Abstract Glutamine metabolism in tumor microenvironments critically regulates antitumor immunity. Using the glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes. We show JHU083-mediated glutamine antagonism in tumor microenvironments induced by TNF, proinflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibited increased tumor cell phagocytosis and diminished proangiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken tricarboxylic acid (TCA) cycle, and purine metabolism disruption. Although the antitumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell–like phenotype in CD8+ T cells and decreased the abundance of regulatory T cells. Finally, JHU083 caused a global shutdown in glutamine-utilizing metabolic pathways in tumor cells, leading to reduced HIF-1α, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key antitumor features. Altogether, our findings demonstrate that targeting glutamine with JHU083 led to suppressed tumor growth as well as reprogramming of immunosuppressive TAMs within prostate and bladder tumors that promoted antitumor immune responses. JHU083 can offer an effective therapeutic benefit for tumor types that are enriched in immunosuppressive TAMs.

JHU083-induced glutamine antagonism affects tumor cell metabolism and induces cell death in urologic tumors. A, Log-fold changes of glutamine utilizing enzymes after JHU083 treatment vs. control tumors in CD45− sorted cells from B6CaP tumors, followed by scRNA-seq. B, Western blot showing qualitative changes in the levels of glutamine synthesizing/utilizing enzymes and transporters in the CD45− fraction of MB49 tumors. C, Percentage of GLUT1+ CD45− live cells determined by flow cytometry in B6CaP tumors (n = 7/group). D, Targeted metabolomic analysis of B6CaP tumors by LC-MS/MS (n = 3/group). E, Volcano plot showing key metabolite levels of JHU083-treated vs. nontreated control tumors based on the metabolomic analysis shown in D. F, Absolute quantification of metabolites by LC/MS-MS (n = 3 or 5/group). G and H, Western blot images showing qualitative changes in c-MYC, phospho-c-MYC, and HIF-1ɑ in MB49 tumors following JHU083 treatment, and (H and I) MTT assay in DON-treated MB49 cells and immunoblot of cleaved caspase 3 quantification in CD45− fraction MB49 tumors (J). Statistical analyses were performed using the unpaired t test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

S5: Outgrowth and CDN treatment of B16F10 is effected in selected knockout animals.

S3: B16F10 is not directly killed by CDN exposure and behaves similarly to other mouse and human cell lines.

S2: CDN injection causes a distinct cytokine and cellular profile in the tumor draining lymph nodes.

S1: Timeline of necrosis after CDN injection.

Antibodies used for flow cytometry (clones and dilutions)

Quantitative analysis of radiation-induced changes in peripheral blood subsets over time.

Cells were first gated on size and singularity by forward scatter and side scatter. Nonviable cells were excluded by live/dead gating. Live cells were gated on CD11c and CD11b, then on CD45 and F4/80 to identify macrophage and dendritic cell phenotypes. Finally myeloid cells were gated for expression of TIM-3. Final populations were (A) F4/80+CD45hiCD11c-CD11bhi (B) F4/80+CD45dimCD11bhi (C) F4/80+CD45hiCD11c+CD11b+ and (D) F4/80-CD45dimCD11c+CD11b-.

S1: Timeline of necrosis after CDN injection. S2: CDN injection causes a distinct cytokine and cellular profile in the tumor draining lymph nodes. S3: B16F10 is not directly killed by CDN exposure and behaves similarly to other mouse and human cell lines. S4: TNFα blockade reduces necrosis after IT CDN. S5: Outgrowth and CDN treatment of B16F10 is effected in selected knockout animals.

S4: TNFα blockade reduces necrosis after IT CDN.

