Abstract 2115: Glutamine blockade via prodrug JHU083 reprograms immunosuppressive tumor associated macrophages (TAMs) and drives tumor immunity in urologic cancers
Authors: Praharaj Monali , Shen Fan , Zhao Liang , Nirschl Thomas R. , Sena Laura , Singh Alok K. , Theodros Debebe , Wang Xiaoxu , Williams Raekwon A. , Thompson Elizabeth , Tam Ada , Yegnasubramanian Srinivasan , Leone Robert D. , Al Jesse , Rais Rana , Slusher Barbara S. , Pardoll Drew M. , Powell Jonathan D. , Zarif Jelani C. 
Published: June 2022

Introduction: Metastatic prostate cancer is currently incurable and kills more than 30,000 men in the United States each year, making it the second leading cancer related cause of death and an area of unmet need. Previously, we reported increased infiltration of immunosuppressive CD206-positive tumor associated macrophages (TAMs) with disease progression in prostate adenocarcinoma.[1] Glutamine metabolism has been implicated in immunosuppressive TAMs as well as metastatic castration resistant prostate cancer (mCRPC)[2] and radioresistant urothelial carcinoma.[3] As such, we hypothesize that rational metabolic interventions could be a promising strategy to simultaneously target both TAMs and cancer cells, resulting in anti-tumor immunity.

Methods: We utilized a novel pro-drug (JHU083) derived from 6-Diazo-5-oxo-L-norleucine (DON) which has significantly lowered toxicity to treat two urological syngeneic immunogenic mouse tumor models in vivo; B6CaP (prostate cancer) and MB49 (bladder cancer). We studied the direct effect on tumor cells, as well as the required immune compartment for drug efficacy in vivo using antibody targeted depletions or adoptively transferred TAMs. Moreover, we used RNA-sequencing and multi-parameter flow cytometry to characterize the effect of JHU083 on TAMs transcriptional programming and proteomic expression profiles in vivo. Lastly, we assessed the phagocytotic capacity of macrophages after treatment with JHU083 with flow cytometry and immunofluorescence (IF) microscopy.

Results: JHU083 treatment showed significant tumor regression in both urologic cancer models. Using in vivo depletion of CD4 or CD8 T cells, or adoptively transferring previously in-vivo JHU083 treated TAMs we established a direct anti-tumor role of TAMs. These TAMs were more inflammatory as shown by the increased TNF-positivity. Strikingly, in the TME we also observed an increase of Glut1 and Hexokinase II indicating metabolic reprogramming of these TAMs towards glycolytic phenotype. Importantly, JHU083-treated TAMs showed significantly increased phagocytic activity of cancer cells, providing direct evidence of functional reprogramming.

Conclusion: We found that JHU083 has two distinct functions in vivo; first, it directly impairs cancer cells which are glutamine dependent and second, it reprograms TAMs from an immunosuppressive to an inflammatory state via antagonism of glutamine metabolism in vivo. These macrophages convert to a highly glycolytic state, have increased TNF production, and have improved phagocytic activity against tumor cells. As urologic cancers are heavily infiltrated with immunosuppressive TAMs, JHU083 is an excellent preclinical candidate for these diseases. It remains to be seen if JHU083 treatment can be effectively combined with checkpoint therapy to elicit durable anti-tumor immunity.

Citation Format: Monali Praharaj, Fan Shen, Liang Zhao, Thomas R. Nirschl, Laura Sena, Alok K. Singh, Debebe Theodros, Xiaoxu Wang, Raekwon A. Williams, Elizabeth Thompson, Ada Tam, Srinivasan Yegnasubramanian, Robert D. Leone, Jesse Al, Rana Rais, Barbara S. Slusher, Drew M. Pardoll, Jonathan D. Powell, Jelani C. Zarif. Glutamine blockade via prodrug JHU083 reprograms immunosuppressive tumor associated macrophages (TAMs) and drives tumor immunity in urologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2115.