Abstract 3414: Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis
Authors: Stiff Andrew , Savardekar Himanshu , Wesolowski Robert , Duggan Megan , Scarberry Luke , Benner Brooke , Wethington Darren , Lapurga Gabby , Sun Steven , Hedberg Jack , Good Logan , Carson William E. 
Published: September 2020

Abstract
Myeloid derived suppressor cells (MDSCs) antagonize antitumor immune responses, and limit the efficacy of immune based therapies for cancer. As a result, MDSCs have garnered attention as therapeutic targets. Unfortunately, there has been limited success in translating agents targeting MDSCs to the clinic. Brd4 is an epigenetic reader and is itself a therapeutic target in oncology due to its ability to regulate the expression of oncogenes such as Myc. In addition, Brd4 is known to regulate inflammatory cytokine production and innate immune responses including myeloid cell function. As a result, it was hypothesized that inhibition of Brd4 would impact MDSC function or expansion. In multiple tumor models (EMT6, 4T1, LLC, and C26) Brd4 inhibitors, both experimental (JQ1) and those in clinical development (PLX51107 and PLX2853) significantly reduced the abundance of total, PMN, and M-MDSC subsets within the tumor and spleen as measured by flow cytometry and IHC (p&lt; 0.05). Furthermore, Nanostring gene expression analysis of EMT6 tumors identified a significant reduction in the abundance signature of neutrophils and macrophages with PLX51107 further confirming a reduction of myeloid cells in the presence of a Brd4 inhibitor (p&lt; 0.01). The Nanostring analysis also showed a significant reduction of IL6 mRNA in PLX51107 treated tumors, and this was confirmed at the protein level by ELISA in the serum of EMT6 and 4T1 tumor bearing mice (p&lt; 0.05). However, exogenous IL6 could not rescue MDSC levels in vivo or in vitro suggesting Brd4 inhibition might be directly affecting MDSCs. This was confirmed by showing that PLX51107 significantly inhibited the expansion of MDSCs from mouse bone marrow cells or healthy donor PBMCs cultured in IL6 and GMCSF as measured by flow cytometry (p&lt;0.05). Furthermore, mouse and FACS sorted MDSCs from patients with melanoma or bladder cancer were found to undergo apoptosis when treated with PLX51107 as measured by flow cytometry (annexin and caspase3, p&lt; 0.05), IF, and western blot. In addition, a pancaspase inhibitor rescued both mouse and patient derived MDSCs from apoptosis in the presence of PLX51107. Further investigation with specific caspase inhibitors showed this effect relied mostly on activation of the intrinsic apoptosis pathway and caspase9. The importance of Brd4 to MDSC survival was further confirmed using a LysMCre Brd4 floxed mouse model. Finally, depletion of MDSCs in vivo with PLX51107 significantly enhanced the efficacy of anti-PDL1 therapy as compared to either agent alone (p&lt; 0.05 in EMT6, 4T1, and LLC tumor models). These results identify Brd4 and the TXNIP/ASK1 apoptosis pathway as novel regulators of MDSC survival, and provide evidence to further investigate Brd4 inhibitors in combination with immune based therapies for solid tumors.
Citation Format: Andrew Stiff, Himanshu Savardekar, Robert Wesolowski, Megan Duggan, Luke Scarberry, Brooke Benner, Darren Wethington, Gabby Lapurga, Steven Sun, Jack Hedberg, Logan Good, William E. Carson. Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3414.