Supplementary Materials and Methods

Tumor metabolism is emerging as a regulator of immune mediated anti-tumor responses. Previously, we reported increased immunosuppressive tumor-associated macrophages (TAMs) infiltration with disease progression in prostate adenocarcinoma (PCa).1 Glutamine metabolism has been implicated in metastatic castration resistant prostate cancer (mCRPC)2. To harness the potent anti-tumor effects of 6-Diazo-5-oxo-L-norleucine (DON), which targets glutamine utilizing enzymes and to mitigate known significant toxicities, we here use a novel pro-drug moiety, i.e., JHU083. We hypothesize that JHU083 will enhance anti-tumor immunity by simultaneously targeting TAMs and cancer cells. Using scRNA-seq dataset from mCRPC patients, we investigated the importance of glutamine metabolism in TAMs in the tumors.3 Enriched expression of glutamine utilizing enzymes was observed in TAMs in the metastasized tumor tissue relative to benign. We used JHU083 to treat two urological syngeneic immunogenic mouse tumor models in vivo; B6CaP (PCa) and MB49 (bladder cancer). JHU083 showed significant tumor regression in both models. Using in vivo depletion of CD4 or CD8 T cells or adoptively transferring in-vivo JHU083 treated TAMs in TME, we established a direct anti-tumor role of TAMs. Moreover, to characterize the effect of JHU083 on TAMs transcriptional, translational signatures and metabolite flux in vivo, we used scRNA-sequencing and bulk RNA sequencing at two different time points, multi-parameter flow cytometry and targeted LC-MS/MS metabolic profiling. Clustering of the transcriptional signatures at both time points using UMAP dimensionality reduction classified 6 discreet differentially expressed clusters of TAMs. It elutes to an overall induction of TNF signaling and increased proliferation in the TAM sub-clusters. Strikingly, this was observed translationally as well. Moreover, translationally in the TME at a later timepoint, an increase of glycolytic transporter and enzyme, i.e., Glut1 and Hexokinase II was also observed. The overall impact of this on the metabolome, specifically glycolytic pathway changes in the sorted TAMs, remains to be elucidated. Lastly, we assessed in-vivo the functional phagocytosis of TAMs with flow cytometry and IF microscopy. JHU083-treated TAMs showed significantly increased phagocytic activity, providing direct evidence of functional reprogramming. Conclusions: We found that JHU083 reprograms TAMs from an immunosuppressive to an inflammatory state which we show has a direct anti-tumor effect. These macrophages convert to a highly proliferative and glycolytic state, have increased TNF production which might be resulting in improved phagocytic activity against tumor cells. As urologic cancers are heavily infiltrated with immunosuppressive TAMs, JHU083 is an excellent preclinical candidate. Citation Format: Monali Praharaj, Fan Shen, Alex J. Lee, Liang Zhao, Thomas R. Nirschl, Xiaoxu Wang, Debebe Theodros, Alok K. Singh, Raekwon A. Williams, Laura A. Sena, Elizabeth A. Thompson, Ada Tam, Srinivasan Yegnasubramanian, Edward J. Pearce, Robert D. Leone, Jesse Alt, Rana Rais, Barbara S. Slusher, Drew M. Pardoll, Jonathan D. Powell, Jelani C. Zarif. Glutamine antagonist prodrug JHU083 reprograms immunosuppressive tumor-associated macrophages to drive tumor immunity in urologic cancers [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A079.

Each mouse was individually imaged with cone beam computed tomography (CBCT) using the SARRP with a 65 kVp and 0.7 mA beam. Using the treatment planning system from the SARRP system (Muriplan) and the CBCT, the target was exactly placed 3mm below the skull's burr hole. The planning system calculated the x-ray beam's (220kVp and 13mA) time of exposure according to the prescribed dose and moved the motorized couch to its target location, after which a 3-mm beam centered on the burr hole and underlying tumor was used to administer a total of 10 Gy radiation per animal at a rate of 1.9 Gy/min (18). The isodose distribution is shown on the figure provided. The dose to adjacent organs or rest of the brain is insignificant as the beam's penumbra demonstrates complete drop off on the edge of the field (17, 50, 51)

Supplementary Figure 1: Radiotherapy suppresses tumor growth. Supplementary Figure 2: Characterization of the TME of B16/F10 tumors post-RT. Supplementary Figure 3: Stereotactic radiation does not change Treg in draining lymph nodes nor in spleens of treated mice. Supplementary Figure 4: Persistently increased Treg Post-RT. Supplementary Figure 5: Stereotactic radiation increases the suppressive markers of Treg in the RENCA tumor model. Supplementary Figure 6: Stereotactic radiation increases the suppressive markers of Treg in the MC38 tumor model. Supplementary Figure 7: Stereotactic radiation does not change the expression of the suppressive markers of Treg in DLNs. Supplementary Figure 8: Stereotactic radiation does not change the expression of the suppressive markers of Treg in spleens. Supplementary Figure 9: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the B16/F10 model. Supplementary Figure 10: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the RENCA model. Supplementary Figure 11: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the MC38 model. Supplementary Figure 12: Expression of 4-1BB on TIL-CD8+ cells. Supplementary Figure 13: The effect of TGF-beta blockade on different T cell subsets. Supplementary Figure 14: TGF-beta expression in the tumor microenvironment. Supplementary Figure 15: The effect of Fingolimod (FTY720) on peripheral blood lymphocytes counts. Supplementary Figure 16: Chemokine/cytokine expression of a selected panel in the tumor microenvironment post-radiation.

