Discovery of a Series of Potent, Selective, and Orally Bioavailable Nucleoside Inhibitors of CD73 That Demonstrates In Vivo Antitumor Activity.

CD73 (ecto-5'-nucleotidase) has emerged as an attractive target for cancer immunotherapy of many cancers. CD73 catalyzes the hydrolysis of adenosine monophosphate (AMP) into highly immunosuppressive adenosine that plays a critical role in tumor progression. Herein, we report our efforts in developing orally bioavailable and highly potent small-molecule CD73 inhibitors from the reported hit molecule 2 to lead molecule 20 and then finally to compound 49. Compound 49 was able to reverse AMP-mediated suppression of CD8+ T cells and completely inhibited CD73 activity in serum samples from various cancer patients. In preclinical in vivo studies, orally administered 49 showed a robust dose-dependent pharmacokinetic/pharmacodynamic (PK/PD) relationship that correlated with efficacy. Compound 49 also demonstrated the expected immune-mediated antitumor mechanism of action and was efficacious upon oral administration not only as a single agent but also in combination with either chemotherapeutics or checkpoint inhibitor in the mouse tumor model.

LKB1 and KEAP1/NRF2 Pathways Cooperatively Promote Metabolic Reprogramming with Enhanced Glutamine Dependence in KRAS-Mutant Lung Adenocarcinoma.

In KRAS-mutant lung adenocarcinoma, tumors with LKB1 loss (KL) are highly enriched for concurrent KEAP1 mutations, which activate the KEAP1/NRF2 pathway (KLK). Here, we investigated the biological consequences of these cooccurring alterations and explored whether they conferred specific therapeutic vulnerabilities. Compared with KL tumors, KLK tumors exhibited increased expression of genes involved in glutamine metabolism, the tricarboxylic acid cycle, and the redox homeostasis signature. Using isogenic pairs with knockdown or overexpression of LKB1, KEAP1, and NRF2, we found that LKB1 loss results in increased energetic and redox stress marked by increased levels of intracellular reactive oxygen species and decreased levels of ATP, NADPH/NADP+ ratio, and glutathione. Activation of the KEAP1/NRF2 axis in LKB1-deficient cells enhanced cell survival and played a critical role in the maintenance of energetic and redox homeostasis in a glutamine-dependent manner. LKB1 and the KEAP1/NRF2 pathways cooperatively drove metabolic reprogramming and enhanced sensitivity to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, these findings elucidate the adaptive advantage provided by KEAP1/NRF2 pathway activation in KL tumors and support clinical testing of glutaminase inhibitor in subsets of KRAS-mutant lung adenocarcinoma. SIGNIFICANCE: In KRAS-mutant non-small cell lung cancer, LKB1 loss results in enhanced energetic/redox stress, which is tolerated, in part, through cooccurring KEAP1/NRF2-dependent metabolic adaptations, thus enhancing glutamine dependence and vulnerability to glutaminase inhibition.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3251/F1.large.jpg.

Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer.

Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839 + erlotinib. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment.

BACKGROUND: Myeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity. METHODS: CB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively. RESULTS: CB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers. CONCLUSIONS: These results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.