Olaparib is the most widely studied third-generation PARP inhibitor (PARPi) in clinical practice with a significant clinical outcome in BRCA deficient tumors such as breast and ovarian cancers. However, innate and adaptive resistance in patients with DNA damage repair (DDR) gene mutations were reported after treatment with PARPi, highlighting the potential implication of other compartments such as the role of immune cells in the mechanism of resistance. Importantly, PARPi is shown to have an immune-modulatory capacity within the tumor microenvironment (TME) by activating the STING pathway and increasing interferon gamma (INF-gamma) and chemokine secretion and hence increasing the recruitment and function of CD8 T cells. In addition, PARPi is shown to affect other cells within the TME such as, macrophages, myeloid-derived suppressor cells (MDSCs) and cancer associated fibroblasts (CAFs), promoting either an immune enhancement or immune suppressor milieu that could indirectly affect CD8 T cell function. However, little is known about the direct effect of PARPi on CD8 T cells. In this study using two cold tumor models, we show that PARPi controls the tumor growth and increases survival in tumor-bearing mice. This effect is due to the enhancement of number and cytotoxicity of antigen-specific CD8 T cells and the maintenance of memory population within the TME. In vitro study shows that PARPi activates the SIRT-1/FOXO1 pathway, leading to the modification of gene expression related to memory and fatty acid oxidation (FAO) metabolism in CD8 T cells. As a result, a unique subpopulation of superior central memory cells with high recall responses and anti-tumor effects are generated after PARPi treatment. Interestingly this unique subpopulation was also generated after PARPi in human cells. Together, our findings highlight the direct effect of PARP inhibition on CD8 T cells and its ability to modulate and reprogram CD8 T cells within the TME, leading to a greater anti-tumor immune response. Citation Format: Wael Traboulsi, Subhadip Kundu, Pankaj Gaur, Zainab Ramlaoui, Dareen Sarhan, Nour Shobaki, Jacob Lee, Nazli Jafarzadeh, Simon T. Barry, Viia E. Valge-Archer, Vivek Verma, Seema Gupta, Samir N. Khleif. PARP inhibition reprograms CD8 T cells, enhancing their function and generation of prolonged memory, leading to greater anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB339.

TNFRSF25, also known as Death Receptor 3 (DR3), is a cell surface receptor of the tumor necrosis factor receptor superfamily (TNFRSF) which is expressed on T cells, innate lymphoid cells, and B cells. Upon binding with TL1A ligand, this co-stimulatory molecule induces T cell activation, leading either to inflammation or cell death. Mouse PTX-35 (mPTX-35) is a TNFRSF25 functional agonist derived from a CDR that was humanized and affinity matured, then fused to a mouse IgG1-Fc backbone. Since costimulatory agonist antibodies have shown limited efficacy in clinical trials, our aims were to (i) determine the anti-tumor efficacy of mPTX-35, (ii) elucidate any mechanisms of resistance, and (iii) identify immune conditions that provide effective anti-tumor immune responses to mPTX-35 treatment. We studied the anti-tumor immune effects of mPTX-35 in both cold and hot tumor models. In a TC-1 cold tumor, we found that treatment with an anti-TNFRSF25 agonist antibody does not affect tumor growth or survival. In contrast, vaccination with an E7 antigen-specific vaccine leads to an environment that reverses primary resistance to the agonist antibody, inhibiting the tumor growth, and enhancing overall survival. Interestingly, we found that similar to other co-stimulatory agonist molecules, the immune effects of the anti-TNFRSF25 agonist are dose-dependent with lower (0.2 mg/kg) and higher (5 mg/kg) doses of mPTX-35 showing a reduced anti-tumor response than a dose of 1 mg/kg, resulting in a bell-shaped response in all tumor models. Similar immune-mediated anti-tumor effects of mPTX-35 were found in a CT26 (hot) tumor model. In conclusion, these results show that anti-TNFRSF25 agonist antibody demonstrates high anti-tumor efficacy in tumors that are infiltrated with T cells while the absence of properly antigen activated CD8 T cells (such as in cold tumors) incurs primary resistance to TNFRSF25 agonist that is reversed by proper priming of CD8 T cells. These results highlight the importance of optimal priming to limit primary resistance mechanisms and drive an efficacious immune response via the TNSFR25 axis. Citation Format: Dareen Sarhan, Lara Rabah, Vikas Tahiliani, Anh M. Trinh, Rahul R. Jasuja, Eric P. Dixon, Matthew Seavey, Vivek Verma, Seema Gupta, Samir N. Khleif. Proper priming of T cells is crucial for the anti-tumor effect of mPTX-35, an anti-TNFRSF25 agonist antibody [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 2881.

