demonstrated significant delay in lymphoma appearance and a 50% reduction in tumor volumes when compared to control mice. The removal of BCATc from T cells did not cause systemic overactivation of the immune system, with no changes in size or appearance of the spleen, liver, or intestines. Splenic Foxp3 expression was reduced by 80% in lymphoma challenged T-BCATcKO mice when compared to T-BCATcfl/fl controls. Hypotolerant and fully activated T-BCATcKO CD4+ T cells released significantly more IFNγ when compared to T-BCATcfl/fl T cells. The addition of NALA reduced the IFNγ levels to those found in control cells. CD8+ T cells from T-BCATcKO mice released significantly more granzyme B and perforin compared to T-BCATcfl/fl T cells. These findings demonstrate that BCATc-deficient T cells provide better systemic response to lymphoma likely due to improved Th1 and effector CD8+ T cell with diminished Treg function. Thus, BCATc may act as an important metabolic checkpoint of the lymphoma TME Citation Format: Tanner J. Wetzel, Lucas Figueroa, Leighton Wheeler, Alexander Martin, Christie Adam, Michael Boyer, Elitsa Ananieva. The cytosolic branched chain aminotransferase is an important metabolic checkpoint of T cell function in the lymphoma microenvironment. [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 5154.

Current immunotherapies rely on modulating host immunity to induce an anti-tumor immune response. The efficacy of such a response depends on the immunogenicity of the tumor, which varies greatly between different cancers or the same cancer in different individuals. While most lymphomas are immunogenic, non-malignant T cells that infiltrate the lymphoma microenvironment are subjected to immunosuppressive signals and areas of nutrient depletion, which leads to T cell exhaustion and inhibition of T cell function. Metabolic enzymes, such as the cytosolic branched chain aminotransferase, BCATc, exert immunosuppressive effects limiting T cells ability to function. Previous research described BCATc as a negative regulator of T cell activation, thus identifying BCATc as a potential target for immunotherapeutic modulation in the tumor microenvironment (TME). In the current study we exploit a low (EL4) and high (EL4-OVA) immunogenic murine lymphoma strains to explore how a loss of BCATc in T cells impacts their ability to produce anti-lymphoma immunity in mice. We used 12 week old male mice with BCATc deleted from T cells (T-BCATcKO) and control T-BCATcfl/fl mice that express the transgenic floxed version of BCATc. Mice were injected with 2.5x105 cells belonging to the low (EL4) or high (EL4-OVA) immunogenic strains and tumor growth, body weight, and food intake were monitored for up to 15 days followed by collection of tumor tissues and organs. Tumors were then lysed, and the protein profile of each tumor was determined by monitoring changes in expression of protein markers of T cell exhaustion (TOX, CD39), apoptosis (BAX, PUMA), or signaling proteins, such as the ribosomal S6 protein, the protein kinase B (AKT), and the energy sensor the AMP-activated protein kinase (AMPK). T-BCATcKO mice showed significant reduction in tumor growth compared to control mice regardless of the lymphoma strain being tested. The mouse cohort with the biggest number of tumor free mice was the T-BCATcKO cohort injected with EL4 cells (13% of the mice were tumor free on day 9). However, EL4-OVA cells grew into smaller tumors across all cohorts. Mice had comparable food intake but T-BCATcKO mice, injected with EL4 cells, had a significantly lower body weight. In T-BCATcKO mice, the reduced EL4 tumor mass correlated with upregulated AMPK and AKT along with higher expression of PUMA. The reduced EL4-OVA tumor masses correlated with downregulated S6 and decreased TOX expression, but high expression of BAX compared to the corresponding tumors isolated from T-BCATcfl/fl mice. These findings suggest that T cells deficient in BCATc can reduce lymphoma growth regardless of the immunogenicity of the lymphoma stain likely by establishing catabolic state and inducing apoptosis. Thus, BCATc may play an immunosuppressive role in the lymphoma TME and the absence of BCATc from T cells may help T cells combat lymphoma. Citation Format: Lucas D. Figueroa, Tanner Wetzel, Leighton Wheeler, Christie Adam, Michael Boyer, Elitsa Ananieva. Mice with T cells deficient in the Cytosolic Branched Chain Aminotransferase reduce the tumor burden of low and high immunogenic strains of 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 5147.

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.

Diffuse large B‐cell Lymphoma (DLBCL) is the most common type of aggressive non‐Hodgkin lymphoma (NHL) worldwide. While therapeutic strategies to combat DLBCL have advanced, these malignancies continue to be challenging to treat due to their heterogeneity and frequently recurring mutations. Aberrations in the oncogene MYC are commonly found in high grade DLBCL and are associated with poor responses to standard therapy. In search of downstream targets of MYC in DLBCL, we found MYC‐binding sites in the promoter regions of BCAT1, BCAT2, and BCKDHA. These genes encode enzymes in the metabolism of the branched chain amino acids (BCAAs), and they have been previously linked to cancer progression. BCAT1 and 2 encode the cytosolic (BCATc) and mitochondrial (BCATm) branched‐chain aminotransferases, while BCKDHA encodes the E1α subunit of the branched‐chain ketoacid dehydrogenase complex. We hypothesized that MYC activates these genes in DLBCL tissues leading to upregulation of BCAA metabolism and lymphoma growth. Human tissues collected from DLBCL patients (n=89) or controls (n=33) were used to analyze the gene expression of MYC, BCAT1, BCAT2 and BCKDHA followed by correlative analysis of the expression of these genes and the survival of DLBCL patients. Next, human DLBCL and B‐lymphoblast (control) cells were treated with the MYC inhibitor 10058‐F4 (0, 25, 50, 100 µM), the BCAA antagonist N‐acetyl leucine amide (NALA, 0, 10, 20, 40 mM) and the BCATc inhibitor gabapentin (0, 5, 10, 20 mM). 24 hours post treatment, the cell viability and cell proliferation, as well as the protein expression of BCATc, BCATm, and E1α, and the total and phosphorylation states of the ribosomal protein S6 (a downstream target of the mammalian target of rapamycin, mTOR) were determined. Results showed that the gene expression of BCAT1, BCAT2and BCKDHA were significantly elevated in tumor tissues from DLBCL patients compared to controls, and there was a positive correlation between their expression and the upregulation of MYC in DLBCL tissues. The high expression of MYC and BCAT2, but not that of BCAT1 and BCKDHA, correlated with poor overall cancer survival. Inhibiting the MYC function by 10058‐F4, the BCAA uptake by NALA, and the BCATc activity by gabapentin led to concentration dependent reductions in the cell viability and proliferation of the DLBCL cells. The protein expression of BCATm and E1α were reduced in the presence of 10058‐F4, which correlated with downregulation of mTOR signaling as measured by the deceased phosphorylation state of S6 in treated DLBCL cells. These results support the hypothesis that MYC is an upstream regulator of genes associated with BCAA metabolism. The results also demonstrate that DLBCL cells need MYC and BCAAs to upregulate mTOR signaling and to survive and may represent a new molecular mechanism via which MYC exerts its effects in DLBCL lymphoma.