Abstract B043: Dual epigenetic therapy combined with anti-PD1 rescues HMA-induced suppressive myeloid phenotype and reduces tumor growth in a PDAC model
Authors: Orlacchio Arturo , Muzyka Stephen , Do Catherine , Simeone Diane M. , Gonda Tamas 
Published: January 2024

Abstract
INTRODUCTION. We previously showed, using the PDAC mouse model KPC (KrasLSL G12D/+; p53 r172H/+; Pdx1-Cre), that sequential treatment with the DNA hypomethylating drug decitabine (5aza-dC; DAC) followed by anti-PD-1 led to increased tumor necrosis, slower tumor growth, increased tumor-infiltrating CD8+ and CD4+ T-cells (TILs), and significantly increased mean survival time. However, despite these positive outcomes, the KPC tumors eventually escaped from treatment. We identified a unique myeloid population as a specific subtype of M2-polarized putatively immunosuppressive Chil3+ macrophages (TAMs). Here we further characterize the recruitment mechansism of these cells and show that HDAC inhibition can further improve the efficacy of DAC+PD-1 treatment. METHODS. Primary macrophages were isolated from bone marrow (BMDM) or the peritoneum of 6-8 weeks mice and RAW264.7 cells were also used and exposed to conditioned media from primary KPC cell lines after HMA treatment (KPC-SN-DAC). Gene set enrichemnt analysis was done on RNASeq data from myeloid cells and validated by RT-qPCR. We used cytokine arrays to identify secreted cytokines from KPC cells. KPC cells were orthtopically injected in C57BL/6 mice, and tumor growth was followed by untrasound. Treatments were started when tumor reaced a size of 1 mm3. RESULTS. Expression profiling reveled that exposure of the myeloid cells to KPC-SN-DAC resulted in an HMA-specific M2-like gene signature. Notably DAC alone had minimal effect on myeloid cells polarization. To assess which factors might trigger this polarization we employed cytokine arrays and identified a number of candidate cytokines induced or secreted from KPC cells specifically following HMA treatment. In order to revert this unfavorable cytokine profile, we have evaluated the combination of DAC with four different HDAC inhibitors with reported immunomodulatory effects: Romidepsin (ROMI), Domatinostat (DOMA), Pracinostat (PRAC) and Entinostat (ENT). In vitro synergy studies through cell viability assays showed that ROMI, PRAC and DOMA synergized at low doses with DAC. Pofiling of supernatants from KPC cells treated with a combination of DAC+HDACis showed that, while ROMI had limited effect, PRAC and DOMA reverted DAC-induced cytokine secretion. Lastly, we tested in vivo the combination of DAC+ROMI/DOMI/PRAC+PD1 using mice orthotopically injected with KPC cells. The combination DAC+PRAC+PD1 showed reduced tumor volume compared to DAC+PD1. Moreover, PRAC+PD1 significantly improved overall survival. Interestingly, HDAC inhibition rescued the increased Chil3 expression in DAC+PD1 treated tumors. CONCLUSIONS. Our results show that paracrine rather than direct effect of hypomethylating therapy is responsible for suppressive myeloid polarization and characterize a specific effect of hypomethylating therapy on immature myeloid cells. Moreover, we have identified HDAC inhibition as a viable strategy to prevent unfavorable myeloid polarization and improve the therapeutic efficacy of the DAC+PD1 paradigm.
Citation Format: Arturo Orlacchio, Stephen Muzyka, Catherine Do, Diane M. Simeone, Tamas Gonda. Dual epigenetic therapy combined with anti-PD1 rescues HMA-induced suppressive myeloid phenotype and reduces tumor growth in a PDAC model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B043.