Therapeutically targeting the CALR/CD47 pathway in MPN to reactivate natural immunosurveillance mechanisms within the body to enhance treatment outcomes.

170 Background: MPNs are a group of related myeloid malignancies characterized by alterations in mature blood cell production/bone marrow environment and have a tendency to evolve into acute myeloid leukaemia. We demonstrated that CD47 blockade in combination with ruxolitinib, increased CALR expression enhancing cytotoxicity in vitro, promoting enhanced neoplastic clone clearance. With this study we describe a potential methodology for assessing effect of new compounds or combinations and the impact of modulating the immunoresponse upon myeloid malignant clones. Methods: Utilising HEL 92.1.7 and SET-2 cells (JAK2 mutated), Marimo cells (CALR mutated) and K562 cell line models; we will incubate with routine therapies (hydroxyurea, anagrelide, ruxolitinib) in combination with a range of anti-CD47 compounds (Anti-CD47 monocloncal antibodies, BET-1 inhibitors, SIRa inhibitor) and will determine the impact upon CD47 and CALR cell surface expression via flow cytometry. Monocyte derived macrophage (MDM) will be produced through Phorbol 12-myristate 13-acetate (PMA) differentiation of a monocytic Thp-1 cell line. Expression of macrophage cell surface markers (CD11b, CD14), as well as intracellular markers (CD68), will be detected by flow cytometry. MDM will then be polarized to either a pro-inflammatory M1, or anti-inflammatory M2 phenotype, with lipopolysaccharide/interferon gamma or interleukins 4 and 13, respectively. Co-culture experiments will then be undertaken to determine how different anti-CD47 therapy combination impact macrophage polarization and destruction of MPN cell lines. Results: We predict that CALR/CD47 levels on each cell line subtype will respond differently highlighting heterogeneity within MPNs and demonstrating the clear need for a more personalised immunotherapy approach. Alterations in JAK/STAT pathway and BET inhibition may hinder macrophage polarisation to the anti-tumorigenic M1 macrophage phenotype. We theorise that indirect co-culture of M1 macrophage with MPN suspension cells under different treatment modalities will enhance MPN cell death and decrease CD47 expression to a greater level than that which we expect to see in M2 polarised macrophage. We also anticipate that the M2 polarised macrophage, which dominate in the tumour microenvironment as MPN progresses, will fail to phagocytose MPN cells and may not be rescued by novel immunotherapeutics. Conclusions: Our group demonstrated the role of CD47 and CALR expression as a key mechanism and a potential target to enhance presentation of the cancer cell to the immune system, and we believe that understanding the interaction of CD47/CALR – macrophages, will drive new therapeutical approaches able to modulate the immune response and improve patients outcomes, potentially leading to complete MPN clone eradiation and cure.