SWI/SNF Chromatin Remodeling Complex Regulates the Expression of PD-L1 in a Spatio-Temporal Manner in Acute Lymphoblastic Leukemia (ALL) in Vitro

Epigenetic changes and interactions between different cells within the tumor microenvironment are critical for the maintenance and progression of cancer. The primary facet of such interactions is to create an immunosuppressive environment for tumor propagation by subverting the host's antitumor response. In this context, one of the critical elements is the Programmed Death 1 (PD-1/CD279), an immune checkpoint protein, which is responsible for the attenuation of the immune response by blocking T-cell function. The interaction between PD-1 on the tumor infiltrating lymphocytes (TILs) and its ligand, PD-L1, on tumor surface is associated with poor prognosis and aggressiveness in several cancers. Interestingly PD-L1 expression on the tumor surface is restricted to regions infiltrated by TILs indicating a direct regulation of PD-L1 - by TIL-secreted proinflammatory cytokines, such as interferon gamma (IFNg) and interleukin 10 (IL-10). This adaptive nature of PD-L1 expression supports the existing concept that epigenetic regulations may serve as a key factor. Nevertheless, there is very little data concerning the PD-L1 expression and the mechanism of immune escape in acute and chronic leukemia.

To understand the mechanism of PD-L1 induction in acute leukemia, we evaluated the baseline expression of PD-L1 in four different ALL cell lines, SD1 (pre B), REH (pre B), CCRM-CEM (T-ALL) and MOLT3 (T-ALL). We found that the baseline levels of both PD-L1 mRNA and protein are very low in these cell lines. However, following the treatment with IFNg, we observed a significant increase in PD-L1, both at the mRNA and protein level. Given the fact that IL-15 is required for lymphocyte growth, and is also implicated as an inducer of PD-L1 in solid tumors, we evaluated the effect of IL-15 on PD-L1. We observed an increase in PD-L1 mRNA and protein levels following IL-15 treatment. To gain further insight if epigenetic regulation is involved in these proinflammatory cytokines- mediated PD-L1 upregulation, we studied the role of SWI/SNF complex in this context. SWI/SNF complex is one of the largest, evolutionary conserved chromatin remodeling complex. It contains two catalytic subunits, BRM and BRG1 that have been demonstrated to be dysregulated in different cancers, including leukemia. We found that BRG1 expression in these ALL cell lines goes up significantly following IL-15 and IFNg treatment. To further ascertain if this upregulation of BRG1 is critical for PD-L1 induction, we did an shRNA mediated knockdown of BRG1. This resulted in no PD-L1 induction upon treatment with either IFNg or IL-15 suggesting BRG1 as a downstream effector. This observation is also recapitulated by overexpressing a dominant negative BRG1 variant. Importantly, we also observed that IFNg or IL-15 mediated upregulation of BRG1 is associated with the inactivation of BRM by acetylation. Pharmacological or genetic reversal of BRM acetylation, could inhibit the BRG1-dependent PD-L1 induction following IL-15 or IFNg treatment.

To determine how BRM and BRG1 regulate the expression of PD-L1 in the presence of proinflammatory signals, we conducted Chromatin Immunoprecipitation (ChIP) assays on the PD-L1 promoter in the presence or absence of IFNg or IL-15. We found that in the absence of any activating signals (IFNg or IL-15), BRM was recruited to the PD-L1 promoter, along with native STAT1. In the presence of IFNg or IL-15, BRM is acetylated and dissociates from the promoter, along with the recruitment of BRG1 and phosphorylated STAT3, resulting in an increase in PD-L1 transcription.

Current immunotherapy involves targeting either PD-1 or PD-L1 by monoclonal antibodies. Although, such therapy has demonstrated promising preliminary results, only a subset of patients has benefited from such therapy in the long term. Furthermore, patients who initially responded to therapy, later developed resistance following relapse. This clinical data suggests that it is imperative to understand the detailed mechanism of PD-L1 regulation to provide better therapeutic options. Our data bestowed a novel insight into the epigenetic regulation of PD-L1 expression on the tumor cells by SWI/SNF chromatin remodeling complex in response to proinflammatory signals. This study provides us with additional therapeutic opportunities, whereby targeting the BRM/BRG1 axis in conjunction with the existing therapy may provide better outcomes.