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The present study explores the role of the cytosolic branched chain amino acid aminotransferase (BCAT1) in CD8+T cell activation, in general, and tumor immunity, in particular, and identifies a non-canonical function of the protein in iron homeostasis. Pharmacologic inhibition of BCAT1 using the novel drug ERG245 abrogates the effector functions of CD8+T cells in vitro and metabolically reprograms the cells towards increased OXPHOS. In vivo, it suppresses activation of CD8+T cells in DSS colitis leading to improved disease outcomes. Remarkably, withdrawal of BCAT1 inhibition further amplifies OXPHOS and gives rise to CD8+T cells with increased cytotoxicity in vitro and in vivo. When combined with an anti-PD-1 treatment, temporal BCAT1 inhibition dramatically increases anti-PD-1 efficacy inducing complete and durable tumor regressions in the moderately immunogenic CT26 tumor model. Single cell RNA-seq data link expression of Bcat genes to exhausted T cells within the tumor microenvironment of human cancer patients, whereas in vitro assays indicate that BCAT1 inhibition partially prevents the adoption of a terminally exhausted phenotype by CD8+T cells. We propose BCAT1 as a target for cancer combinatory therapies.SIGNIFICANCEThe study explores for the first time the role of BCAT1 in CD8+T cell activation and proposes novel strategies for using BCAT1 inhibitors in cancer and beyond. It demonstrates that BCAT1 exerts its function without significantly altering branched chain amino acid (BCAA) levels through a mechanism that controls iron homeostasis, a novel non-canonical mechanism of action, and implicates BCAT1 in the adoption of an exhausted phenotype by T cells found in human cancers. While the majority of metabolic drugs temper OXPHOS, it demonstrates that an agent that increases OXPHOS in CD8+T cells can be used successfully as an immune-oncology drug.

We have previously demonstrated that BCAT1, the enzyme responsible for the reversible transamination of leucine in the cytosol, is a druggable target in immune-oncology: the small molecule inhibitor ERG245 dramatically increases the efficacy of anti-PD-1 treatment in the moderately immunogenic CT26 colon cancer model. We have also shown that Bcat1 gene expression is enriched in exhausted CD4+ and CD8+ intratumoral cells in cancer patients from diverse cancers. Here, we illustrate that the adoptive cell transfer (ACT) of murine CD8+ T cells treated with ERG245 increased the median survival of CT26 tumor-bearing mice from 25 days, observed with the ACT of untreated CD8+ T cells, to 32 days. This suggested that inhibition of BCAT1 prevented the known exhaustion of adoptively transferred CD8+ T cells, induced by the tumor microenvironment of CT26 tumors. To confirm this hypothesis, we developed an in vitro model of generating terminally exhausted (PD-1+TIM-3+GZMB+CXCL13+) CD8+ T cells by subjecting the cells to consecutive cycles of αCD3/αCD28 activation in the presence of TGF-β. Addition of ERG245 in the last activation cycle decreased terminal exhaustion of the cells. Mechanistically, bulk RNAseq experiments indicated and ICP analysis confirmed that inhibition of BCAT1 interfered with metal homeostasis leading to increased concentrations of Fe in the mitochondria and decreased concentrations of Zn in the nucleus of newly activated CD8+ T cells. The immediate impact of ERG245 treatment on the cells included a general decrease in the genes of processes depending on proper metal equilibrium such as cholesterol biosynthesis and ribosome biogenesis. Withdrawal of BCAT1 restored or reversed the cell phenotype leading to epigenetically altered CD8+ T cells with decreased histone acetyltransferase and histone demethylase presence, increased cytotoxicity, and increased ribosome biogenesis. Collectively, the data suggest that temporal inhibition of BCAT1 inhibition induces profound effects on CD8+ T cells including increased cytotoxicity and suppression of terminal exhaustion, thus rendering the cells rescuable by anti-PD-1 therapy. Citation Format: Adonia E. Papathanassiu, Weixing Li, Hong A. Vu. Temporal inhibition of BCAT1 alters metal homeostasis leading to reversal of terminal exhaustion of CD8 T cells, increased cytotoxicity, and increased efficacy of checkpoint inhibition in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4236.