Growing evidence suggests that hypoxia is one of the most important phenomena of solid tumors that supports tumor progression as well as therapeutic resistance. When T cells infiltrate the tumor microenvironment (TME), they encounter hypoxia and, in this condition, cells upregulate an evolutionary conserved Hypoxia-inducible factor 1 (HIF-1) transcription factor to adapt to the hypoxic stress. Hypoxia has been reported to alter specific aspects of CD8+ T cell biology through various mechanisms but in large part by the HIF1-α, leading to immune suppression. Therefore, identification and development of drugs that target hypoxia is critical to augment immune cells' function and enhance the anti-tumor response of immunotherapy. Over the past decades, several cellular and pre-clinical studies have shown that ascorbic acid (AA) possesses anti-tumor properties and acts as an adjuvant to various cancer therapies. AA, as a proven redox potential modulator, has proved to also be effective in targeting oxygen sensing regulator (reducing Hif1-α levels) and epigenetic reprogramming (increasing ten-eleven translocation (TET) methylcytosine dioxygenase enzymatic activity) in cancer cells. Most recently, immunomodulating potential of AA has been recognized as it enhanced the efficacy of checkpoint blockade by increasing immune cell infiltration within the TME. However, the exact mechanisms by which AA exerts immunotherapeutic effects in cancer have not been fully understood. Here, we hypothesized that AA exerts its immunotherapeutic effects by targeting tumor-induced hypoxic stress in CD8 T cells. Indeed, we found that AA induces a robust anti-tumor response by decreasing tumor volume and increasing mice survival. Immune profiling in the TME revealed that AA increases the infiltration of CD8+ T cells with enhanced cytotoxic cytokine production and proliferation. To further evaluate the underlying mechanism, we challenged CD8+ T cells with a cognate antigen ex vivo under the hypoxic/normoxic conditions. We found that hypoxia negatively affected the effector functions and proliferation of CD8 T cells. Interestingly, AA could reverse these effects, bringing the function and proliferation of cells back to the normoxic level by increasing the expression of Granzyme B, Interferon gamma and Ki-67. This reversal of hypoxia-induced adverse effects on CD8 T cells was achieved by AA-mediated decrease in the Hif1-α levels (a hypoxia mediator) and increase in TET-2 protein expression and function (characterized by increased 5-hydroxymethylcytosine (5-hmc) levels). Moreover, AA treatment could significantly enhance the metabolic fitness of CD8+ T cells (characterized by improved glycolytic ability and mitochondrial function) that were exposed to severe hypoxia. In conclusion, our data indicate that AA treatment shows enhanced anti-tumor effects via restoring cytotoxic T cell functions under hypoxic TME. These results also provide new insights into the immunomodulatory effects of AA via targeting hypoxia in CD8 T cells. Given that hypoxia in the TME is a barrier for generation of effective immune response, our results suggest that AA treatment is an effective strategy against cancer and may further enhance the anti-tumor effects of immunotherapeutic agents. Citation Format: Nazli Jafarzadeh, Pankaj Gaur, Dareen Sarhan, Nour Shobaki, Wael Traboulsi, Zainab Ramlaoui, Vivek Verma, Seema Gupta, Samir N. Khleif. Ascorbic acid rescues antitumor immune response by reversing hypoxia-induced CD8+T cell dysfunction [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 LB217.