Hirdler, Jacob

Abstract Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)

Abstract Although immune checkpoint blockade that targets the PD-L1 has revolutionized the treatment of human cancers, only a small proportion of patients positively respond to this form of treatment. A limitation of these therapies is that they only target and inhibit PD-L1’s extracellular function, which is to bind its receptor on PD-1 on T cells and inhibit T cell cytotoxicity. Recent discoveries have shown that PD-L1 expressed on tumor cells can signal intrinsically and independently of PD-1 ligation to promote tumor cell survival. Besides tumor cells, host myeloid cells have been shown to have high expression of PD-L1, therefore contributing to the immunosuppressive nature of the tumor. However, PD-L1 intrinsic signaling in human myeloid cells has not been studied. Therefore, the purpose of our work is to determine the intrinsic functions of PD-L1 in myeloid cells and how this impacts anti-tumor immunity. To study this, we used a novel PD-L1 antibody (H1A) that does not block PD-L1’s binding with PD-1, but directs PD-L1 for degradation, thereby decreasing total PD-L1 expression. Additionally, we developed a chimeric mouse model in which the murine PD-1/PD-L1 has been replaced with the human version. This allows us to evaluate PD-L1’s intrinsic function in vivo. In our preliminary studies, the degradation of PD-L1 in human monocytes led to increased chemokine release as well as increased conversion of effector T cells. Additionally, H1A treatment of tumor-bearing mice led to lower tumor burden in a CD8 T cell dependent manner. Based on these results, PD-L1 intrinsic signaling has the potential to regulate the immune-stimulatory response of monocytes, thereby limiting effector T cell responses against cancer. Supported by grants from NIH (R0192389) and Mayo Clinic graduate school of biomedical sciences

Abstract Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection. R01CA256927

Abstract The immune response to anti-PD-1 therapy in cancer patients is not completely defined due to lack of reliable biomarkers that can determine T cell function. Bim, a member of the Bcl-2 family, was recently identified as a downstream signaling molecule in the PD-1 pathway. Within CD8 T cells, Bim appears to have a dual function, and can be pro-apoptotic or promote effector activity. Our clinical data has shown that high levels of Bim in CD8 T cells are predictive of response to anti-PD-1 therapy in metastatic melanoma whereas in colorectal cancer, a low level of Bim is associated with treatment response. Thus, we sought to determine what molecular partners might influence the role of Bim. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2), an inner membrane protein of mitochondria, as a protein that interacts with Bim. Accordingly, we found CD8 T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratios, and a low ANT2/Bim ratio was present in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival function of ANT2, our results indicate that ANT2 may balance the pro-apoptotic and effector roles of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interactions in CD8 T cells may provide a novel marker to predict clinical response to anti-PD-1 therapy.

