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.

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.

Comparison of AMG 416 and cinacalcet in rodent models of uremia.

BACKGROUND: AMG 416 is a novel peptide agonist of the calcium-sensing receptor (CaSR). This report describes the activity of AMG 416 in two different rodent models of uremia, compared in each case to cinacalcet, an approved therapeutic for secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease on dialysis. METHODS: AMG 416 was administered as a single intravenous (IV) bolus in a severe, acute model of renal insufficiency (the "1K1C" model) and plasma parathyroid hormone (PTH) and serum calcium levels were monitored for 24 hours. In a chronic, less severe model of renal dysfunction, the 5/6 nephrectomy (5/6 Nx) model, AMG 416 was administered as a once-daily IV bolus for 28 days. Both studies included a control (vehicle) group and a comparison cinacalcet group (po dosing at 30 mg/kg and 10 mg/kg for the 1K1C and 5/6 Nx studies, respectively). RESULTS: Administration of AMG 416 by IV bolus injection into rats with acute renal dysfunction (1K1C model) resulted in a sustained reduction in plasma PTH from the initial elevated values. Following a single IV bolus (0.5 mg/kg), AMG 416 caused a substantial drop in PTH levels which remained approximately 50% below their initial level at 24 hrs. In the same model, oral treatment with cinacalcet (30 mg/kg) resulted in an acute drop in PTH which almost returned to the starting level by 24 hours after dosing. In the 5/6 Nx chronic uremia model, daily IV dosing of AMG 416 over 4 weeks (1 mg/kg) resulted in a sustained reduction in PTH, with approximately 50% of the initial level observed 48 hours post treatment throughout the study. Cinacalcet treatment (10 mg/kg) in the same model resulted in acutely lowered plasma PTH levels which returned to placebo levels by 24 hours post-dose. Consistent with the reductions in plasma PTH, reductions in serum calcium were observed in both AMG 416- and cinacalcet-treated animals. CONCLUSIONS: As a long-acting CaSR agonist suitable for administration by the IV route, AMG 416 is a potential new therapy for the treatment of CKD patients with SHPT receiving hemodialysis.

Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer.

Glutamine serves as an important source of energy and building blocks for many tumor cells. The first step in glutamine utilization is its conversion to glutamate by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 had antiproliferative activity in a triple-negative breast cancer (TNBC) cell line, HCC-1806, that was associated with a marked decrease in glutamine consumption, glutamate production, oxygen consumption, and the steady-state levels of glutathione and several tricarboxylic acid cycle intermediates. In contrast, no antiproliferative activity was observed in an estrogen receptor-positive cell line, T47D, and only modest effects on glutamine consumption and downstream metabolites were observed. Across a panel of breast cancer cell lines, GAC protein expression and glutaminase activity were elevated in the majority of TNBC cell lines relative to receptor positive cells. Furthermore, the TNBC subtype displayed the greatest sensitivity to CB-839 treatment and this sensitivity was correlated with (i) dependence on extracellular glutamine for growth, (ii) intracellular glutamate and glutamine levels, and (iii) GAC (but not KGA) expression, a potential biomarker for sensitivity. CB-839 displayed significant antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2(+) cell line model, JIMT-1, both as a single agent and in combination with paclitaxel. Together, these data provide a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in patients with TNBC and other glutamine-dependent tumors.

Pharmacology of AMG 416 (Velcalcetide), a novel peptide agonist of the calcium-sensing receptor, for the treatment of secondary hyperparathyroidism in hemodialysis patients.

A novel peptide, AMG 416 (formerly KAI-4169, and with a United States Adopted Name: velcalcetide), has been identified that acts as an agonist of the calcium-sensing receptor (CaSR). This article summarizes the in vitro and in vivo characterization of AMG 416 activity and the potential clinical utility of this novel compound. AMG 416 activates the human CaSR in vitro, acting by a mechanism distinct from that of cinacalcet, the only approved calcimimetic, since it can activate the CaSR both in the presence or the absence of physiologic levels of extracellular calcium. Administration of AMG 416 in vivo into either normal or renally compromised rats results in dose-dependent reductions in parathyroid hormone (PTH) levels and corresponding decreases in serum calcium, regardless of the baseline level of PTH. Treatment of 5/6 nephrectomized rats with AMG 416 resulted in dramatic improvements in their metabolic profile, including lower PTH and serum creatinine levels, reduced amounts of vascular calcification, attenuated parathyroid hyperplasia, and greater expression of the parathyroid gland regulators CaSR, vitamin D receptor, and FGF23 receptor compared with vehicle-treated animals. No drug accumulation was observed under this dosing regimen, and the terminal half-life of AMG 416 was estimated to be 2-4.5 hours. As a long-acting CaSR agonist, AMG 416 is an innovative new therapy for the treatment of hemodialysis patients with secondary hyperparathyroidism.

A shed form of LDL receptor-related protein-1 regulates peripheral nerve injury and neuropathic pain in rodents.

