Therapeutic impact of BET inhibition on immune cell death checkpoints in MPN.

Abstract

e18595 Background: Myeloproliferative neoplasms (MPNs) are haematological cancers characterized by excessive bone marrow cell growth and have the potential to progress to acute myeloid leukaemia (AML). Current therapies, such as chemotherapy and Ruxolitinib, are effective in only about 50% of patients, underscoring the need for improved treatment options. CD47, expressed on cancer cells, acts as an anti-phagocytic signal, while Calreticulin (CALR) serves as a pro-phagocytic marker, promoting cancer cell elimination by macrophages. Emerging therapies, including Bromodomain and Extra Terminal (BET) inhibitors, target key pathways involved in cell proliferation and immune signalling, such as CD47/CALR, offering innovative approaches to cancer immunotherapy. This study aimed to evaluate the effects of BET inhibition, alone or combined with standard therapies, on CD47/CALR signalling and cell death in MPN cell models. Methods: The SET-2 and HEL.92 cell lines, harbouring heterozygous and homozygous JAK2 V617F mutations, respectively, were treated for 48 hours with a novel BET inhibitor either alone or in combination with conventional therapies. Flow cytometry was used to measure cell surface CD47, extracellular, and intracellular CALR expression. Results: In SET-2 cells, treatment with BET inhibitor, alone or combined with Ruxolitinib, led to a significant reduction in CD47 expression (61% for BET inhibitor alone and 58% for BET inhibitor + Ruxolitinib compared to 94% in untreated controls; p<0.0001). Intracellular CALR levels increased significantly from 17% in untreated samples to 38% with BET inhibitor alone (p<0.01) and 43% with Ruxolitinib combination (p<0.05). However, extracellular CALR expression decreased (untreated 32%, BET inhibitor 27%, p=0.8, BET inhibitor + Ruxolitinib 15%, p=0.06). In HEL.92 cells, a similar trend was observed for CD47, with CD47 expression decreasing from 88% in controls to 76% with BET inhibitor, although this was not significance (p=0.4). In contrast to the results seen in SET-2 cells, intracellular CALR dropped from 57% to 43% (p=0.4). The combination of BET inhibitors and Ruxolitinib did not yield significant results in this cell line. Conclusions: These findings indicate that BET inhibition affects immune signalling mechanisms in the SET-2 cell line, suggesting its potential as a therapeutic approach for MPN immunotherapy. However, the lack of significant response in the HEL.92 cell line points to the potential importance of allele burden in shaping treatment outcomes. Future research will focus on examining how BET-inhibition influences CD47/CALR expression ratios and its impact on macrophage-mediated phagocytosis to further define its role in anti-cancer immune responses.