The Effects of SB1518, a Novel Oral JAK2 Inhibitor, On Ex Vivo Expanded PV Erythroid Progenitors Correlate with Clinical Observations.
Authors: Goh Kee Chuan , Hart Stefan , Tan Yong Cheng , Chithra Amalini , Ong Kiat H. , Wood Jeanette 
Published: October 2019

Abstract
Abstract 2913
Poster Board II-889

Introduction:
Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) in which the patients' erythroblastic lineage acquires a hyper-proliferative potential but retains normal maturation, leading to elevated hematocrit and its attendant risks. The discovery of an activating mutation (JAK2V617F) in almost all PV patients and about half of the other Ph-negative MPNs prompted the development of small-molecule JAK2 inhibitors as novel therapeutics. In preclinical translational studies, these compounds have been tested against primary hematopoietic cells in ex vivo assays such as the endogenous erythroid colony (EEC) assay or proliferation assay with erythroid progenitors (EP). Results have been mixed, with some compounds showing selective activity against JAK2V617F-bearing cells while others do not discriminate between wild-type and mutant. SB1518 is a JAK2 inhibitor being evaluated in Phase I/II studies on myelofibrosis patients. We report here the effects of SB1518 on ex vivo expanded EPs from PV patients and healthy volunteers.

Patients and methods:
Peripheral blood mononuclear cells (PBMCs) were isolated from 11 PV patients and 8 healthy volunteers. EPs were expanded ex vivo from the PBMCs using a published protocol which involves culturing in an erythropoietin (EPO)-containing medium over a 12-day period [Cancer Sci. (2008) Vol 99, 1265]. Flow cytometry for co-expression of CD71 and CD235a confirmed that >98% of the expanded cells belonged to the basophilic erythroblast lineage. The EPs were treated with SB1518 and other JAK2 inhibitors to determine the effects of JAK2 inhibition on downstream signaling, cell viability and JAK2V617F allele frequency.

Results:
SB1518 inhibited phospho-STAT5 levels in a dose-dependent manner (IC50 < 200 nM) and reduced the viability of expanded erythroid progenitors from all sources; normal volunteers with JAK2WT (mean IC50 = 260 nM) or PV patients with JAK2V617F (mean IC50 = 230 nM). The difference in IC50 between the 2 groups was not statistically significant. Moreover, SB1518 treatment had no effect on the JAK2V617F allele frequency in the EPs from PV patients, indicating similar drug sensitivity for EPs from the same patient, regardless of the presence of mutation. This suggests that the constitutively activated JAK2 mutant and the EPO-activated wild-type JAK2 exert equivalent quantitative impact on proliferative pathways in EPs. Similar effects were observed with other JAK2 inhibitors of different chemical classes and selectivity profiles. These data are consistent with current observations in JAK2 clinical trials, namely that clinical benefit is not dependent on JAK2 genotype and there is little or no effect on allele frequency in blood cells.

Conclusions:
In summary, we demonstrated that current JAK2 candidates, being inhibitory against both WT and mutant JAK2 kinases, will exert similar effects in biological systems sustained by JAK2 activation, whether constitutive or ligand-stimulated. Our data are consistent with current experience in myelofibrosis trials where clinical benefit is attainable independent of JAK2 mutation status and indicate that activation of JAK2 signaling either by an activating mutation or by other means, contributes to the symptoms of the disease.

Disclosures:
Goh: S*BIO PTE LTD: Employment. Hart:S*BIO PTE LTD: Employment, Equity Ownership. Tan:S*BIO PTE LTD: Employment. Chithra:S*BIO PTE LTD: Employment. Wood:S*BIO PTE LTD: Employment.