Immune responsive gene 1 (Irg1) is an LPS-inducible gene within macrophages (MF) whose protein product is responsible for the synthesis of itaconate from aconitate through a break in the TCA cycle. Although primarily present in proinflammatory macrophages, IRG1 expression has been linked with certain cancers (glioblastoma, ovarian carcinoma) and its tumor-driven upregulation in peritoneal resident macrophages (pResMF) has been shown to contribute to peritoneal tumor growth. Here, we report Irg1 gene expression at the single cell level (scRNAseq) in samples obtained from colorectal cancer patients (n=7; ~11,000 single cells). Transcriptomic analysis showed that Irg1 was primarily expressed by cancer cells, although other tumoral cell types also showed substantial levels of the gene. To determine if IRG1 is a druggable target in cancer, we synthesized a series of small MW inhibitors and selected ERG344 for in vivo testing based on its ability to inhibit itaconate synthesis in a rat glioblastoma cell line (C6) engineered to stably express Irg1. The IC50 value of inhibition was ~150 μM. In vivo, ERG344 suppressed tumor growth and increased survival probability in a syngeneic colon cancer model (CT26). Drug administration was initiated when the tumors reached an average tumor volume of ~100 mm3 and involved daily ERG344 doses of 0.2 mg/kg ip or 0.3 mg/kg po. Complete tumor regressions (~17%) were observed in the same model when ERG344 was administered ip at 3 mg/kg. Immunophenotyping of CT26 tumors treated with either vehicle or 0.2 mg/kg ERG344 ip indicated that IRG1 inhibition renders the tumor microenvironment less immunosuppressive by decreasing the frequency of monocytic myeloid suppressor cells (m-MDSCs), increasing the frequency of cytotoxic CD8+ T cells, and decreasing the CD8+ expression of the negative co-stimulatory molecule PD-1. To determine if ERG344 is also able to reduce pResMF-driven peritoneal tumor growth, murine melanoma B16F15 cells were injected into the peritoneal cavity of C57BL/6 mice treated with vehicle or 1 mg/kg ERG344 (n=8/group). Seven days after tumor inoculation, mice treated with ERG344 showed reduced tumor growth in the peritoneal cavity, which led to increased survival probability of the animals. The studies collectively suggest that IRG1 is a novel target for the treatment of primary and metastatic tumor growth in colorectal cancer and highlight the therapeutic potential of ERG344. Citation Format: Adonia E. Papathanassiu, Francesca Lodi, Hong A. Vu, Diether Lambrechts. ERG344: A novel IRG1 inhibitor for the treatment of colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1202.

BCAT1, the enzyme responsible for the reversible transamination of leucine in the cytosol, has recently been implicated in the development and growth of various types of cancer. The limited expression of BCAT1 in adult tissues under physiological conditions suggests that the enzyme is a sensible target for drug development. To better understand the role of BCAT1 in cancer, we examined its gene expression at the single cell level (scRNAseq) in specimens obtained from lung, colorectal, breast, and ovarian cancer patients (n=36; >200,000 single cells). In those samples, Bcat1 gene expression was primarily associated with myeloid cells and fibroblasts with the exception of ovarian cancer in which robust Bcat1 presence was also seen in cancer cells. In tumor infiltrating lymphocytes (TILs), enrichment in Bcat1 gene expression was observed in exhausted CD4+ and CD8+ T cells compared to other T cell subtypes; this was independent of the cancer type examined. We speculated that inhibition of BCAT1 in TILs reverses exhaustion and has a therapeutic impact in cancer. In the syngeneic CT26 colon cancer model, combination treatment of a BCAT1 inhibitor (ERG245; ip administration of 5 mg/kg, given at days 7, 8 and 9 after tumor cell inoculation) and an anti-PD1 antibody (ab) (clone RMP1-14; ip administration given at days 7, 11, 14, and 18) led to eradication of established tumors. In the same model, limited treatment of large tumors (average size of ~600 mm3) with ERG245+anti-PD1 ab suppressed tumor growth and increased the frequency of CD8+ T cells expressing Granzyme B and IFNγ by ~50% compared to anti-PD1 monotherapy. Mechanistically, in vitro treatment of newly activated hCD8+ T cells with ERG245 impaired translocation of the lactate transporter MCT1 to the cell surface and increased the levels of intracellular lactate within 30 min of activation leading to upregulation of transcription factor ATF3. ERG245-driven metabolic reprogramming of CD8+ T cells towards an oxidative phenotype (increased OXPHOS) was also observed at 24h of treatment. Withdrawal of ERG245 restored intracellular lactate levels without reversing the metabolic reprogramming of the cells. We propose that these are elements of a mechanism that links temporal inhibition of BCAT1 to the rise of highly energetic CD8+ T cells with increased cytotoxicity and proliferative capacity in vitro and in vivo. Citation Format: Adonia E. Papathanassiu, Francesca Lodi, Hagar Elkafrawy, Michelangelo Certo, Hong Vu, Jeong Hun Ko, Jacques Behmoaras, Claudio Mauro, Diether Lambrechts. BCAT1 as a druggable target in immuno-oncology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3402.

