Background: acquisition of somatic mutations including JAK2V617F or MPLW515L/K results in constitutive activation of JAK-STAT signaling that commonly characterizes myeloproliferative disorders (MPD). JAK2 is the central signaling mediator for most hematopoietic cytokines in this signaling pathway.

Hypothesis: small molecule JAK2 inhibitors may have a therapeutic role in MPD regardless of JAK2 mutation status.

Methods: We studied effects of the JAK2 inhibitor, TG101209, in a cell-free kinase assay, relevant cell lines, an in vivo phosphorylation assay, and primary cells from MPD patients carrying JAK2V617F or MPLW515L/K mutations.

Results: in a cell-free kinase assay, TG101209 inhibited JAK2 potently (IC50=6nM) and displayed selectivity relative to JAK3, ABL, and VEGFR2 kinases (IC50=170nM, 820nM, and 150nM, respectively). In cell proliferation assays, TG101209 inhibited JAK2-expressing Ba/F3 cells (IC50=280nM) and JAK2V617F-homozygous human erythroleukemia (HEL) cells (IC50=300nM), but not BCR-ABL-carrying K562 cells (IC50>2μM). At 24 hours, TG101209 (600nM) induced apoptosis of HEL cells but not K562 cells (40% and 5%, respectively). SCID mice injected intravenously with Ba/F3-JAK2V617F cells developed significant disease burden including massive splenomegaly due to tumor infiltration (day 12). We observed marked decrease in STAT5 phosphorylation in splenic tumors 5 hours after administration of 2 oral doses of TG101209 (50 mg/kg) in this in vivo model. Based on the above data, we plated patient-derived CD34+ cells in methylcellulose (0nM, 300nM, and 600nM TG101209) with and without cytokines. We studied 2 controls and 9 MPD patients: PV=5 (all JAK2V617F+) and AMM=4 (3 were MPLW515L/K+) for colony number, colony size, and colony mutation burden, under each assay condition. TG101209 inhibited overall colony growth from MPD patients (IC50=300–600nM), and it preferentially suppressed growth of mutant colonies relative to wild type in both of 2 MPLW515K+ patients (AMM1 and AMM2) and in 3 of 5 JAK2V617+ patients (PV1-PV3) (Table).

Conclusions: TG101209 potently inhibits cell growth that is dependent on constitutive JAK-STAT signaling. Furthermore, for MPD patient-derived cells, TG101209 preferentially suppresses growth of progenitors carrying JAK-activating mutations (MPLW515K>JAK2V617F>MPLW515L). Hence, our data supports the strategy of targeting aberrant JAK-pathway signaling in MPD as a viable therapeutic approach.

PatientMutationIC50 (nM)No Drug (% colonies mutation-positive)TG101209 (% colonies mutation-positive)
ErythroidMyeloidErythroidMyeloidErythroidMyeloid
Normal WT 1000 600 n/a    
PV1 JAK2V617F ~600 300–600 82 90 36 70 
PV2 JAK2V617F ~300 70 55 60 20  
PV3 JAK2V617F NA  20 30 30 0 
PV4 JAK2V617F >600 ~600 7 10 17 10 
PV5 JAK2V617F ~300 300–600 64 92 82 100 
AMM1 MPLW515K <300 ~300 100 90 0 20 
AMM2 MPLW515K 300–600 ~300 100 90 25 36 
AMM3 MPLW515L 300–600 300–600 100 100 92 90 
AMM4 WT >600 300–600 n/a    
PatientMutationIC50 (nM)No Drug (% colonies mutation-positive)TG101209 (% colonies mutation-positive)
ErythroidMyeloidErythroidMyeloidErythroidMyeloid
Normal WT 1000 600 n/a    
PV1 JAK2V617F ~600 300–600 82 90 36 70 
PV2 JAK2V617F ~300 70 55 60 20  
PV3 JAK2V617F NA  20 30 30 0 
PV4 JAK2V617F >600 ~600 7 10 17 10 
PV5 JAK2V617F ~300 300–600 64 92 82 100 
AMM1 MPLW515K <300 ~300 100 90 0 20 
AMM2 MPLW515K 300–600 ~300 100 90 25 36 
AMM3 MPLW515L 300–600 300–600 100 100 92 90 
AMM4 WT >600 300–600 n/a    
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Topics:
carrying, cytokine, jak2 gene v617f, kinase inhibitors, mutation, phosphotransferases, acute erythroblastic leukemia, bcr-abl tyrosine kinase, massive splenomegaly, mechlorethamine

Disclosures: JH, GN, CCM, HZ, and RS are employed by TargeGen Inc.; JH, GN, CCM, HZ, and RS have stock options inTargeGen Inc.; NO research funds were provided to AP, TL, CF, RM, or AT from TargeGen Inc. for scientific experiments.; AT provided expert testimony regarding myeloproliferative disorders to TargeGen Inc. on one occasion for which he received financial compensation.

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2006, The American Society of Hematology
2006
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