Background. Malignant cells frequently exhibit dysregulation of nutrient/energy metabolism that can be exploited for development of novel targeted molecular therapy. Transcriptional repression of ASS, an enzyme essential for arginine production, in certain cancers and not normal cells makes these tumor cells selectively auxotrophic (dependent on external source) of this semi-essential amino acid and extremely susceptible to arginine depletion. One of the clinically applicable strategies to deplete extracellular arginine is systemic administration of the enzyme arginine di-iminase (ADI) conjugated with polyethylene glycol (PEG20) for optimal bioavailability. While well characterized in malignant pleural mesothelioma, melanoma and hepatocellular carcinoma, the status of ASS expression and sensitivity to ADI-PEG20 has not previously been investigated in NSCLC and this is the overarching objective of this study. Materials and Methods. ASS expression in 12 NSCLC cell lines and normal cells is determined by quantitative RT-PCR, western blots. ASS expression in lung cancers is quantified by immunohistochemistry (IHC) of tissue microarray of 72 NSCLC tumors. ADI-PEG20-mediated inhibition of cell viability, autophagy activation, induction of apoptosis and cell cycle arrest are assayed using MTT, fluorescence microscopy, AnnexinV/PI staining with flow cytometry respectively. Methylation of the promotor region of the ASS gene in ASS-negative cells is evaluated by methylation-specific PCR. Results. Six of 12 NSCLC cells have no or extremely low basal expression of ASS mRNA and protein and are, thus, exquisitely sensitive to ADI-PEG20 treatment (IC50 values: 45± 5 to 90±8 ng/ml (mean ± standard deviation, n=6)). ASS+ NSCLC cells and normal cells (primary or immortalized human fibroblasts) are totally resistant to ADI-PEG20. About 80% of NSCLC tumors do not express ASS by IHC using a well-validated anti-ASS antibody (Santa Cruz, clone H231). ADI-PEG20 treatment strongly activates autophagy with accumulation of autophagic vacuoles detected by fluorescence microscopy. Significant induction of apoptosis (15±3% to 55±6%, n=4) is noted in 5 NSCLC cells treated with ADI-PEG20 (100 or 200 ng/ml for 4 or 7 days) as well as cell cycle arrest. Two cell lines have methylated ASS promotor (one complete, one partial) as the cause of gene silencing. Conclusion. Up to 50% of NSCLC cell lines and 80% of NSCLC tumors lack ASS expression. ASS- NSCLC are selectively and significantly susceptible to ADI-PEG20-mediated arginine depletion in vitro. Induction of autophagy, apoptosis and cell cycle arrest collectively contribute ADI-PEG20-mediated cytotoxicity. As ADI-PEG20 is currently in phase II clinical trials for other carcinomas with encouraging results, it can be used to develop novel targeted molecular therapy for NSCLC tumors lacking ASS. Citation Format: Min You, Medhi Wangpaichitr, Jonathan D. Nguyen, Jennifer R. Chapman, Maureen Cioffi-Lavina, Niramol Savaraj, Dao M. Nguyen. Exploiting obligate arginine auxotrophy in tumor cells lacking arginino-succinate synthetase (ASS) expression to develop targeted molecular therapy for non-small cell lung cancer (NSCLC). [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 1435. doi:10.1158/1538-7445.AM2014-1435
The impact of non-small cell lung cancer (NSCLC) tumor metabolism on the immune microenvironment is not well understood in the context of platinum resistance. We have identified and characterized crucial metabolic differences between cisplatin-resistant (CR) and cisplatin-sensitive (CS) NSCLC cells in which CR cells possessed higher indoleamine 2,3-dioxygenase-1 (IDO1) activity as determined by an increase in extracellular kynurenine concentration in vitro and in vivo. Utilizing in vitro co-culture models (n=6), we showed that IDO-mediated kynurenine production from CR cells suppressed natural killer group 2 member D (NKG2D) on natural killer (NK) and CD8+ T cells, but enhanced immunosuppressive regulatory T cell (Treg) and myeloid-derived suppressor cells (MDSC) populations. Utilizing syngeneic (n=7) and humanized (n=5) mouse models, mice with CR tumors exhibited a more highly immunosuppressive environment that blocked the antitumor immune response. Inhibition of IDO1 by the selective IDO1 inhibitor epacadostat (200 mg/kg P.O. once a day for 15 days) attenuated tumor growth in these CR tumor-bearing mice. Interestingly, we found an induction of tryptophan 2,3-dioxygenase-2 (TDO2) enzyme in epacadostat-treated mice. To overcome this compensatory effect induced by IDO1 inhibition, mice were treated with a novel IDO1/TDO2 dual inhibitor (AT-0174; Antido Therapeutics Pty Ltd, Melbourne, Australia) at 170 mg/kg P.O. once a day for 15 days. Dual inhibitors suppressed tumor growth to a greater degree than IDO1 inhibitors in CR conditions, and significantly enhanced immune effector populations as well as suppressed immunosuppressive Tregs and MDSC population in CR tumors. In addition, increased expression of programmed death-ligand 1 (PD-L1) was observed in CR cells, we assessed the effects of combination treatment of AT-0174 + anti-PD1 antibody. This combined therapy produced a profound anti-tumor effect in CR tumors and extended survival in mice. Our data support an increase in the therapeutic efficacy of blocking both IDO1/TDO2 in treating CR lung cancer by enhancing tumor immune surveillance. Thus, inhibiting the kynurenine pathway may be a more suitable therapeutic approach in a subgroup of lung cancer patients who failed platinum therapy and lead to improved outcomes in the treatment and management of NSCLC. Support by Dept. of Veterans. Citation Format: Sydney Spector, Chunjing Wu, George Theodore, Jonathan Dan Nguyen, Emily Kim, Ashley Garcia, Niramol Savaraj, Diane Lim, Lynn Feun, Medhi Wangpaichitr. Targeting IDO1 and TDO2 is crucial for the modulation of immune effector in platinum resistant non-small cell lung cancer [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 5886.
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