The arginase inhibitor Nω−hydroxy−nor−arginine (nor−NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target

Abstract

Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1−α and HIF2−α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω−hydroxy−nor−arginine (nor−NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor−NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor−NOHA overcame hypoxia-mediated resistance towards BCR−ABL1 kinase inhibitors. While nor−NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor−NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor−NOHA on cellular respiration. In conclusion, we show that nor−NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor−NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor−NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.

A Novel Oral Arginase 1/2 Inhibitor Enhances the Antitumor Effect of PD-1 Inhibition in Murine Experimental Gliomas by Altering the Immunosuppressive Environment

“…Dietary supplementation with L-arginine altered the spectrum of TILs and enhanced cytotoxicity in human colorectal and breast cancers ( 66 , 67 ). Elevation of arginine levels exerted immune-stimulatory effects in various cancers, for example blocking arginase activity with nor-NOHA in leukemic cells induced cell death ( 68 ) and treatment with another inhibitor CB-1158 had antitumor effects in several non-CNS cancers in mice ( 24 ). Here we demonstrate that a novel, oral ARG1/2 inhibitor, which increases L-arginine levels in the brain and restores the functionality of GAMs and NK cells, sensitizes murine gliomas to the PD-1 inhibition.…”

Arginase as a Potential Biomarker of Disease Progression: A Molecular Imaging Perspective

“…30 min [ 126 ]). Despite the short half-life, the potential of nor-NOHA to inhibit arginase was successfully evaluated in several tumors [ 127 , 128 ], airway [ 129 , 130 ], and cardiovascular [ 131 , 132 , 133 , 134 ] disease models. Other L-arginine-like molecules containing guanidine derivatives showed a lack of specificity to arginase and also some inhibitory effect on NOS [ 135 ], which would hamper potential therapeutic applications directed to the regulation of L-ornithine/NO • levels.…”

OATD-02 Validates the Benefits of Pharmacological Inhibition of Arginase 1 and 2 in Cancer

“…Extensive research has provided strong evidence that dysregulated arginase activity accompanies various types of neoplastic diseases [ 51 ], and the inhibition of arginases contributes to the restoration of the effective immune response against cancer [ 4 , 5 , 15 , 24 , 42 , 52 ]. Moreover, targeting arginases with small-molecule inhibitors was reported to affect the malignant phenotype of cancerous cells through multiple mechanisms, including induction of apoptosis and cell cycle arrest, reduction of invasiveness, or cell migration [ 5 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ]. On the other hand, defective l -arginine synthesis, due to the silencing of argininosuccinate synthase 1 (ASS1), is a common metabolic weakness in cancer and results in an intrinsic dependence on extracellular l -arginine due to an inability to synthesise l -arginine for growth [ 60 , 61 ].…”

“…Several synthetic and natural arginase inhibitors have been studied and developed to date [ 27 , 51 , 52 , 53 , 54 ]. The most advanced is CB-1158 (numidargistat) developed by Calithera Biosciences and Incyte.…”