Preclinical Rationale for the Use of Crizanlizumab (SEG101) in Myelofibrosis
Authors: Verachi Paola , Martelli Fabrizio , Zingariello Maria , Chaturvedi Shalini , Wilke Celine , Campello-Iddison Valerie , Migliaccio Anna Rita 
Published: November 2020

Myelofibrosis (MF), the end stage of Philadelphia-negative Myeloproliferative Neoplasms, is still an unmet clinical need. Treatment with the JAK1/2 inhibitor ruxolitinib ameliorates symptoms and improves overall survival but it is unclear whether it is effective in improving the clinical progression of these patients. The bone marrow (BM) and spleen from MF patients contain great numbers of impaired megakaryocytes (MK) which have been hypothesized to be responsible for their clinical manifestation by expressing on their surface high levels of the adhesion receptor P-selectin which triggers a process of pathological neutrophil emperipolesis leading to increased bioavailability of the pro-fibrotic transforming growth factor-β (TGF-β) in the microenvironment1,2. We have peviously reported that ablation of P-selectin cures myelofibrosis in Gata1low mice, a bona fide model for MF3. Using these data as a foundation, we tested whether Crizanlizumab, an antibody targeting human P-selectin that effectively prevents pain crisis in patients with Sickle Cell Disease4, represents an effective option to treat MF. To this aim, we treated Gata1low mice with RB40.34, an antibody against murine P-selectin used in the pre-clinical study that supported the clinical trial with crizanlizumab5, alone or in combination with ruxolitinib. Twenty-four 11-months old Gata1low mice were microchipped, randomly separated in four groups (3 males and 3 females/group) and treated as follow: Group 1: vehicle; Group 2: biotinylated RB40.34 (30 μg/mouse/day per 3 days/week by tail vein injection); Group 3: ruxolitinib (45mg/Kg twice per day by gavage); Group 4: biotinylated RB40.34 and ruxolitinib in combination. Mice were treated for 2 cycles of 5 consecutive days with two days of rest in between and sacrificed on day 5 (I end-point) and 12 (II end-point). End-points: Day 5: all mice were weighed and bled for blood counts determinations and presence of labeled-RB34.40 on platelets. Males were sacrificed for signaling studies and determination of the presence of RB40.34 on MK from BM and spleen. Day 12: females were weighed, bled for blood counts determinations and sacrificed for histopathology analyses. None of the treatments induced changes in body weight that remained similar to those observed before treatment, suggesting that overall all the treatments were well tolerated. None of the treatments rescued the platelet deficiency of Gata1low mice, which remained thrombocytopenic, nor induced anemia by day 12, inspite they had been bleed 1 week earlier. This lack of anemia indicates that the treatments do not impair the ability of the mice to recover from erythroid stress. Atday 5, high levels of RB40.34 were observed on the platelets and on the MK of mice treated with the antibody, alone or with ruxolitinib. However, only the two drugs in combination significantly normalized the TGF-β signaling in BM by reducing the abnormally high content of TFGβRII and SMAD2/3 and the phosphorylation levels of ERK in this organ. These results indicate that when used in combination, the two drugs are effective in rescuing the abnormal TGF-β signaling of BM from these mice. Byday 12, high levels of RB40.34 were observed on BM and spleen of mice treated with the antibody alone or with ruxolitinib but again only treatment with the two drugs in combination greatly improved the cellularity of the BM medulla (Hematoxilin-eosin staining), which is typically reduced in Gata1low mice, and reduced the massive fibrosis (Gomory staining) and high TGF-β content (immunostaining) observed in the diaphysis area of the long bones (Fig. 1). Treatment with the two drugs in combination also reduced fibrosis and TGF-β content in spleen restoring the architecture of this organ which regained a normal appearance with well recognizable red and white pulp zones. When used alone, neither RB40.34 nor ruxolitinib had any effect of the MF phenotype of this mouse model.
In conclusion, RB40.34 in combination with ruxolitinib rescues the MF phenotype of Gata1low mice with limited toxicity providing pre-clinical evidence that treatment with crizanlizumab in combination with ruxolitinib may be more effective than that with ruxolitinib alone to cure MF patients.
References: 1Schmitt et al Blood 2000;96:1342; 2Centurione et al Blood 2004; 104:3573; 3Spangrude et al Stem Cells 2016; 34:67; 4Ataga et al NEJM 2017; 376:429; 5Embury et al Blood 2004; 104:3378.

Disclosures
Wilke: Novartis Pharma AG: Current Employment, Other: Stock owner. Campello-Iddison:Novartis: Current Employment. Migliaccio:Novartis: Research Funding.