Injury to the peripheral nervous system (PNS) initiates a response controlled by multiple extracellular mediators, many of which contribute to the development of neuropathic pain. Schwann cells in an injured nerve demonstrate increased expression of LDL receptor-related protein-1 (LRP1), an endocytic receptor for diverse ligands and a cell survival factor. Here we report that a fragment of LRP1, in which a soluble or shed form of LRP1 with an intact alpha-chain (sLRP-alpha), was shed by Schwann cells in vitro and in the PNS after injury. Injection of purified sLRP-alpha into mouse sciatic nerves prior to chronic constriction injury (CCI) inhibited p38 MAPK activation (P-p38) and decreased expression of TNF-alpha and IL-1beta locally. sLRP-alpha also inhibited CCI-induced spontaneous neuropathic pain and decreased inflammatory cytokine expression in the spinal dorsal horn, where neuropathic pain processing occurs. In cultures of Schwann cells, astrocytes, and microglia, sLRP-alpha inhibited TNF-alpha-induced activation of p38 MAPK and ERK/MAPK. The activity of sLRP-alpha did not involve TNF-alpha binding, but rather glial cell preconditioning, so that the subsequent response to TNF-alpha was inhibited. Our results show that sLRP-alpha is biologically active and may attenuate neuropathic pain. In the PNS, the function of LRP1 may reflect the integrated activities of the membrane-anchored and shed forms of LRP1.

Cytokine regulation of MMP-9 in peripheral glia: implications for pathological processes and pain in injured nerve.

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced in Schwann cells hours after peripheral nerve injury and controls axonal degeneration and macrophage recruitment to the lesion. Here, we report a robust (90-fold) increase in MMP-9 mRNA within 24 h after rat sciatic nerve crush (1 to 60 days time-course). Using direct injection into a normal sciatic nerve, we identify the proinflammatory cytokines TNF-alpha and IL-1beta as potent regulators of MMP-9 expression (Taqman qPCR, zymography). Myelinating Schwann cells produced MMP-9 in response to cytokine injection and crush nerve injury. MMP-9 gene deletion reduced unstimulated neuropathic nociceptive behavior after one week post-crush and preserved myelin thickness by protecting myelin basic protein (MBP) from degradation, tested by Western blot and immunofluorescence. These data suggest that MMP-9 expression in peripheral nerve is controlled by key proinflammatory cytokine pathways, and that its removal protects nerve fibers from demyelination and reduces neuropathic pain after injury.

The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells: possible implications in peripheral nerve injury.

Schwann cells undergo phenotypic modulation in peripheral nerve injury. In the adult rodent, Schwann cells are resistant to death-promoting challenges. The responsible receptors and signaling pathways are incompletely understood. In this study, we demonstrate that low-density lipoprotein receptor-related protein-1 (LRP-1) is expressed in adult sciatic nerve. After crush injury, LRP-1 is lost from the axoplasm and substantially upregulated in Schwann cells. Increased LRP-1 mRNA expression was observed locally at the injury site in multiple forms of sciatic nerve injury, including crush injury, chronic constriction injury, and axotomy. Endogenously produced tumor necrosis factor-alpha (TNF-alpha) was mostly responsible for the increase in LRP-1 expression; this activity was reproduced by direct injection of TNF-alpha into injured nerves in the TNF-alpha gene knock-out mouse. TNF receptor II was primarily involved. TNF-alpha also increased LRP-1 mRNA in Schwann cells in primary culture. Silencing of Schwann cell LRP-1 with siRNA decreased phosphorylated Akt and increased activated caspase-3. Equivalent changes in cell signaling were observed in LRP-1-deficient murine embryonic fibroblasts. Schwann cell death was induced in vitro by serum withdrawal or TNF-alpha, to a greater extent when LRP-1 was silenced. Schwann cell death was induced in vivo by injecting the LRP-1 antagonist, receptor-associated protein, into axotomy sites in adult rats. These results support a model in which LRP-1 functions as a pro-survival receptor in Schwann cells.

Omega-3 polyunsaturated fatty acid impairment of early host resistance against Listeria monocytogenes infection is independent of neutrophil infiltration and function.

The primary objective of this study was to determine whether the n-3 PUFA-mediated changes in host response to a Listeria monocytogenes infection (e.g., cytokine production and bacterial clearance) were dependent upon neutrophils. Balb/c mice were fed one of two semi-purified diets that contained either 0 or 41 g of n-3 PUFA/kg. After 4 week, mice were injected with a neutrophil-depleting (RB6-8C5) or isotype-control antibody 24 h prior to infection. Bacterial clearance from the liver and spleen at 3 days post-challenge was measured and the concentration of five pro-inflammatory cytokines in sera 24 h post-infection were determined using a novel protein multiplexing kit. We found that neutrophil depletion impaired bacterial clearance independent of the effect of n-3 PUFA. Interestingly, we observed a rather complex interaction between neutrophil-depletion and n-3 PUFA intake on in vivo pro-inflammatory cytokine production. For example, neutrophil depletion elevated circulating IL-6 and MCP-1 (2- to 5-fold; p<0.05) in n-3 PUFA-fed mice, but less so or not at all in mice fed the control diet. In summary, our data suggest that n-3 PUFA-mediated reduction of host resistance to L. monocytogenes is independent of neutrophil activity.