Effects of NVP-BSK805, a Novel JAK2 Inhibitor, on BCR-ABL and JAK2^V617F Positive Cell Lines

Janus kinases are critical components of cytokine signaling pathways that regulate hematopoiesis, growth, immunity, inflammation, and development. Oncogenic mutations of the non-receptor tyrosine kinase JAK2 are found in many Philadelphia chromosome negative myeloproliferative neoplasms. The V617F mutation in JAK2 occurs in 95% of patients with polycythemia vera, 50% of those with essential thrombocythemia and 50% of primary myelofibrosis patients. Preclinical results strongly support that JAK2 inhibitors could be effectively used in these three indications. Replacement of valine 617 with phenylalanine upregulates the tyrosine kinase activity of JAK2, causing constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. JAK2 has also been postulated to play an important role in BCR-ABL signal transduction. Therefore, inhibitors of the tyrosine kinase activity of JAK2 are under investigation as new therapy strategies for CMPNs. In this study the role of the novel JAK2 inhibitor, NVP-BSK805 (Novartis Pharmaceuticals), has been investigated in cells expressing either BCR-ABL or mutant JAK2. Possible synergistic effects between NVP-BSK805 and the already established tyrosine kinase inhibitors imatinib and nilotinib were assessed.

The in vitro activity of NVP-BSK805 was analyzed in 12 hematopoietic cell lines, including 7 BCR-ABL positive (K562, KCL22, KU812, Lama87, BV173, EM3, SUP-B15), 4 JAK2 mutated (CHRF288, SET2, UKE1, HEL), the T-cell leukemia cell line Jurkat, and the neuroendocrine colonic tumour line LCC-18. Concentration kinetics from 0 up to 25 μM were established using XTT proliferation assays and flow cytometry for measuring apoptosis. Protein levels of JAK2, phospho-JAK2, STAT5, phospho-STAT5 and BCR-ABL were analyzed using Western blotting. NVP-BSK805 was also tested in combination with imatinib and nilotinib. JAK2 was sequenced in all cell lines in order to detect possible mutations in the gene.

Of the JAK2 mutated cell lines tested, 3 of 4 (CHRF288, SET2, UKE1) showed a significant reduction of proliferation, as well as viability, compared to the other cell lines. CHRF288 responded best to NVP-BSK805 with an IC₅₀ value of 0.22 ± 0.04 μM. UKE1 and SET2 had similar values of 0.35 ± 0.03 μM and 0.37 ± 0.05 μM. Interestingly, HEL (V617F positive) cells showed only an IC₅₀ value (1.8 ± 0.17 μM) for NVP-BSK805, comparable with that of the non-mutated BCR-ABL positive cell lines (1.5 to 2.7 μM). LCC-18 showed the weakest response of all cell lines tested, with an IC₅₀ value of 9.93 ± 0.202 μM. Each cell line responded to concentrations higher than 5 μM with a strong reduction of proliferation due to inhibition of various kinases. Combination of the JAK2 inhibitor with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL positive cell lines responded well to both CML therapeutic agents. Western blotting of proteins of the JAK-STAT pathway confirmed the results of the proliferation and apoptosis tests showing a strong reduction of phoshorylated STAT5 in CHRF288 cells after a 30 min incubation even with NVP-BSK805 concentrations as low as 0.01 μM. UKE-1 and SET-2 showed reduction of pSTAT5 from 0.1 μM. Levels of total STAT5 were not affected. In all the other cell lines no changes were detected in any of the proteins tested.

Here, we tested a novel JAK2 inhibitor in cells carrying the V617F mutation. Interestingly, not every cell line with the JAK2 V617F mutation showed a good response upon JAK2 inhibition, indicating that there are additional factors determining response. On the other hand, clinical trials with JAK inhibitors in myelofibrosis have shown responses in V617F-mutated and non-mutated patients, warranting further research to identify predictors of response. In BCR-ABL mutant cells not harbouring JAK2 mutations no significant inhibition of proliferation or apoptosis was detected following JAK2 inhibition, indicating that there are JAK2 independent signal transduction pathways of BCR-ABL to avoid apoptosis.

le Coutre:Novartis Pharmaceuticals: Honoraria, Research Funding.