The cystine/glutamate antiporter xCT is a tumor-associated antigen that has been newly identified in many cancer types. By participating in glutathione biosynthesis, xCT protects cancer cells from oxidative stress conditions and ferroptosis, and contributes to metabolic reprogramming, thus promoting tumor progression and chemoresistance. Moreover, xCT is overexpressed in cancer stem cells. These features render xCT a promising target for cancer therapy, as has been widely reported in the literature and in our work on its immunotargeting. Interestingly, studies on the TP53 gene have revealed that both wild-type and mutant p53 induce the post-transcriptional down modulation of xCT, contributing to ferroptosis. Moreover, APR-246, a small molecule drug that can restore wild-type p53 function in cancer cells, has been described as an indirect modulator of xCT expression in tumors with mutant p53 accumulation, and is thus a promising drug to use in combination with xCT inhibition. This review summarizes the current knowledge of xCT and its regulation by p53, with a focus on the crosstalk of these two molecules in ferroptosis, and also considers some possible combinatorial strategies that can make use of APR-246 treatment in combination with anti-xCT immunotargeting.

Breast cancer is the most frequent cancer in women. Despite recent clinical advances, new therapeutic approaches are still required. The cystine-glutamate antiporter xCT, encoded by the SLC7A11 gene, which imports cystine in exchange with glutamate, is a potentially new target for breast cancer therapy, being involved in tumor cell redox balance and resistance to therapies. xCT expression is regulated by the oncosuppressor p53, which is mutated in many breast cancers. Indeed, mutant p53 (mut-p53) can induce xCT post-transcriptional down modulation, rendering mut-p53 tumors susceptible to oxidative damage. Interestingly, the drug APR-246, developed to restore the wild-type function of p53 in tumors harboring its mutation, alters the cell redox balance in a p53-independent way, possibly rendering the cells more sensitive to xCT inhibition. Here, we propose a combinatorial treatment based on xCT immunetargeting and APR-246 treatment as a strategy for tackling breast cancer. We demonstrate that combining the inhibition of xCT with the APR-246 drug significantly decreased breast cancer cell viability in vitro and induced apoptosis and affected cancer stem cells’ self-renewal compared to the single treatments. Moreover, the immunetargeting of xCT through DNA vaccination in combination with APR-246 treatment synergistically hinders tumor progression and prevents lung metastasis formation in vivo. These effects can be mediated by the production of anti-xCT antibodies that are able to induce the antibody dependent cellular cytotoxicity of tumor cells. Overall, we demonstrate that DNA vaccination against xCT can synergize with APR-246 treatment and enhance its therapeutic effect. Thus, APR-246 treatment in combination with xCT immunetargeting may open new perspectives in the management of breast cancer.

Background: Chimeric antigen receptor (CAR) T cells have become a well-established treatment option for patients, with six products approved for different hematologic diseases and new approvals allowing for their therapeutic use as early as second line. However, relapse rates of around 50% have been observed in all patient subsets, with one major mechanism associated with CAR T failure being cancer cell resistance to apoptosis. A form of cancer therapeutic named BH3-mimetics has been designed to inhibit members of the anti-apoptotic B cell lymphoma-2 (Bcl-2) family and, therefore, directly activate the apoptotic machinery in malignant cells. We hypothesized that integration of these anti-apoptotic molecules into CAR T cells would induce resistance towards the BH3 mimetics and allow combinational therapeutic approaches. Methods: 4-1BB and CD28 CAR constructs were designed to overexpress one of four anti-apoptotic proteins: wildtype Bcl-2, a Venetoclax-resistant Bcl-2 variant (G101V), B cell extra-large (Bcl-xL), or myeloid cell leukemia-1 (Mcl-1). CAR T cells made from these constructs were tested against leukemia (Nalm6) and lymphoma (JeKo-1) cell lines in combination with three different BH-3 mimetics: Venetoclax (ABT-199, an FDA-approved Bcl-2 inhibitor), Navitoclax (ABT-263, a Bcl-2/Bcl-xL inhibitor), and AZD5991 (an Mcl-1 inhibitor). Results: CAR T cells with a 4-1BB costimulatory domain tended to have increased killing over CARs with CD28 and were less susceptible to apoptosis; therefore, 4-1BB CARs were used for all further testing. Integration of Bcl-2 family proteins into CAR T cells didn’t impair or even increase tumor cell clearance in vitro; however, in combination with Venetoclax, Navitoclax, or AZD5991, killing capacity significantly increased compared to control CAR T cells. Even without combination with drugs, CAR T cells overexpressing Bcl-xL and Bcl-2 (both wildtype and mutant) provided higher anti-tumor activity and prolonged survival against JeKo-1 cells in vivo, whereas only Bcl-xL overexpression showed increased tumor control compared to regular 4-1BB CARs against Nalm6 cells. Conclusion: Of the tested antiapoptotic proteins, Bcl-xL overexpressing CAR T cells proved superior, having higher proliferation and increased anti-tumor activity in combination with or without BH3 mimetics, providing a new strategy to optimize CAR T cell function for the treatment of leukemia and lymphoma. Citation Format: Felix Korell, Michael Olson, Diego Salas-Benito, Mark B. Leick, Rebecca C. Larson, Harrison Silva, Alessandro Gasparetto, Trisha R. Berger, Amanda Bouffard, Michael C. Kann, Markus Mergen, Tamina Kienka, Marc Wehrli, Stefanie R. Bailey, Anthony Letai, Marcela V. Maus. Chimeric antigen receptor (CAR) T cells overexpressing Bcl-xL increase proliferation and antitumor activity alone and in combination with BH3 mimetics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4098.