ALBERT

Description: 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 Paient Mutation IC50 (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

Description: Introduction: Clinical resistance to kinase inhibitors is an emerging theme in targeted, oncology therapies. For example, Gleevec resistance results due to a host of mutations in bcr-abl, the kinase associated with chronic myelogenous leukemia (CML), and have necessitated the development of 2nd generation inhibitors dasatinib and notilinib. More recently, mutations in JAK2 kinase have recently been identified as the key molecular drivers for the majority of patients with the chronic MPDs polycythemia vera, essential thrombocythemia and myelofibrosis with myeloid metaplasia. TG101348 is a selective inhibitor of JAK2 kinase and represents a novel therapy for these MPDs. Methods: In order to better understand what mutations may occur in clinic as a result of TG101348 treatment, we used N-ethyl-N-nitrosourea (ENU)-exposed human erythroleukemia (HEL) cells to evaluate resistance mutations to TG101348. In brief, ENU pretreated HEL cells were exposed to 3 uM TG101348, a dose that is 10X the cellular IC50, for 28 days followed by genotyping to determine the incidence and location of kinase domain mutants that arose in resistant cells. Results: Eleven different mutations were identified, with the most prevalent mutations in associated with the ATP binding pocket. In cells bearing these mutations TG101348 did not inhibit STAT5 phosphorylation, cellular proliferation or induced apoptosis at concentrations up to 3 uM, whereas in wildtype HEL cells it did. Consistent with the mutations arising specific for TG101348, JAK2 inhibitors which are derived from chemically distinct series were equally potent on wildtype or TG101348 resistant HEL cells. Conclusions: A series of JAK2 point mutations were identified which generate resistance to the JAK2 inhibitor TG101348 in JAK2V617F-bearing cells. None of these mutations corresponded to any mutations that have been previously reported for MPD patients and therefore the clinical relevance of these mutations is not yet known. While these ENU induced mutations do not yet have clinical correlates, monitoring for their presence in any patients who do not respond to TG101348 in clinical trials may shed light on mechanisms of resistance as they occur.

Description: Introduction Polycythemia vera (PV) is characterized by excessive production of erythroid cells and in most cases a point mutation (V617F) in the Jak2 cytokine signaling kinase. We investigated whether a selective JAK2 inhibitor decreased Jak2 V617F induced erythroid differentiation. Methods Wild-type and mutant Jak2 V617F genes were excised from the retroviral Jak2-mus-MSCV-neo vector (Levine et al), cloned into the lentiviral vector pLV CMV IRES2 GFP and their presence verified by DNA sequencing. Lentiviral vectors bearing the wild-type and mutant Jak2 genes or vector alone were used to transduce human peripheral blood CD34+ cells, which were then divided for plating into megacult medium for megakaryocytic colony growth and methylcellulose culture for enumeration of all other progenitor cell types. Normal cord blood HSC (CD34+/CD38−/CD90+) were clone sorted with the FACS Aria and transduced with no vector, backbone vector, wild-type JAK2 or mutant JAK2 vector in methocult media (Stem Cell Technologies Inc, GF+ H4435) +/− 300 nM of a selective JAK2 inhibitor, TG101348. Colonies were scored at day 14. RNA was isolated from the colonies (Qiagen RNeasy kit) and RT-PCR was performed with wild-type and mutant JAK2 allele specific primers. Results Transduction of cord blood HSC with the mutant Jak2 vector resulted in skewed erythroid colony formation compared to wild-type Jak2, vector alone and untransduced HSC (Figure 1; n=3). RT-PCR with murine Jak2 specific primers resulted in ~900 bp fragments corresponding to murine Jak2 from colonies transduced with the wild-type and mutant Jak2 and confirmed by sequencing, but not those from colonies transduced with the vector alone or the untransduced cells. Like the results in cord blood cells, adult peripheral blood CD34+ cells transduced with the mutant Jak2 developed a skewed developmental pattern, with far greater erythroid colony formation compared to wild-type Jak2 or vector alone. In megacult assays, CD34+ cells transduced with the mutant Jak2 had similar megakaryocytic potential as wild-type Jak2 or vector alone. Addition of TG101348 (300 nM), inhibited mutant kinase-induced erythroid colony formation (Figure 1) in 3 experiments while 100– 300 nM was inhibitory to PV (n=2 patients) HSC and progenitors. Current experiments focus on inhibition of Jak2 in a bioluminescent highly immunocompromised mouse model of Jak2V617F-induced myeloproliferation (Figure 2). Conclusion JAK2 V617F skews differentiation of HSC toward the erythroid lineage and may be inhibited with a selective JAK2 inhibitor - TG101348. Figure 1. In vitro JAK2 Inhibition. Figure 1. In vitro JAK2 Inhibition. Figure 2. Bioluminescent JAK2 V617F-induced Myeloproliferation Model. Figure 2. Bioluminescent JAK2 V617F-induced Myeloproliferation Model.