Activation and differentiation of adaptive and innate immune cells require metabolic reprogramming involving diverse metabolic processes. Understanding these processes affords the opportunity to influence and ultimately re-direct the function and fate of these cells. Here, we present data suggesting that exposure of newly activated human CD8+ T cells to a small molecule inhibitor (ERG245; 50-400 μM) of BCAT1 (the cytosolic isoform of the enzyme responsible for the first step in the catabolism of leucine, isoleucine, and valine) resulted in inhibition of T cell proliferation, inhibition of IFNγ and Granzyme B production, and upregulation of PD-1. The antiproliferative effect, as determined by CFSE dilution, required inhibition of BCAT1 during the first 24 hrs of T cell activation and it was reversible upon early withdrawal of the inhibitor. Early withdrawal of ERG245 reversed the observed immunosuppression, ultimately giving rise to CD8+ T cells with higher proliferative capacity and increased cytotoxicity compared to untreated control cells. Exposure of CD8+ T cells to ERG245 resulted in a distinct metabolic phenotype including reduced concentrations of α-ketoglutaric acid (αKG), and lower levels of trimethylation of the lysine 4 (K4) and lysine 27 (K27) sites of histone 3 (H3), which implies that BCAT1 inhibition has epigenetic effects on human CD8+ T cells by modulating αKG-dependent histone demethylases. Similar observations were made with CD8+ T cells, isolated from the spleens of Bcat1 KO mice. In vivo, ERG245 (given at 5 mg/kg ip, bid at days 0, 1 and 2 of treatment), dramatically increased the efficacy of an anti-PD-1 antibody (clone RMP1-14; given at 10 mg/kg at days 0, 4, 7, and 11 of treatment) in the CT26 colon cancer model. Specifically, the combination of ERG245 and anti-PD-1 eradicated established tumors within a week of treatment initiation (cure rate of >80%), whereas the monotherapies (ERG245 or anti-PD-1 alone) did not improve the disease outcome. The data suggest that the temporal exposure of moderately immunogenic tumors to BCAT1 inhibition sensitizes the tumors to checkpoint inhibition. Citation Format: Adonia E. Papathanassiu, Dong-Wook Kim, Kwon-Sik Park, Jeong Hun Ko, Jacques V. Behmoaras. Immunotuning CD8+ T cells with BCAT1 inhibition to increase the efficacy of anti-PD-1 therapy and to eradicate moderately immunogenic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3200.

Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions., BCATs catabolize leucine and other BCAAs. Here the authors show that BCAA metabolism affects the broken Krebs cycle, reprogramming macrophages to be less proinflammatory, and show that BCAT1 inhibitor ERG240 can treat arthritis in mice and glomerulonephritis in rats.

Inhibition of heat shock protein 90 (Hsp90) has emerged as a novel intervention for the treatment of solid tumors and leukemias. Here, we report that F(1)F(0)-ATP synthase, the enzyme responsible for the mitochondrial production of ATP, is a co-chaperone of Hsp90. F(1)F(0)-ATP synthase co-immunoprecipitates with Hsp90 and Hsp90-client proteins in cell lysates of MCF-7, T47D, MDA-MB-453, and HT-29 cancer cells. Inhibition of F(1)F(0)-ATP synthase by efrapeptins results in the disruption of the Hsp90 complexing with its substrate proteins and, in most cases, in the degradation of the latter. Hsp90-client proteins affected by the inhibition of F(1)F(0)-ATP synthase included ERalpha, mutated p53 (m.p53), Hsp70, Hsp27, and caspase-3 but not Raf-1. This is the first report identifying caspase-3 as a substrate protein of Hsp90. Unlike typical Hsp90 inhibitors, efrapeptin treatment triggers Hsp70 downregulation in parallel with depletion of Hsp90. This suggests that suppression of Hsp90 chaperone function through inhibition of F(1)F(0)-ATP synthase does not result in activation of transcription factor HSF-1, a generally unfavorable consequence of anti-cancer treatments based on Hsp90 inhibition.