Abstract NRG-1 signals through epidermal growth factor receptor (EGFR) family members. Despite promising preclinical data, HER2 blockade in prostate cancer has failed to improve outcomes in clinical trials.1-4 We have discovered a novel complex systemic role of NRG-1 that drives sensitivity and resistance to androgen deprivation therapy (ADT) in castration-sensitive (CSPC) and castration resistant prostate cancer (CRPC), respectively. Significantly, sensitivity to ADT can be restored in CRPC by manipulating NRG-1 signaling. Our data posit NRG-1 as a systemic “switch” in prostate cancer. Plasma NRG-1 is elevated in our cohort of patients with metastatic CRPC (mean 4.9ng/ml) versus healthy controls (mean 3.2ng/ml, p<0.0001) or metastatic CSPC (mean 3.4ng/ml, p<0.0001). Paradoxically, high systemic NRG-1 predicted superior OS and PFS in CSPC but inferior clinical outcomes in CRPC. Consistently, opposite cell survival between LNCaP (CSPC) vs Du145 & PC3 (CRPC) was observed with recombinant human NRG-1 and enzalutamide (ENZ). ENZ combined with increasing NRG-1 concentration induced LNCaP cell death in an additive manner, but NRG-1 rescued Du145 & PC3 from ENZ-induced death in a dose-dependent manner. We next hypothesized that differential NRG-1 downstream signaling is mediated by engagement with distinct EGFR receptor dimers. While HER2 expression is similar, LNCaP express higher HER3 than Du145 & PC3. High ratio of HER2 to HER3 in Du145 & PC3 mirrors clinical reports of HER2 upregulation in CRPC5,6 and also predicts patient survival in the Stand Up to Cancer cohort.7 Significantly, we recapitulated the CRPC phenotype in LNCaP by inducing HER2 overexpression, HER3 suppression, and chemically-mediated HER2/HER3 dimerization. Conversely, HER2 knockdown sensitized Du145 cells to ENZ. Next, we queried composite single-cell whole human8 and CRPC9 transcriptomes for NRG-1 expression and discovered that the source of NRG-1 is the patients’ own mesenchymal stem cells (MSCs). Bone-derived MSC outgrowth cells produced copious NRG-1, which was increased substantially by coincubation with prostate cancer cell supernatants. Importantly, we were able to manipulate MSCs to reduce their NRG-1 production as a possible clinical translation. Similarly, patients treated with these compounds showed significant reduction in plasma NRG-1 levels after treatment. Additional clinical studies are pending. Together, our observations confirm that NRG-1 has differential signaling: HER3 homodimerization leads to prostate cancer cell death, whereas heterodimerization of HER3 with HER2 leads to prostate cancer cell survival via ADT resistance in a plasma NRG-1 dependent manner. Consequently, HER2 and HER3 receptor availability determine the molecular switch of NRG-1 in prostate cancer. Therefore, NRG-1 can be both a prognostic marker and therapeutic target in CRPC, and multiple clinical trials are pending. Citation Format: Jacob Orme, Roman Thaler, Dejie Wang, Siven Chinniah, Jacob Hirdler, Fernando Quevedo, Lance Pagliaro, Kwon Eugene, Alan Bryce, Brian Costello, Haojie Huang, Sean Park. Mesenchymal stem cell-derived systemic NRG-1 mediates a targetable molecular switch in metastatic prostate cancer [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 397.

Abstract Cytotoxic T lymphocytes (CTL's) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. However, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, we sought to identify key factors associated with failure of PD-1 blockade in order to develop strategies that could significantly improve response rates. We performed single-cell RNA-seq analysis of peripheral CD8+ T cells from responding and non-responding patients and found that the non-responders had less NKG7 expression. Functional assays revealed that knock-down of NKG7 compromised cytolytic granule localization and trafficking within CTL's. Since decreasing NKG7 reduced the cytolytic efficiency of CD8+ T cells, we next hypothesized that by increasing expression of NKG7 we could promote better T cell killing of tumor and enhance the effects of immune checkpoint inhibitors. To test this, we utilized peripheral blood samples from melanoma patients who failed to respond to anti-PD-1 treatment and transfected the PBMC's with NKG7 mRNA. The addition of NKG7 mRNA significantly increased T cell killing of melanoma tumor cells in an in vitro cytotoxicity assay, and this effect was even more robust in combination with anti-PD-1 or anti-PD-L1 pre-treatment. To confirm that over-expression of NKG7 could improve CD8+ T cell killing in an antigen-specific manner, we transfected OT-1 transgenic T cells with murine sequence of NKG7 mRNA or control mRNA. OT-1 CD8+ T cells with increased NKG7 expression exhibited enhanced cytolytic ability when challenged with either EL4 cells pulsed with OVA peptide or B16-OVA tumor cells in vitro. In addition, NKG7 mRNA-transfected OT-1 CD8+ T cells suppressed the aggressive growth of B16-OVA cells in vivo significantly better than control OT-1 CD8+ T cells. These results indicate that deficits in a T cell-intrinsic cytolytic factor can lead to failure of PD-1/L1 blockade and establish NKG7 as a new therapeutic target for enhancing response to cancer immunotherapy. Citation Format: Whitney J. Barham, Ti Wen, Ying Li, Henan Zhang, Cindy Liu, Jacob Hirdler, Joanina Gicobi, Fabrice Lucien-Matteoni, Yiyi Yan, Haidong Dong. NKG7 mRNA therapy increases the anti-tumor cytotoxicity of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 504.