Long-acting Erwinia chrysanthemi, Pegcrisantaspase, induces alternate amino acid biosynthetic pathways in a preclinical model of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease without meaningful therapeutic options beyond the first salvage therapy. Targeting PDAC metabolism through amino acid restriction has emerged as a promising new strategy, with asparaginases, enzymes that deplete plasma glutamine and asparagine, reaching clinical trials. In this study, we investigated the anti-PDAC activity of the asparaginase formulation Pegcrisantaspase (PegC) alone and in combination with standard-of-care chemotherapeutics. METHODS: Using mouse and human PDAC cell lines, we assessed the impact of PegC on cell proliferation, cell death, and cell cycle progression. We further characterized the in vitro effect of PegC on protein synthesis as well as the generation of reactive oxygen species and levels of glutathione, a major cellular antioxidant. Additional cell line studies examined the effect of the combination of PegC with standard-of-care chemotherapeutics. In vivo, the tolerability and efficacy of PegC, as well as the impact on plasma amino acid levels, was assessed using the C57BL/6-derived KPC syngeneic mouse model. RESULTS: Here we report that PegC demonstrated potent anti-proliferative activity in a panel of human and murine PDAC cell lines. This decrease in proliferation was accompanied by inhibited protein synthesis and decreased levels of glutathione. In vivo, PegC was tolerable and effectively reduced plasma levels of glutamine and asparagine, leading to a statistically significant inhibition of tumor growth in a syngeneic mouse model of PDAC. There was no observable in vitro or in vivo benefit to combining PegC with standard-of-care chemotherapeutics, including oxaliplatin, irinotecan, 5-fluorouracil, paclitaxel, and gemcitabine. Notably, PegC treatment increased tumor expression of asparagine and serine biosynthetic enzymes. CONCLUSIONS: Taken together, our results demonstrate the potential therapeutic use of PegC in PDAC and highlight the importance of identifying candidates for combination regimens that could improve cytotoxicity and/or reduce the induction of resistance pathways.

Erwinia asparaginase (crisantaspase) increases plasma levels of serine and glycine.

The impact of asparaginases on plasma asparagine and glutamine is well established. However, the effect of asparaginases, particularly those derived from Erwinia chrysanthemi (also called crisantaspase), on circulating levels of other amino acids is unknown. We examined comprehensive plasma amino acid panel measurements in healthy immunodeficient/immunocompetent mice as well as in preclinical mouse models of acute myeloid leukemia (AML) and pancreatic ductal adenocarcinoma (PDAC) using long-acting crisantaspase, and in an AML clinical study (NCT02283190) using short-acting crisantaspase. In addition to the expected decrease of plasma glutamine and asparagine, we observed a significant increase in plasma serine and glycine post-crisantaspase. In PDAC tumors, crisantaspase treatment significantly increased expression of serine biosynthesis enzymes. We then systematically reviewed clinical studies using asparaginase products to determine the extent of plasma amino acid reporting and found that only plasma levels of glutamine/glutamate and asparagine/aspartate were reported, without measuring other amino acid changes post-asparaginase. To the best of our knowledge, we are the first to report comprehensive plasma amino acid changes in mice and humans treated with asparaginase. As dysregulated serine metabolism has been implicated in tumor development, our findings offer insights into how leukemia/cancer cells may potentially overcome glutamine/asparagine restriction, which can be used to design future synergistic therapeutic approaches.

Venetoclax and pegcrisantaspase for complex karyotype acute myeloid leukemia.

Complex karyotype acute myeloid leukemia (CK-AML) has a dismal outcome with current treatments, underscoring the need for new therapies. Here, we report synergistic anti-leukemic activity of the BCL-2 inhibitor venetoclax (Ven) and the asparaginase formulation Pegylated Crisantaspase (PegC) in CK-AML in vitro and in vivo. Ven-PegC combination inhibited growth of multiple AML cell lines and patient-derived primary CK-AML cells in vitro. In vivo, Ven-PegC showed potent reduction of leukemia burden and improved survival, compared with each agent alone, in a primary patient-derived CK-AML xenograft. Superiority of Ven-PegC, compared to single drugs, and, importantly, the clinically utilized Ven-azacitidine combination, was also demonstrated in vivo in CK-AML. We hypothesized that PegC-mediated plasma glutamine depletion inhibits 4EBP1 phosphorylation, decreases the expression of proteins such as MCL-1, whose translation is cap dependent, synergizing with the BCL-2 inhibitor Ven. Ven-PegC treatment decreased cellular MCL-1 protein levels in vitro by enhancing eIF4E-4EBP1 interaction on the cap-binding complex via glutamine depletion. In vivo, Ven-PegC treatment completely depleted plasma glutamine and asparagine and inhibited mRNA translation and cellular protein synthesis. Since this novel mechanistically-rationalized regimen combines two drugs already in use in acute leukemia treatment, we plan a clinical trial of the Ven-PegC combination in relapsed/refractory CK-AML.

Asparaginase Erwinia chrysanthemi effectively depletes plasma glutamine in adult patients with relapsed/refractory acute myeloid leukemia.

Depletion of glutamine (Gln) has emerged as a potential therapeutic approach in the treatment of acute myeloid leukemia (AML), as neoplastic cells require Gln for synthesis of cellular components essential for survival. Asparaginases deplete Gln, and asparaginase derived from Erwinia chrysanthemi (Erwinaze) appears to have the greatest glutaminase activity of the available asparaginases. In this Phase I study, we sought to determine the dose of Erwinaze that safely and effectively depletes plasma Gln levels to ≤ 120 µmol/L in patients with relapsed or refractory (R/R) AML. Five patients were enrolled before the study was halted due to issues with Erwinaze manufacturing supply. All patients received Erwinaze at a dose of 25,000 IU/m2 intravenously three times weekly for 2 weeks. Median trough plasma Gln level at 48 h after initial Erwinaze administration was 27.6 µmol/L, and 80% (lower limit of 1-sided 95% CI 34%) of patients achieved at least one undetectable plasma Gln value (< 12.5 µmol/L), with the fold reduction (FR) in Gln level at 3 days, relative to baseline, being 0.16 (p < 0.001 for rejecting FR = 1). No dose-limiting toxicities were identified. Two patients responded, one achieved partial remission and one achieved hematologic improvement after six doses of Erwinaze monotherapy. These data suggest asparaginase-induced Gln depletion may have an important role in the management of patients with AML, and support more pharmacologic and clinical studies on the mechanistically designed asparaginase combinations in AML.