Introduction: Several groups are developing orally bioavailable small molecule JAK2 inhibitors as an approach to targeted therapy of myeloproliferative disorders. We previously reported that one such inhibitor (TG101209) potently inhibits JAK2V617F in vitro, and was effective in reducing tumor growth in a mouse xenograft model (Leukemia, 2007 Aug;21(8):1658–68). Here, we expand our analysis using TG101348, a highly selective JAK2 inhibitor currently in preclinical development, using cell lines and primary cells harboring JAK2V617F, MPLW515L/K, or JAK2 exon 12 mutations.

Methods: Cell proliferation was assessed using the XTT assay. For primary cell experiments, PBMCs were plated in methylcellulose medium at 0, 300nM, 600nM, and 1200nM TG101348, both with and without erythropoietin (Epo). Hematopoietic colonies were scored after 12–16 days culture, and harvested for genotyping.

Results: TG101348 inhibits growth of human erythroleukemia (HEL) and Ba/F3-V617F cells (IC50=300nM and 580nM, respectively), as well as Ba/F3-(MPL)W515L cells (IC50=620nM). JAK2:JAK3 selectivity of TG101348 was demonstrated by its 10-fold more potent inhibition of CHRF (JAK2T875N) as compared to CMK (JAK3A572V) cell proliferation. TG101348’s inhibitory effect on ex vivo hematopoietic colony growth was studied in 12 MPD patients (JAK2V617F=9, MPLW515L/K=1, JAK2 exon 12 mutation [N542-E543del]=1, no mutation=1), and 2 healthy controls (Table).

Conclusions: (1) TG101348 inhibits colony growth from MPD patients more potently than healthy controls, reflecting its potential therapeutic window. (2) Endogenous colony growth is inhibited at nanomolar concentrations in all cases, with some alleles showing greater sensitivity than others (MPLW515K = exon 12 mutation > JAK2V617F). The corresponding Epo-supported erythroid colonies are less potently inhibited, reflecting the generally lower prevalence of JAK2 mutations in such colonies. (3) EEC growth in the absence of known JAK2 or MPLW515 mutations is also inhibited, indicating a similar dependence on dysregulated JAK-STAT signaling in such cases. (4) The ability of TG101348 to selectively suppress mutation-positive hematopoietic colonies will be discussed.

Inhibitor effect on ex vivo hematopoietic colony growth

PaientMutationIC50 (nM)% Mutation-positive colonies
  Epo No Epo Epo No Epo (EEC) 
Normal 1 None >1200 n/a n/a n/a 
Normal 2 None >1200 n/a n/a n.a 
PV1 V617F 600 <300 5 40 
PV2 V617F 600 300 33 50 
PV3 V617F 600–1200 300–600 9 70 
PV4 V617F 300–600 600 0 15 
PV5 V617F 600–1200 300 80 100 
PV6 V617F 600 300–600 7 19 
PV7 V617F 600–1200 600 32 36 
PV8 V617F 600–1200 <300 45 37 
PV9 V617F 600 600–1200 20 12 
PV10 Exon 12 (N542–E543del) 900 <300 9 60 
PMF1 W515K 300–600 <300 100 33 
PMF2 None >1200 300 n/a n/a 
PaientMutationIC50 (nM)% Mutation-positive colonies
  Epo No Epo Epo No Epo (EEC) 
Normal 1 None >1200 n/a n/a n/a 
Normal 2 None >1200 n/a n/a n.a 
PV1 V617F 600 <300 5 40 
PV2 V617F 600 300 33 50 
PV3 V617F 600–1200 300–600 9 70 
PV4 V617F 300–600 600 0 15 
PV5 V617F 600–1200 300 80 100 
PV6 V617F 600 300–600 7 19 
PV7 V617F 600–1200 600 32 36 
PV8 V617F 600–1200 <300 45 37 
PV9 V617F 600 600–1200 20 12 
PV10 Exon 12 (N542–E543del) 900 <300 9 60 
PMF1 W515K 300–600 <300 100 33 
PMF2 None >1200 300 n/a n/a 
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Topics:
fedratinib, jak2 gene exon 12, mutation, myeloproliferative disease, acute erythroblastic leukemia, erythropoietin, genotype determination, leukemia, mechlorethamine, molecular targeted therapy

Author notes

Disclosure: Employment: John Hood, Richard Soll, and Glenn Noronha are employees of TargeGen. Ownership Interests: John Hood, Richard Soll, and Glenn Noronha own stock options in TargeGen. Research Funding: Animesh Pardanani, Ayalew Tefferi, and Catriona Jamieson have received research funfing from TargeGen Inc.

2007, The American Society of Hematology
2007
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