Eckard, Jonathan

Abstract Introduction: D/L-alpha-metyrosine (SM-88, racemetyrosine) is a clinical stage chemically modified racemic amino acid therapy with broad anticancer activity in patients across 15 different indications. While amino acid metabolism has been leveraged in oncology imaging for decades, SM-88 offers a novel therapeutic approach to selectively disrupt cancer cells. This study explored phenotypic and mechanistic effects of exposing cancer cells to D/L-alpha-metyrosine as well as potential immunomodulation of the in vivo tumor microenvironment related to tyrosine hydroxylase inhibition and catecholamine depletion. Methods: In vitro experiments were conducted in a range of human and mouse cancer cell lines, including MCF-7, 4T1, HCT116, CAPAN2, PAN02, PANC01. The effect of SM-88 methyl-ester (SM-88 ME) alone on viability, apoptosis, migration/invasion, protein synthesis inhibition, autophagy, cell cycle arrest, and ROS generation was assessed. In vivo experiments included xenograph studies of HCT116 with monotherapy consisting of either IP injections up to 200 mg/kg/day of SM-88 ME, or orally administered SM-88 up to 324 mg/kg/day. Orthotopic xenographs of 4T1 and PAN02 models using monotherapy doses up to 200 mg/kg/day are ongoing. Ongoing immunohistochemistry and flow cytometry is focused on correlation with LAT1 expression and efficacy, alteration in macrophage polarization (M1/M2 ratio), Tregs, and various T cell populations in the tumor microenvironment. Results: Exposure to SM-88 ME exhibited a dose dependent increase in ROS, as well as decreases in caspase 3/7 activation. An increase in ROS following exposure to SM-88 ME was also observed in PAN02 cells. Similar decreases in caspase 3/7 activation were observed in CAPAN2, HCT116, and MCF7 cells. Interestingly, across a variety of cell lines including PAN02, PANC1, and 4T1 cell morphology changes suggested the formation of autophagic vacuoles. Viability across all cell lines was reduced in a dose and time dependent manner. In a mouse HCT116 SC xenograft model, mice receiving 324 mg/kg/day SM-88 ME showed significantly reduced tumor size compared to control mice. Additional in vitro and in vivo experiments are ongoing. Conclusions: The importance of amino acid metabolism in cancer has gained greater awareness over the past decade, however potential therapeutic approaches in this area remain limited. SM-88 has been dosed in over 180 cancer patients with encouraging efficacy and safety findings to date. Early data suggest increases in ROS generation and decreases in apoptosis may occur in certain cancer types (PAN02 pancreatic cancer), suggesting a shift away from apoptosis and towards autophagy. Potential immune modulatory effects of SM-88, including alterations in macrophage polarization related to tyrosine hydroxylase inhibition and subsequent reductions in catecholamine production are being examined. Citation Format: Alexander Vandell, Jonathan Eckard, Steve Hoffman, Giuseppe Del Priore, Martin Fernandez-Zapico. In vitro and in vivo anticancer effects of D/L-alpha-metyrosine (SM-88), a novel metabolism-based therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5998.