Description: 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 600 300–600 n/a

Description: The molecular pathogenesis of the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocytosis, and myelofibrosis with myeloid metaplasia has been conclusively linked to a single mutation in JAK2 (Janus Associated Kinase 2). A G-T transversion event in exon 14 that translates into a substitution of phenylalanine for valine at amino residue 617 leads to constitutive activation of JAK2V617F in a majority of these MPD cases. Assays in several laboratories indicate that between 90–100% of patients with polycythemia vera harbor this allele. Because the JAK2.V617F gain-of-function mutant affects such a large patient population, pharmacologically targeting the mutated JAK2 is highly clinically relevant. In order to address this unmet clinical need we designed and synthesized a series of structurally novel compounds for their capacity to inhibit JAK2.V617F. Compounds were identified which potently inhibited JAK2 enzyme, with enzymatic IC50s as low as 1 nM. Subsets of this group were subsequently identified which were highly selectivity against undesirable off-target kinases, including up to 100X selectivity versus the highly homologous JAK3 kinase. Compounds were then advanced into in vitro assays in JAK2.V617F transformed cell lines in which compounds were identified with proliferation EC50s as low as 60 nM (representative compounds listed in Table below). Compounds from this series were subsequently shown to be orally available and efficacious in rodent models of MPD driven disease. Conclusion: TargeGen has synthesized a series of JAK2.V617F inhibitors with promising potency, selectivity and pharmaceutical properties for utility in the treatment of myeloproliferative disorders. Representative JAK2 Inhibitors Compound Primary Target Selectivity Profile Cell EC50 JAK2 IC50 JAK2 vs JAK3 (X-fold selectivity) BaF3:JAK2.V617F TG101192 3.8 15 140 TG101209 6 28 279 TGs101348 12.5 83 297 JAK Inhibitor I (Calbiochem) 10 1.5 320

Description: 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.

Description: 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.

Description: A number of genetic mutations have been identified in the development of acute myeloid leukemia (AML), including mutations in the proto-oncogene N-RAS, p53, c-Kit and the FMS-like receptor tyrosine kinase 3 (FLT3). FLT3 plays an important role in the formation of early stem/progenitor cells during hematopoietic development. FLT3 is mutated in ∼30% of AMLs, with internal tandem duplications (ITD) in the juxtamembrane region being the most common (FLT3-ITD). This results in constitutive activation of FLT3 kinase activity and patients harboring such mutations do not respond well to conventional treatments and demonstrate poor clinical prognosis. There is growing evidence that the JAK/STAT pathway may also contribute to the etiology of AML. For instance, the JAK2 V617F point mutation driving various chronic myeloproliferative disorders (MPDs) has recently been detected in de novo AMLs and STAT3/5 phosphorylation is commonly elevated in AML patients. We have developed TG101348, a selective JAK2/FLT3 small molecule inhibitor which has the potential to treat both MPDs and AML. In in vitro based assays, TG101348 was capable of inhibiting the proliferation of human AML cell lines representing a variety of mutations. In AML cell lines carrying mutations in N-RAS such as KG-1, HL-60, and THP-1, EC50s ranged from ∼300 nM-1000 nM. In TF-1a AML cells, which harbor a mutation in p53, TG101348 demonstrated an EC50 of 493 nM. Most interestingly, for these cell lines, FLT3/VEGFR2 specific small molecule inhibitors such SU5416 were significantly less effective, demonstrating EC50s that were generally 〉10-fold higher. TG101348 was most potent against the FLT3-ITD positive cells lines, MV411 (EC50=57 nM) and MOLM13 (EC50=69 nM). In MV411 cells, TG101348 functionally inhibited FLT3 autophosphorylation, and phosphorylation of downstream intermediates including STAT5, ERK, and AKT. In a human MV411 xenograft models, oral administration (120mg/kg, BID) of TG101348 resulted in complete regression of the subcutaneously implanted MV411 tumors. Taken together, these data suggest that the dual-acting JAK2/FLT3 kinase inhibitor TG101348 may have a therapeutic advantage in that it has the capacity to block multiple pathways critical for dysregulated cell proliferation and survival in AML.

Description: The T315I point mutation situated at the ATP-binding site of BCR/ABL tyrosine kinase remains to be the major unmet medical need in treating CML patients. None of the marketed CML drugs, including Imatinib or the recently approved Dasatinib, are able to inhibit this clinically most prevalent mutant of BCR/ABL kinase. To overcome the T315I mutation-presented hurdle, a novel small molecule BCR/ABL inhibitor, TG101223, has been synthesized and characterized in vitro and in vivo. Enzymatic activity of T315I mutant kinase and proliferation of BaF3:BCR/ABLT315I cells were inhibited with an IC50 of 50 nM and EC50 of 500 nM respectively. However, little effect of TG101223 was observed on the proliferation of the control BaF3 cells (IC50≥5,000 nM). Consistent with these observations, 24 hour treatment with 2,000 nM TG101223 induced DNA fragmentation in BCR/ABLT315I cells, but not in the control BaF3 cells. Furthermore, caspase-3 activation was detected in BCR/ABLT315I cells by an in-cell caspase assay following 4 h treatment with 2,000 nM TG101223. The above results strongly suggest that TG101223 inhibits BCR/ABLT315I-mediated cell proliferation and survival. In contrast, Imatinib did not inhibit AblT315I enzyme nor BCR/ABLT315I cell proliferation. The kinase selectivity of TG101223 was determined using a commercial enzyme assay with a panel of 49 phylogentically diverse protein kinases. Only Abl, Flt3 and the mutants of those two kinases were inhibited by more than 80% with 500 nM TG101223. Importantly, TG101223 demonstrated a desirable pharmacokinetic profile in mouse (oral bioavailability: 40%, clearance: 23 mL/min/kg and T½: 5 h). The tumorogenicity of BCR/AblT315I cells were confirmed by the observation that tail vein infusion with BCR/ABLT315I cells induced splenomegaly in SCID mice and death by day 14. To assess the in vivo activities of TG101223, mice were dosed orally once with TG101223 on day 13, followed by harvesting of their spleens 7 hours after dosing for Western blot-based protein phosphorylation analysis. A significant induction in phosphorylation of both CrkL and BCR/ABL was observed in the spleens of T315I cell recipient mice, supporting the notion that splenomegaly is a result of the infiltration of proliferating BCR/ABLT315I cells. The above induction in spleen CrkL and BCR/ABL phosphorylation was inhibited by TG101223. These data suggest that TG101223 is a promising candidate for treating the drug resistant CML patients.