Abstract 6176: Selective HDAC6 inhibitor ITF3756 induces IL-2 production and CD4 and NK cells activation in MLR assay
Authors: Gianluca Fossati, Chiara Ripamonti, Valeria Spadotto, Michela Bottani, Christian Steinkuhler
Journal: Cancer Research (2025)

Abstract Over the recent years, it has become increasingly clear that epigenetics plays a key role in regulating the anti-tumor immune response. Modification of the altered epigenetic status generated by tumors is a new and promising approach to induce an efficient antitumor immune response. Epigenetic drugs could represent a new avenue for cancer immunotherapy, particularly in combination with immune checkpoint inhibitors. Acetylation of proteins is one of the epigenetic modifications that has a recognized role in shaping the tumor microenvironment and the antitumor immune response. Among the histone deacetylase family, HDAC6 is a peculiar enzyme involved in tumor growth and in the modulation of the immune functions, that has recently taken hold. We have developed ITF3756, a selective HDAC6 inhibitor, currently in Phase I clinical trial, which exerts in vivo antitumor activity that is strictly dependent on a functional immune system. In vitro, it reduces the immunosuppressive function of myeloid cells and promotes their T cell stimulating activity. T cell development is also influenced by driving CD8 T cells towards a more central memory, less exhausted phenotype with an increased effector function upon tumor cells co-culture. These data derived from studies done with purified cells from human Peripheral Blood Mononuclear Cells (huPBMC). To shed light on the activity of ITF3756 in a more complex environment, Mixed Lymphocyte Reaction (MLR) was chosen as a model. MLR is largely used for investigating the activity of immunotherapy compounds including immune checkpoint inhibitors and can give insights on the possible in vivo mechanism of action of the investigated compounds. HuPBMC from different donors were cocultured in the presence or absence of ITF3756. Supernatants and cells were collected at different time points and analyzed by ELISA and flow cytometry respectively. ITF3756 enhanced the production of IL-2 in the supernatants at 48 and 72h. Flow cytometry analysis showed that CD4 T cells and B cells, but not CD8 T cells, were the major populations where IL-2 was significantly increased in the presence of ITF3756. The increase of IL-2 production by ITF3756 at the earlier time points is correlated with a significant increase of CD4+CD25+ cells at day 6 and with a significant enhancement of the expression of the activator receptor NKG2D in Natural Killer cells (NK). Further analysis of CD4+CD25+ cells showed that ITF3756 activated CD4+CD25+CD127+ cells, but not the T regulatory cells (CD4+CD25+CD127-). Our data indicate that in a multicellular in vitro immune microenvironment, ITF3756 activates CD4 and NK cells and increases the production of IL-2. Given the importance of IL-2 in the tumor microenvironment, these new results, in addition to those previously collected on isolated myeloid and CD8 T cells, support the notion of the use of a HDAC6i such as ITF3756 for a promising immune checkpoint co-treatment therapy. Citation Format: Gianluca Fossati, Chiara Ripamonti, Valeria Spadotto, Michela Bottani, Christian Steinkuhler. Selective HDAC6 inhibitor ITF3756 induces IL-2 production and CD4 and NK cells activation in MLR assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6176.