Combination of Ruxolitinib and Magrolimab Significantly Increases Calreticulin Expression in Myelofibrosis CD34+ Cells in Vitro. Proof of Concept for Combination Therapy

Abstract

Background Primary myelofibrosis (MF), as part of Myeloproliferative neoplasms (MPN), is a chronic myeloid cancer characterized by bone marrow fibrosis and either overproduction or depletion of blood cells, and a high tendency to evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. Upregulation of cell membrane CALR and CD47 has been observed to increases in response to chemotherapy; however, their role in myeloid malignancies is poorly understood. We have previously found that CD47, not CALR, is overexpressed on the membrane of patients with MPN. Anti-CD47 monoclonal antibody Magrolimab, is currently being evaluated in clinical trials in AML, MDS and some solid tumours but have yet to be tested, either in monotherapy or in combination in MF Aims To investigate the alterations in expression of CALR and CD47 in MF patients when treated with standard therapies and/or CD47 inhibitors, in comparison with healthy controls. Methods Mononuclear cells were collected by Ficoll separation from bone marrow of 6 MF patients and from 2 controls. CD34+ cells and monocytes were purified using Dynabeads kits from Thermofisher. Cells expression of CALR and CD47 was measured by flow cytometry, before and after incubation with ruxolitinib (Jak inhibitor) alone and in combination with magrolimab. Cells survival was calculated after combining CD34 with monocytes. Informed Consent was obtained for all samples collected. Results CD47 membrane expression was 57% in untreated MF cells compared to 12% in the control samples and CALR membrane expression was 51.8% in untreated MF cell compared to 12.5% in the control samples. After incubation with ruxolitinib, MF CD34+ cell survival decreased to 98.2%. Following Incubation with magrolimab alone, MF CD34+ cells show no changes in survival (100% alive). The combination of ruxolitinib and magrolimab reduced slightly the MF CD34+ cell survival to 92%. A substantial membrane overexpression of CALR was observed in MF CD34+ cells when incubated with Ruxo and Magrolimab, 74.1%, vs Ruxo alone 52.6%, Magrolimab alone 53.4% and in untreated (51.8%). Interestingly, in the healthy controls, the exposure to combination therapy did not cause the same degree of CALR overexpression (20.7% vs 12.5%), indicating a much stronger pro-phagocytic effect following magrolimab/ruxolitinib combo in MF cancer cells. Conclusion Previous studies have shown that CD47, not CALR, is overexpressed on the membrane of patients with MPN, suggesting a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. We previously reported treating MF patients with cyto-reductive agents, increases CD47 expression leading to a stronger anti-phagocytic signal. In vitro, treatment with ruxolitinib or ruxolitinib plus magrolimab, slightly reduce the overall cell survival rate in CD34+ MF samples, comparing with no significant changes in single agent magrolimab. However, the combination of Ruxolitinib and Magrolimab significantly increases CALR membrane expression comparing with Ruxo or Magrolimab alone, signalling a much stronger pro-phagocytic message in MF cells comparing with controls. The induced CALR over-expression, could stimulate a stronger immune response in vivo, where the presence of macrophages would allow removal of CALR rich cells and potentiate the cytoreductive activity towards MF clone. These data support the rational of combining Ruxolitinib with Magrolimab in the next clinical trial stage in myelofibrosis.