Tissue factor pathway inhibitor (TFPI) contains three Kunitz-type proteinase inhibitor domains and is a potent inhibitor of tissue factor-mediated coagulation. Here, we report that TFPI inhibits the proliferation of basic fibroblast growth factor-stimulated endothelial cells. A truncated form of TFPI, containing only the first two Kunitz-type proteinase inhibitor domains, has very little antiproliferative activity, suggesting that the carboxyl-terminal region of TFPI is responsible for this activity. Binding studies revealed that full-length TFPI, but not the truncated TFPI molecule, is recognized by the very low density lipoprotein receptor (VLDL receptor) indicating that this receptor is a novel high affinity endothelial cell receptor for TFPI. The antiproliferative activity of TFPI on endothelial cells is inhibited by the receptor-associated protein, a known antagonist of ligand binding by the VLDL receptor, and by anti-VLDL receptor antibodies. These results confirm that the antiproliferative activity of TFPI is mediated by the VLDL receptor and suggest that this receptor-ligand system may be a useful target for the development of new anti-angiogenic and antitumor agents.

Efrapeptins (EF), a family of fungal peptides, inhibit proteasomal enzymatic activities and the in vitro and in vivo growth of HT-29 cells. They are also known inhibitors of F(1)F(0)-ATPase, a mitochondrial enzyme that functions as an Hsp90 co-chaperone. We have previously shown that treatment of cancer cells with EF results in disruption of the Hsp90:F(1)F(0)-ATPase complex and inhibition of Hsp90 chaperone activity. The present study examines the effect of EF on breast cancer growth in vitro and in vivo. As a monotherapy, EF inhibited cell proliferation in vitro with an IC(50) value ranging from 6 nM to 3.4 μM. Inhibition of Hsp90 chaperone function appeared to be the dominant mechanism of action and the factor determining cellular sensitivity to EF. In vitro inhibition of proteasome became prominent in the absence of adequate levels of Hsp90 and F(1)F(0)-ATPase as in the case of the relatively EF-resistant MDA-MB-231 cell line. In vivo, EF inhibited MCF-7 and MDA-MB-231 xenograft growth with a maximal inhibition of 60% after administration of 0.15 and 0.3 mg/kg EF, respectively. 2-Deoxyglucose (2DG), a known inhibitor of glycolysis, acted synergistically with EF in vitro and antagonistically in vivo. In vitro, the synergistic effect was attributed to a prolonged endoplasmic reticulum (ER) stress. In vivo, the antagonistic effect was ascribed to the downregulation of tumoral and/or stromal F(1)F(0)-ATPase by 2DG.

Recent evidence suggests that the cytosolic presence of branched chain amino acid aminotransferase (BCAT1) in adult tissues is associated with diverse pathological conditions including cancer and inflammatory diseases such as rheumatoid arthritis; in these diseases, BCAT1 appears to modulate the expression of many key proteins and to be a new druggable target. In cancer, BCAT1 physically associates with CD147 and regulates the levels and the cellular localization of the protein in a number of different cell lines in vitro and in vivo. Silencing of Bcat1 gene downregulates the total expression of CD147 in MDA-MB-435 cells, while inhibition of BCAT1 by sodium 4-methyl-5-oxo-hexanoate (ERG240) leads to suppression of the extracellular CD147 in the same cells. In turn, suppression of extracellular CD147 results in downregulation of CD147-associated proteins such as MCT4, matrix metalloproteinases (MMPs), cyclophilin A, and GAPDH. Furthermore, inhibition of BCAT1 is associated with prevention of cancer cell migration and reduction in secretion of metabolic by-products such as lactate. Surprisingly, treatment of MDA-MB-231 tumor-bearing animals with 2-deoxyglucose (2DG), a well-known inhibitor of glycolysis, leads to a decrease in the levels of c-MYC and BCAT1, an increase in the total expression of CD147, and the increased shedding of the extracellular CD147 into the circulation. This observation suggests that BCAT1-driven regulation of CD147 in cancer cells involves a complex mechanism. Inhibition of tumoral BCAT1 is expected to adversely affect the metastatic potential of various tumors not only through a reduction in cell motility but also through alterations in the tumor microenvironment, driven by inhibition of extracellular CD147. Citation Format: Adonia E. Papathanassiu, Hong A. Vu. Inhibition of BCAT1 suppresses the expression of pro-metastatic proteins and reduces cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2014-2683

