Targeting BRD4 to Counteract Cytokine-Induced Expression of PD-L1 and PD-L2 on Vascular, Osteoblastic and Stromal Niche Cells

Abstract

The stem cell niche plays a crucial role in promoting the expansion of leukemic stem cells (LSC) and in mediating resistance of LSC against various anti-neoplastic drugs in different types of leukemias. Recent data suggest that targeting niche cells may be an effective approach to overcome niche-mediated drug resistance of LSC. However, niche cells themselves are considered to be often resistant against therapeutic intervention. This may especially be relevant in those myeloid neoplasms where niche cell expansion is a pathognomonic feature and the resulting neo-angiogenesis, bone marrow fibrosis and/or sclerosis are important prognostic variables. We and others have recently shown that several of the anti-neoplastic drugs used to treat myeloid leukemias can also counteract niche cell expansion. So far, however, little is known about mechanisms underlying resistance of niche cells against anti-neoplastic drugs. Recent studies have shown that certain immune checkpoint antigens are overexpressed in cancer cells and/or their microenvironment and that these checkpoint antigens contribute to drug resistance. Other studies have shown that checkpoint molecules, including PD-L1 and CD47, are displayed by LSC in acute myeloid leukemia, chronic myeloid leukemia and other myeloid neoplasms. We asked whether niche-related cells also display these checkpoint molecules. Furthermore, we screened for cytokines that promote expression of checkpoint antigens on niche cells and asked for drugs that counteract growth and cytokine-induced checkpoint expression in these cells. The following niche cells were examined: primary human umbilical vein endothelial cells (HUVEC), the human endothelial cell line HMEC-1, the human osteoblast-like cell line CAL-72, primary osteoblasts, and the human stromal cell line HS5. Expression of the checkpoint antigens PD-L1, PD-L2, PD1, CD28, CD47, CD80, CD83, CD86 and TIM-3 was determined by flow cytometry. All niche cells were found to constitutively express PD-L1, PD-L2, and CD47. The other checkpoint antigens were expressed at low or undetectable levels in these cells. Expression of PD-L1, PD-L2 and CD47 in niche cells was confirmed by quantitative PCR and by immunocytochemistry. Of all cytokines tested (n=12), interferon-alpha (IFN-A; effective range 50-500 ng/ml) and interferon-gamma (IFN-G; 10-500 ng/ml) were found to promote surface expression of PD-L1 and PD-L2 in HMEC-1, CAL72, and HS5 cells in our immunocytochemical and flow cytometry experiments. Moreover, we found that tumor necrosis factor-alpha (TNF-A; 100 ng/ml) enhances IFN-G-induced expression of PD-L1 and PD-L2 in HMEC-1, CAL-72 and HS5 cells. The BRD4-targeting drug JQ1 and the BRD4 degraders dBET1 and dBET6 suppressed cytokine-induced upregulation of surface expression of PD-L1 and PD-L2 in HMEC-1, HUVEC, CAL-72 and HS5 cells, suggesting a role for BRD4 and MYC in checkpoint regulation. In all cell lines examined, dBET6 was found to be a more effective drug compared to JQ1 and dBET1 (IFN-G-induced expression of PD-L1 in: HMEC-1: IC 50 for JQ1 0.5 µM vs dBET1 1 µM vs dBET6 0.05 µM; CAL-72: IC 50 for JQ1 >0.5 µM vs dBET1 1 µM vs dBET6 <0.05 µM). Together, we show that endothelial cells, osteoblasts and stromal cells display PD-L1, PD-L2 and CD47 in a constitutive manner. Our data also show that the pro-inflammatory cytokines IFN-G and IFN-A augment expression of PD-L1 and PD-L2 in these cells, and that TNF-A can further enhance IFN-A/IFN-G-induced expression of PD-L1 and PD-L2 in niche cells. In addition, we show that the BRD4-targeting drugs JQ1, dBET1 and dBET6 suppress cytokine-induced PD-L1 and PD-L2 expression on niche cells. Whether checkpoint expression in niche cells is associated with drug resistance in myeloid neoplasms and whether drug-induced suppression of checkpoint expression in niche cells can overcome resistance remains unknown.