Abstract A42: Bromodomain inhibitors suppress Nrf2-dependent HO-1 positive macrophage accumulation in murine models of KRAS-mutated pancreatic cancer
Authors: Leal Ana S. , Liby Karen T. 
Published: June 2021

The Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress responses and can drive cancer progression, metastasis and chemoresistance. Nrf2 also attenuates inflammation through elimination of reactive oxygen species (ROS) and regulation of macrophage specific genes involved in oxidative/stress responses. The tumor microenvironment (TME) in pancreatic cancer is rich in macrophages that promote cancer progression and chemoresistance. Tumor cells can use epigenetic modulation to evade immune recognition and to shape the TME toward an immunosuppressive phenotype. Bromodomain inhibitors are a class of drugs that target BET (bromodomain and extra-terminal) proteins, impairing their ability to bind to acetylated lysines and therefore interfering with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell growth, apoptosis and inflammation. Recent studies suggest that BET proteins may play a key role in the regulation of Nrf2-dependent antioxidative and inflammatory genes. Therefore, we hypothesized that the use of small BRD4 inhibitors could prevent the activation of Nrf2 –dependent macrophages in the pancreas. LSL-KrasG12D/+;Pdx-1-Cre (KC) and Nrf2 whole body knockout (KO) mice were stimulated with caerulein (acute protocol - 75 ug/kg injected i.p. every hour for 8 hours for 2 consecutive days) to induce pancreatitis. 72 hours later, macrophage infiltration into the pancreas was analyzed by flow cytometry. KC mice showed a significantly higher infiltration of macrophages when compared with Nrf2 KO or wild-type mice (51.1% vs 10.7% vs 21.5%). Treatment with I-BET 762 (60 mg/Kg BID once a day, starting 72 hours before caerulein stimulation and continued until the end of the study) did not reduce the infiltration of macrophages. However, I-BET 762 treatment did reduce the expression of HO-1, a downstream effector of Nrf2, as observed by Western blot and IHC. Nrf2 KO mice were almost devoid of HO-1 expression. Additionally, KC mice stimulated with caerulein and maintained on I-BET 762 treatment (60 mg/kg of diet) for 9 weeks showed a downregulation of CD206 expression in the pancreas, a marker of M2 macrophages. KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mice will succumb to pancreatic cancer at an average of 23 weeks of age. I-BET 762 treatment for 12 weeks significantly reduced the levels of HO-1 expression in the pancreas microenvironment by 60% without altering the percentage of macrophages. HO-1 expression increased in RAW 267.4 cells or bone marrow-derived macrophages (BMDM) from wild-type mice stimulated with conditioned media from murine pancreatic cancer cells, but not in BMDM from Nrf2 KO mice. Moreover, the increase in HO-1 is decreased by treatment with I-BET 762 or N-acetyl-L-cysteine. This study suggests that bromodomain inhibitors can regulate Nrf2 activation in macrophages, with or without Kras mutations. Future studies will address the role of Nrf2 on macrophage function in vivo.

Citation Format: Ana S. Leal, Karen T. Liby. Bromodomain inhibitors suppress Nrf2-dependent HO-1 positive macrophage accumulation in murine models of KRAS-mutated pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A42.