Background Initial systemic therapy for patients with metastatic colorectal cancer (mCRC) is usually based on two- or three-drug chemotherapy regimens with fluoropyrimidine (5-fluorouracil (5-FU) or capecitabine), oxaliplatin and/or irinotecan, combined with either anti-VEGF (bevacizumab) or, for RAS wild-type (WT) tumors, anti-EGFR antibodies (panitumumab or cetuximab). Recommendations for patients who are not eligible for intensive combination therapies are limited and include fluoropyrimidine plus bevacizumab or single agent anti-EGFR antibody treatment. The use of a monochemotherapy concept of trifluridine/ tipiracil in combination with monoclonal antibodies is not approved for first-line therapy, yet. Results from the phase II TASCO trial evaluating trifluridine/ tipiracil plus bevacicumab in first-line treatment of mCRC patients and from the phase I/II APOLLON trial investigating trifluridine/ tipiracil plus panitumumab in pre-treated mCRC patients suggest favourable activity and tolerability of these new therapeutic approaches. Methods FIRE-8 (NCT05007132) is a prospective, randomized, open-label, multicenter phase II study which aims to evaluate the efficacy of first-line treatment with trifluridine/tipiracil (35 mg/m2 body surface area (BSA), orally twice daily on days 1–5 and 8–12, q28 days) plus either the anti-EGFR antibody panitumumab (6 mg/kg body weight, intravenously on day 1 and 15, q28 days) [arm A] or (as control arm) the anti-VEGF antibody bevacizumab (5 mg/kg body weight, intravenously on day 1 and 15, q28 days) [arm B] in RAS WT mCRC patients. The primary objective is to demonstrate an improved objective response rate (ORR) according to RECIST 1.1 from 30% (control arm) to 55% with panitumumab. With a power of 80% and a two-sided significance level of 0.05, 138 evaluable patients are needed. Given an estimated drop-out rate of 10%, 153 patients will be enrolled. Discussion To the best of our knowledge, this is the first phase II trial to evaluate the efficacy of trifluridine/tipiracil plus panitumumab in first-line treatment of RAS WT mCRC patients. The administration of anti-EGFR antibodies rather than anti-VEGF antibodies in combination with trifluridine/tipiracil may result in an increased initial efficacy. Trial registration EU Clinical Trials Register (EudraCT) 2019-004223-20. Registered October 22, 2019, ClinicalTrials.govNCT05007132. Registered on August 12, 2021.

Amino acids are integral components of cancer metabolism. The non-essential amino acid asparagine supports the growth and survival of various cancer cell types. Here, different mass spectrometry approaches were employed to identify lower aspartate levels, higher aspartate/glutamine ratios and lower tricarboxylic acid (TCA) cycle metabolite levels in asparagine-deprived sarcoma cells. Reduced nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide hydride (NADH) ratios were consistent with redirection of TCA cycle flux and relative electron acceptor deficiency. Elevated lactate/pyruvate ratios may be due to compensatory NAD+ regeneration through increased pyruvate to lactate conversion by lactate dehydrogenase. Supplementation with exogenous pyruvate, which serves as an electron acceptor, restored aspartate levels, NAD+/NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Chemicals disrupting NAD+ regeneration in the electron transport chain further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion. We speculate that reductive stress may be a major contributor to the growth arrest observed in asparagine-starved cells.

Background. Amino acids are integral components of cancer metabolism. Adequate availability of the non-essential amino acid asparagine is necessary to support growth and survival of various cancer cell types, including sarcoma cells. Methods. Different mass spectrometry approaches were employed to determine changes in the metabolome of asparagine-deprived mouse and human sarcoma cells, which may be responsible for arrested growth and apoptosis due to asparagine depletion. Results. Lower aspartate levels, higher aspartate/glutamine ratios and lower levels of tricarboxylic acid (TCA) cycle metabolites in asparagine-deprived sarcoma cells indicated a redirection of TCA cycle flux and were accompanied by reduced NAD+/NADH ratios, consistent with relative deficiency of electron acceptors in asparagine-starved cells. Elevated lactate/pyruvate ratios may be due to compensatory regeneration of NAD+ through increased pyruvate to lactate conversion by lactate dehydrogenase in asparagine-deprived cells. Supplementation with exogenous pyruvate, which was previously shown to serve as electron acceptor in cells challenged by reductive stress, restored aspartate levels, NAD+/NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Treatment with chemicals disrupting regeneration of NAD+ in the electron transport chain (e.g. metformin) further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion. Conclusions. We conclude that asparagine deprivation, through metabolic reprogramming, causes reductive stress, which, in turn, results in lower aspartate levels in asparagine-starved cells. These changes are major contributors to the growth arrest observed in asparagine-starved cells. Future studies are needed to further elucidate the cellular processes that contribute to lack of electron acceptors in asparagine-starved tumor cells. Citation Format: Christoph Bauer, Meret Quante, Carla Regina, Michaela Schneider, Geoffroy Andrieux, Oliver Gorka, Olaf Groß, Melanie Boerries, Bernd Kammerer, Simone Hettmer. Lack of electron acceptors contributes to redox stress and growth arrest in asparagine-starved sarcoma cells [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 275.