Mak, Chi Ching

Abstract The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

Abstract Myeloproliferative disorders (MPDs), diagnosed in 15,000 individuals annually, are characterized by overproduction of lineage-committed blood cells, thrombotic events, occasionally marrow fibrosis or progression to acute myelogenous leukemia and frequently a point mutation (V617F) in the JAK2 kinase. Mutant JAK2 expression results in erythrocyte overproduction in almost all cases of polycythemia Vera (PV). We investigated the molecular mechanisms driving erythroid skewed differentiation and the capacity of a selective JAK2 inhibitor (TG101348) to normalize PV progenitor differentiation. Both JAK2 V617F+ transduced cord blood and PV progenitor erythroid colony formation was potently inhibited by TG101348 (300 nM). Bioluminescent immunocompromised mouse transplantation studies revealed that TG101348 (150 mg/kg) treatment significantly reduced human erythroid engraftment by both PV and JAK2 V617F+ cord blood progenitors. Moreover, the imbalance between GATA-1 and PU.1 transcripts typical of JAK2 V617F+ progenitors normalized after treatment with TG101348. In addition, TG101348 inhibited both JAK2-mediated STAT5 phosphorylation and AKT-regulated GATA-1 phosphorylation in an erythropoietin responsive cell line suggesting a dual mechanism for the erythroid inhibitory effects of TG101348. Because of selective inhibition of JAK2 V617F+ progenitors both in vitro and in vivo, TG101348 may be an effective molecularly targeted inhibitor of JAK2 V617F+ driven MPDs in clinical trials.

Abstract 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. Patient Mutation IC50 (nM) No Drug (% colonies mutation-positive) TG101209 (% colonies mutation-positive) Erythroid Myeloid Erythroid Myeloid Erythroid Myeloid 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