Abstract 5274: Targeting retinoblastoma protein in tumor-associated macrophages suppresses ovarian cancer progression
Authors: Tcyganov Evgenii N. , Kwak Taekyoung , Yang Xue , Poli Adi N. , Hart Colin , Bhuniya Avishek , Cassel Joel , Kossenkov Andrew , Auslander Noam , Sharma Paridhima , Cadungog Mark G. , Jean Stephanie , Chatterjee-Paer Sudeshna , Weiner David , Donthireddy Laxminarasimha , Bristow Bryan , Zhang Rugang , Salvino Joseph M. , Montaner Luis J. 
Published: March 2024

Abstract
Ovarian cancer is characterized by an immunosuppressive tumor microenvironment (TME) maintained by tumor-associated M2-like macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. An effective approach to target TAMs in ovarian cancer treatment is currently not available, but would be expected to release immunosuppressive pressure, enable a robust T cell response and improve immunotherapy outcomes. Retinoblastoma protein (Rb) is a well-known tumor suppressor and regulator of tumor cell proliferation. Accumulating evidence demonstrates its role in immune cells, in myeloid cells, in particular. However, these mechanisms remain poorly understood.
Based on our previous data on Rb role in myeloid cell viability, we decided to test the effects of Rb modulation in macrophages. We used the small molecule, AP-3-84 compound, and newly developed analogs which bind the LxCxE motif of Rb and disrupt Rb interaction with its binding adaptor proteins. AP-3-84 induced cell death preferentially in macrophages, but not in T cells or tumor cells (without effects on cell proliferation). Gene and protein expression analysis revealed that Rb targeting induced major intracellular stress response programs and p53/mitochondria-related cell death pathways in TAMs. Moreover, we found that M2 type polarized macrophages expressed higher levels of Rb compared to M1 macrophages. In agreement with that, M2 type macrophages were significantly more sensitive to AP-3-84 treatment than M1 polarized cells. Next, we demonstrated that low dose therapeutic use of AP-3-84 in mice bearing ovarian cancer significantly re-shaped the immune composition of the TME by depleting TAMs and inducing remarkable T cell infiltration. Importantly, these AP-3-84 effects were immune-mediated, since identical AP-3-84 treatment in NSG immunodeficient mice had no effect on tumor growth. Similarly, anti-CD4/CD8 depletion also reverted AP-3-84 effects on cancer inhibition. An analysis of activation and differentiation markers in myeloid and T cell subsets in the TME showed a shift of the remaining macrophages towards an M1-like cell type accompanied by activation of T cells. Using ovalbumin-overexpressing ovarian cancer cell line, we documented the accumulation of antigen-specific T cells in the TME associated with a substantial delay of ovarian cancer upon AP-3-84 therapeutic Rb targeting.
Ex vivo, we observed an analogous cell death induction by AP-3-84 treatment in TAMs from post-surgery ascites from ovarian cancer patients. Using available datasets, we further documented that an increase in Rb expression in the TME myeloid cells is associated with poorer prognosis.
Overall, our data supports that therapeutic targeting of the Rb LxCxE motif is a promising approach for ovarian cancer treatment due to depletion of TAMs and re-shaping the ovarian cancer TME.
Citation Format: Evgenii N. Tcyganov, Taekyoung Kwak, Xue Yang, Adi N. Poli, Colin Hart, Avishek Bhuniya, Joel Cassel, Andrew Kossenkov, Noam Auslander, Paridhima Sharma, Mark G. Cadungog, Stephanie Jean, Sudeshna Chatterjee-Paer, David Weiner, Laxminarasimha Donthireddy, Bryan Bristow, Rugang Zhang, Joseph M. Salvino, Luis J. Montaner. Targeting retinoblastoma protein in tumor-associated macrophages suppresses ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5274.