Gating strategy to assess for surface expression of TIM-3 on (A) CD4+ and (B) CD8+ T cells isolated from peripheral lymph nodes, lungs, livers, spleens, and brains.

Abstract 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.

Renal Cell Carcinoma (RCC) is one of the most commonly diagnosed cancers world wide with research efforts dramatically improving understanding of the biology of the disease. To investigate the role of the immune system in treatment naive clear cell Renal Cell Carcinoma (ccRCC), we interrogated the immune infiltrate in patient-matched ccRCC tumor samples, benign normal adjacent tissue (NAT) and peripheral blood mononuclear cells (PBMCs isolated from whole blood, focusing our attention on the myeloid cell infiltrate. Using flow cytometric, MS, and ExCYT analysis, we discovered unique myeloid populations in PBMCs across patient samples. Furthermore, normal adjacent tissues and ccRCC tissues contained numerous myeloid populations with a unique signature for both tissues. Enrichment of the immune cell (CD45(+)) fraction and subsequent gene expression analysis revealed a number of myeloid-related genes that were differentially expressed. These data provide evidence, for the first time, of an immunosuppressive and pro-tumorigenic role of myeloid cells in early, clinically localized ccRCC. The identification of a number of immune proteins for therapeutic targeting provides a rationale for investigation into the potential efficacy of earlier intervention with single-agent or combination immunotherapy for ccRCC.

Brain metastasis is common in patients with breast cancer, and those with triple negative status have an even higher risk. Stereotactic radiosurgery (SRS) is preferred to whole brain radiation therapy (WBRT) in most patients. However, triple negative status is currently not considered when determining optimal radiation therapy. Given the aggressive nature of triple negative breast cancer, we evaluated a role for WBRT for all patients in this cohort. We conducted a single-institution retrospective cohort study to determine whether triple negative patients with brain metastases have a higher burden of intracranial disease and whether type of initial radiation therapy affects overall survival for this cohort of patients. 85 patients met the inclusion criteria for this study. 25% of patients had triple negative breast cancer, of which 91% received SRS and 53% of patients received WBRT. The average number of new brain metastases from time of initial brain imaging to radiation therapy was 0.67 (St.Dev:1.1) in the non-triple negative status patients and 2.6 (St. Dev:3.7) in the triple negative status patients (p=0.001). Using a cox proportional hazards model, it was found that whole brain radiotherapy does not significantly affect overall survival in patients with triple negative breast cancer (p = 0.96). Our findings highlight the highly aggressive intracranial nature of triple negative breast cancer. Indeed, the rate of increase in brain metastases is significantly higher for triple negative patients compared to non-triple negative patients. As a result, we evaluated whether triple negative patients would benefit from whole brain radiation regardless of findings on initial brain imaging. Despite 53% of patients receiving WBRT, our investigation found that there is no additional benefit to WBRT in triple negative breast cancer patients. These results suggest a need to re-evaluate the role of WBRT in the management of triple negative breast cancer.

Abstract Tumor progression to metastasis is not cancer cell autonomous, but rather involves the interplay of multiple cell types within the tumor microenvironment. Here we identify asporin (ASPN) as a novel, secreted mesenchymal stromal cell (MSC) factor in the tumor microenvironment that regulates metastatic development. MSCs expressed high levels of ASPN, which decreased following lineage differentiation. ASPN loss impaired MSC self-renewal and promoted terminal cell differentiation. Mechanistically, secreted ASPN bound to BMP-4 and restricted BMP-4–induced MSC differentiation prior to lineage commitment. ASPN expression was distinctly conserved between MSC and cancer-associated fibroblasts (CAF). ASPN expression in the tumor microenvironment broadly impacted multiple cell types. Prostate tumor allografts in ASPN-null mice had a reduced number of tumor-associated MSCs, fewer cancer stem cells, decreased tumor vasculature, and an increased percentage of infiltrating CD8+ T cells. ASPN-null mice also demonstrated a significant reduction in lung metastases compared with wild-type mice. These data establish a role for ASPN as a critical MSC factor that extensively affects the tumor microenvironment and induces metastatic progression. Significance: These findings show that asporin regulates key properties of mesenchymal stromal cells, including self-renewal and multipotency, and asporin expression by reactive stromal cells alters the tumor microenvironment and promotes metastatic progression.