BVB808 rap- idly and potently blocked JAK2-dependent phosphorylat

BVB808 rap- idly and potently blocked JAK2-dependent phosphorylation of STAT5 and induced PARP cleavage in JAK2 V617F-dependent MB-02 and SET-2 cells. Inhibition of pSTAT5 required an10-fold higher dose of BVB808 in CMK cells compared with MB-02 and SET-2 cells, consistent together with the preferential activity against JAK2. To determine the in vivo activity of BVB808, we employed a bone marrow transplant model of Jak2 V617F-driven MPN. Bone marrow from BALB c mice was transduced with Jak2 V617F and transplanted into congenic recipients. Upon de- velopment of polycythemia, mice have been randomized to treat- ment with 50 mg kg of either vehicle or BVB808 twice daily. Just after 3 wk of therapy, mice had been sacrificed and assessed for pharmacodynamic and clinical endpoints. Compared with controls, BVB808-treated mice had decreased reticulocyte and WBC counts.
BVB808 lowered bone marrow hypercellularity, normalized spleen weight, and suppressed pSTAT5 in both selelck kinase inhibitor spleen and bone marrow. Point mutations in the JAK2 kinase domain confer resistance to JAK inhibitors Mutations in tyrosine kinases are a widespread cause of genetic resistance to enzymatic inhibitors. To identify resistance mutations in JAK2, we modi- fied an approach that was previously applied to determine BCR ABL1 mutations that confer resistance to imatinib. Expression of CRLF2 having a JAK2 R683G renders murine Ba F3 cells capable of development within the absence of IL-3. We randomly mutagenized human JAK2 R683G cDNA and transduced the mutagenized cDNA library into Ba F3 cells expressing CRLF2. The transduced popula- tion was selected in 1 M BVB808 in the absence of IL-3. Within two 3 wk, a number of BVB808-resistant clones expanded from single cells. We sequenced the mutagenized JAK2 R683G cDNA from genomic DNA of person BVB808-resistant clones and identified several clones with E864K, Y931C, or G935R mutations.
Even within the absence of a transforming oncogene, trans- duction of Ba F3 cells can sometimes result in individual clones which have escaped IL-3 independence via non- JAK2 mediated signaling. If this occurred, the surviving IL-3 independent cells would selleck be resistant to JAK2 inhibitors but not dependent on JAK2. Thus, we took 3 approaches to confirm that the cells expressing E864K, Y931C, or G935R in cis with a JAK2 gain-of-function allele are dependent on JAK2 function and resistant to enzymatic inhibitors. First, we recloned the mutations into human JAK2 R683G cDNA by site-specific mutagenesis and confirmed their capability to confer BVB808 resistance when expressed in combination with independently in cis with mouse Jak2 V617F and expressed them with all the erythropoietin receptor in Ba F3 cells.