Effects of ruxolitinib and magrolimab on calreticulin in myelofibrosis CD34+ cells in vitro: Proof of concept for combination therapy.

person Ciro Roberto Rinaldi University of Lincoln, Lincoln, United Kingdom info_outline Ciro Roberto Rinaldi, Kristian Boasman, Matthew Simmonds

Authors person Ciro Roberto Rinaldi University of Lincoln, Lincoln, United Kingdom info_outline Ciro Roberto Rinaldi, Kristian Boasman, Matthew Simmonds Organizations University of Lincoln, Lincoln, United Kingdom Abstract Disclosures Research Funding Pharmaceutical/Biotech Company Gilead Background: 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. We have previously found that CD47, not CALR, is overexpressed on the membrane of patients with MPN. Anti-CD47 Magrolimab, is currently being evaluated in clinical trials in AML, MDS but have yet to be tested, either in monotherapy or in combination in MF. 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. Conclusions: 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.

Pre-clinical