Abstract Introduction: TYME-18 is a combination of an ambiphilic steroid acid, tauroursodeoxycholic acid (TUDCA) and surfactants (N-methyl pyrrolidone, polidocanol, and linoleic acid) administered intratumorally to induce regression without injuring adjacent tissue. Methods: Balb/C mice were inoculated with CT-26 cancer (106 cells) until a tumor was established to approximately 100 mm3. An initial feasibility semi-quantitative assessment was performed with 10 animals (table 1). A larger confirmatory experiment testing the individual components of TYME-18 was also conducted (tables 2 & 3). Mice received intra-tumoral injections of TYME-18 with surfactants; or surfactants without TUDCA, or vehicle with measurements 3 times/wk. Intra-tumoral injections were administered 6 times at 3-day intervals over a total of 4 weeks. Animals were sacrificed when tumors reached 2000 mm3 or at the end of the study (16 weeks). Table 1.HISTOPATHOLOGIC FINDINGSFinal tumor sizes & intratumoral necrosis. TYME-18 vs Control. CT-26 model. (Fisher's Exact Test p=0.001)GroupAnimal IDTumor SizeIntratumoral NecrosisControlG1A-1>1 cmModerateG1A-2>1 cmMildG1B-2>1 cmMildG1B-3>1 cmModerateG1B-4>1 cmModerate-TreatedG2A-1<1 – 5 mmSevereG2A-3<1 – 5 mmSevereG2B-1<1 – 5 mmSevereG2B-3<1 – 5 mmSevereG2B-55 – 6 mmSevere- Table 2.Days Post Implementation average tumor volume mm3 (ANOVA P<0.0001)0369121520Vehicle83.1157.9263.4401.4613.9849.41368.9Surfactant Alone76.4131.1166.3209.6274.3345.7642.8TYME-1880.528.228.934.235.854.457.2- Table 3.Tumor Free Mice at Study End: Day 112 (Fisher's Exact Test p < 0.0001)Treatment# of Mice# Tumor Free Mice% Tumor Free MiceVehicle2000Surfactant Alone1218.33TYME-18121191.6 Results: TYME-18 demonstrated a significant difference between treated and untreated animals (see Tables 1, 2, 3). A minor reduction in tumor cell mass was observed in response to the surfactant, with the greatest response (ANOVA p=.001) observed in the TYME-18 (surfactant + TUDCA) (table 2). Discussion: The local administration of an ambiphilic steroid acid along with surfactants, impeded the ability of tumors to increase in size. Both the surfactants and the ambiphilc steroid acid contributed to this effect with an apparent synergistic effect. Aside from the physical chemical acid effect on tissue, ambiphilic steroid acids are now recognized as important regulators of energy metabolism. Their effects on FXR, LXR, PPAR-α, PPAR-γ are known to regulate important changes in energy metabolism including insulin utilization, the sensitivity of the insulin receptor, the transcription of the genes that mediate glucose and lipid metabolism as well as various immune effects; all of which could be useful in the treatment of cancer. Currently, TYME-18 is under investigation as a simple and nontoxic treatment for tumors for which systemic therapy is not indicated. Citation Format: John Rothman, Steve Hoffman, Allyson Ocean, Jonathan Eckard, Giuseppe Del Priore, Martin Fernandez-Zapico. In vivo mouse model data demonstrating reduction in tumor cell proliferation following intratumoral administration of TYME 18 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2764.

Abstract Background: A wide range of cancers, including pancreatic cancer, display altered expression of amino acid transporter LAT-1. LAT-1 overexpression has been shown to be an important prognostic factor and predicts chemo resistance in pancreatic cancer (Kaira K, 2012; Altan B, 2018). Radiolabeled amino acids for PET imaging, including 3-18F-l-α-methyl-tyrosine ([18F] FAMT), have successfully leveraged cancer’s aberrant LAT1 expression and amino acid uptake for decades. SM-88 is a denatured D/L racemic form of α-methyl-tyrosine designed for selective uptake and disruption of protein synthesis in cancer cells, as well as disruption of catecholamine production via inhibition of tyrosine hydroxylase. Catecholamines have been reported to potentially have a role in driving pancreatic cancer progression and changes in the tumor immune status (Renz B, Cancer Cell 2018; Calvani M, Oncotarget 2014). Initial clinical and preclinical results with SM-88 indicate that this agent could pose a novel approach in the treatment of pancreatic cancer. Methods and Results: Initial xenograft models of human gastrointestinal cancers using monotherapy SM-88 showed a >50% decrease in tumor growth at three weeks of treatment (p<0.05). Ongoing preclinical experiments are testing the full pharmacodynamic effects of SM-88 on pancreatic cancer cells, as well as potential effects on the tumor microenvironment due to catecholamine interruption. In results from the ongoing phase 2 trial of SM-88 in heavily pretreated metastatic pancreatic cancer patients, SM-88 has shown monotherapy antitumor activity based on RECIST 1.1 criteria. Additionally, 16/24 patients demonstrated a reduction in circulating tumor cells, with the median nadir decline of 63%. LAT1 has been reported to be important in EMT transition and mesenchymal cell survival (Holldorsson S, Cancer letters 2017); examination of the impact of SM-88 on this important emerging biomarker is ongoing. Conclusion: Altered amino acid (LAT1) metabolism has been shown to be a negative prognosticator in pancreatic cancer outcomes and progression. Targeting LAT1 and amino acid pathways has been identified as a therapeutic approach in multiple cancers (Fuchs BC, Semin Ca Biol 2005). Initial and ongoing studies of SM-88 may clarify the potential utility of this approach in the treatment of pancreatic cancer. Citation Format: Martin Fernandez-Zapico, Dae Won Kim, Philip Philip, Alexander Vandell, Jonathan Eckard, Ron Korn, Giuseppe Del Priore, Diane Simeone. Therapeutic potential of targeting amino acid metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B15.