Apart from a recent report suggesting that hypoxia upregulates CD147, little is known about the mechanisms responsible of the tumoral overexpression of CD147, a transmembrane and secretory glycoprotein known to promote metastasis through multiple mechanisms. Here, we report that the total and surface concentration of CD147 appear to be under metabolic control. Glucose (Glc) deprivation in MDA-MB-231 and MDA-MB-435 tumor cells leads to a decrease in the levels of total CD147 and a dramatic increase in the expression of cell surface CD147. A similar mobilization of CD147 to the plasma membrane was observed with glutamine (Gln) deprivation in MDA-MB-435 cells; when the cells were deprived of both nutrients (Glc and Gln) a dramatic increase (∼6-fold) in the cell surface expression of CD147 was observed. Plasma membrane associated CD147 is known to promote metastasis through homotypic and heterotypic interactions between cancer and stromal cells. An 80% reduction in the membrane found CD147 was observed when Gln-deprived cells were treated with ERG-240, a small molecule that inhibits the cytosolic form of BCAT, the enzyme responsible for the reversible transamination of leucine (Leu). On the other hand, Leu deprivation was shown to decrease CD147 by 6-7 fold in both cancer cell lines tested here. Treatment of the cells with ERG-240 partially reversed the increase in the levels of CD147 seen after supplementation of the Leu-deprived cells with 105 mg/L Leu. CD147 is known to possess 3 N-glycosylation sites and to exist in forms with various degrees of glycosylation; the fully glycosylated protein is associated with an increased prometastatic potential. Inhibitors of N-glycosylation, such as 2-deoxyglucose (2DG), are known to possess anticancer properties. In the present study, treatment of MDA-MB-231 cells in vitro with 10 mM 2DG led to 80% reduction of the cell surface expression of highly glycosylated CD147 without the concomitant reduction in the total concentration of CD147. In contrast, treatment of athymic female nude mice, bearing orthotopically implanted MDA-MB-231 tumors, with 125 mg/Kg 2DG twice a week led to tumors that expressed markedly increased levels of the highly glycosylated CD147 variant (Control/Treated or T/C=2.3, p=0.01). The increased expression of the protein in the 2DG-treated tumors was accompanied with an increase in the levels of circulating human CD147 (T/C=1.7, p=0.01) normalized per tumor volume. Collectively, the data point towards a metabolic control of CD147 and suggest that inhibition of tumor growth due to nutrient deprivation may initiate pathways that promote metastasis. Citation Format: Adonia E. Papathanassiu, Hong A. Vu. Metabolic control of the prometastatic protein CD147. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2013-5416

Branched chain amino acid metabolism begins with the reversible transamination of the amino acids to the corresponding α-keto acids. The reaction is catalyzed by the enzyme Bcat. The cytosolic form of this enzyme, also known as Bcat1, is thought to be expressed only in embryonic tissues, in select adult organs (brain, ovary), and in c-myc induced brain tumors. In the current study, Bcat1 is shown to be expressed by a variety of tumor cell lines including MCF-7, MDA-MB-231, MDA-MB-435, and HT-29. Its expression appears to be associated with the ability of the cells to uptake glucose since treatment of MDA-MB-231 with 2-deoxyglucose leads to the suppression of the cellular Bcat1 levels in vitro and in vivo. In the cytosol, Bcat1 appears to complex with CD147, a protein known to promote cancer growth and metastasis through multiple pathways. Silencing of Bcat1 in MDA-MB-231 and MDA-MB-435 cells results in the downregulation of CD147, while inhibition of Bcat1 by ERG-240, a small molecule structurally related to leucine, leads to downregulation of the cell surface expressed CD147 and its transmembrane partner monocarboxylate transporter-4 (MCT-4) in MDA-MB-231 breast cancer cells. MCT-4 is known to mediate the expedient removal of lactate, a cytotoxic by-product of cancer glycolysis, from the cytosol. Furthermore, treatment of MDA-MB-435 cells with 10 mM ERG-240 results in downregulation of matrix metalloproteinase 9 (MMP-9) production, while treatment of normal adult fibroblasts with either ERG-240 or conditioned media of MDA-MB-435 cells previously exposed to ERG-240 results in downregulation of MMP-2 production. The later is attributed to the ability of ERG240 to suppress the presence of soluble CD147 in the conditioned media. Exocytosis of CD147 has been implicated in the formation of the pre-metastatic niche through inductions of MMPs and VEGF. Inhibition of Bcat1 by ERG-240 suppresses cell proliferation in various tumor cell lines with an IC50 value of 5 to 10 mM. ERG240-induced inhibition of MDA-MB-231 cell migration is also observed. The results from an on-going in vivo study are presented. In summary, our experiments suggest that: a) metabolism of branched chain amino acids is intimately linked with the expression of proteins controlling metastatic activities of tumor cells and b) Bcat1, which is not expressed by normal adult tissues, is an attractive target for the development of new, non-toxic anticancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3216. doi:1538-7445.AM2012-3216