Primary Cell Experiments with TG101348, a JAK2-Selective Inhibitor, in the Presence of Myeloproliferative Disorder-Associated JAK2V617F, MPLW515L/K, and JAK2 Exon 12 Mutations.

Abstract

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 growthPaientMutationIC50 (nM)% Mutation-positive coloniesEpoNo EpoEpoNo Epo (EEC)Normal 1None>1200n/an/an/aNormal 2None>1200n/an/an.aPV1V617F600<300540PV2V617F6003003350PV3V617F600–1200300–600970PV4V617F300–600600015PV5V617F600–120030080100PV6V617F600300–600719PV7V617F600–12006003236PV8V617F600–1200<3004537PV9V617F600600–12002012PV10Exon 12 (N542–E543del)900<300960PMF1W515K300–600<30010033PMF2None>1200300n/an/a