Info
Background:
Aggressive cancers, including triple-negative breast cancer (TNBC) and pancreatic ductal adenocarcinoma (PDAC) show resistance to immune checkpoint inhibitor (ICI) therapy. Recent evidence shows that these tumors display accelerated glutamine metabolism, which can be inhibited as an anti-tumor target. Sirpiglenastat (DRP-104), a prodrug of 6-diazo-5-oxo-L-norleucine (DON), is a potent glutamine antagonist currently under clinical investigation. In this study, we evaluated JHU083, a preclinical analog of DRP-104, in immunocompetent murine TNBC and PDAC models. Previous studies showed that TNBC and PDAC are characterized by abundant myeloid-derived suppressor cells (MDSCs), and treatment with glutamine antagonist DON/JHU083 decreases blood and tumor MDSC and improves the tumor immune microenvironment in murine TNBC. Here we test the hypothesis that in both PDAC and TNBC tumors, glutamine antagonism significantly inhibits tumor growth accompanied by immune activation detected by immunoPET using [Zr-89] labeled anti-CD69 antibody (Cancer Immunol Res. 2022;10:1084). We corroborated PET findings with FACS-based immune profiling of dissociated tumors and an array of experiments to elucidate the underlying mechanism.
Hypothesis/Objective:
We hypothesized that glutamine antagonism with JHU083 inhibits tumor growth by remodeling the immunosuppressive tumor microenvironment and enhancing T-cell activation, and that these changes can be noninvasively detected using [Zr-89]CD69 immunoPET.
Methods:
Orthotopic TNBC models (4T1, EO771 or MMTV-PyMT) were established in the mammary fat pad and PDAC models (clone 2838c3 and 6419c5, both derived from KPC tumor) subcutaneously in immunocompetent mice. For in vivo studies, TNBC models were randomized into four groups: control (untreated), JHU083, ICI (anti–PD-1 and anti–CTLA-4 mAb), and combination (ICI plus JHU083) while PDAC models into control (untreated) and JHU083 groups. Tumor growth was monitored for 2-3 weeks on treatment. After one week of treatment, a cohort of mice in each model underwent CD69 PET at 48 hours post-injection of [Zr-89] labeled DFO-H1.2F3 (full length anti-murine CD69 mAb). The tumors were harvested for immune profiling by FACS. To further elucidate mechanism, we measured oxidative stress in cancer cells, MDSCs and T cells and changes in T cell activation markers in the co-culture with MDSCs, performed T cell depletion studies and profiled cytokines in tumor interstitial fluid.
Results:
Glutamine antagonism by JHU083 induced a remarkable inhibition of tumor growth in both PDAC and TNBC models accompanied by increased tumor Zr-89 PET activity (Figure 1). The increased PET signals were associated with increased fraction of CD69⁺ cells and CD8⁺ T cells but reduced MDSCs in the tumor by FACS (SI-Figure 1). Exposing cultured cells to DON increased intracellular ROS production and lipid peroxidation in PDAC, TNBC cells and MDSCs, but not in T cells; co-culture of MDSCs with T cells significantly suppressed the number of CD69⁺ cells and granzyme B⁺ CD8⁺ T cells and such suppression was reversed by adding DON in culture media (SI-Figure 2). Cytokine profiling suggests JHU083 treatment altered TME by depleting MDSC-promoting cytokines (G-CSF and GM-CSF) while increasing IFNγ⁺ CD8⁺ T cells in 4T1 tumors; the observation that T-cell depletion in 4T1-bearing mice aborted JHU083-mediated tumor growth inhibition (SI-Figure 3) is consistent with T-cell dependent effect through interaction with MDSCs.
Conclusion:
Our data suggest that glutamine antagonism spares T cells but selectively targets the metabolic vulnerability of cancer cells and immunosuppressive myeloid cells hence lifting their suppression on T cells. Such divergent effect of JHU083 leads to improved tumor immune microenvironment that underlines the tumor growth inhibition in both TNBC and PDAC models. Importantly, [Zr-89]CD69 PET provides a noninvasive imaging biomarker of intratumoral immune activation associated with glutamine antagonism.
Acknowledgement
This study was supported by NIH/NCI R01CA266285, R01CA301356 and a grant from Ludwig Institute for Cancer Research.
Image/Figure:
Click to view full size
Click to view full size
Image/Figure Caption:
Figure 1. In vivo immunoPET using [Zr-89] labeled anti-CD69 monoclonal antibody and tumor growth delay in pancreatic ductal carcinoma (PDAC) murine models and in a triple negative breast cancer (TNBC) murine model. Tumor growth, CD69 PET image of control and treated mice at 48h post tracer injection, and PET ROI quantification of [Zr-89]CD69 in mean % ID with control, JHU083 in 2838c3 PDAC (A-C) and 6419c5 PDAC (D-F) and with control, ICI, JHU083, ICI plus JHU083 in 4T1 TNBC (G-I). [Zr-89]-DFO-H1.2F3 (full length monoclonal antibody anti-murine CD69) was used. Both 2838c3 and 6419c5 clones were derived from KPC tumor.
Author
Hoon Choi
University of Pennsylvania