Shorr, Jolene

Abstract Background: Systemic mastocytosis (SM) and hypereosinophilic syndrome (HES) are JAK2V617F-negative orphan diseases without effective long-term therapeutic options. Both of these diseases share clinical and laboratory features with FIP1L1-PDGFRA-positive chronic eosinophilic leukemia (CEL). SM is associated with KITD816V or other KIT mutations. HES is currently not molecularly characterized. The exquisite sensitivity of FIP1L1-PDGFRA to inhibition by imatinib mesylate (IM) underlies the efficacy of this agent in treating FIP1L1-PDGFRA-positive CEL. In contrast, KITD816V is resistant to inhibition by IM, and is only moderately sensitive to dasatinib (cellular IC50=micromolar concentrations), which may explain the relatively disappointing clinical responses in SM with these agents. TG101348 is an orally bioavailable JAK2-selective inhibitor that is currently being tested in a Phase 1 clinical trial for the treatment of myelofibrosis. The aim of the current study was to evaluate TG101348 (relative to the tyrosine kinase inhibitors IM, dasatinib, and sorafenib) for its ability to: inhibit growth of leukemic cell lines that carry KITD816V and FIP1L1-PDGFRA; and inhibit the in vitro growth of eosinophil colonies derived from progenitor cells from HES patients. Methods: The following cell lines were used for in vitro experiments: HMC-1, a mast cell leukemia line (KITD816V-positive); EOL-1, a CEL derived line (FIP1L1-PDFRA-positive), Ba/F3 T674I, a Ba/F3 line that express the IM-resistant FIP1L1-PDFRAT674I mutation, and HEL, a human erythroleukemia line (JAK2V617F-positive). Cell proliferation assays were performed in triplicate using the XTT assay (Leukemia. 2007 21:1658). Drug concentrations in these experiments ranged from 2.9 × 10−12 M to 10−4 M. JAK Inhibitor I (Calbiochem) is a non-selective JAK inhibitor tool compound. Eosinophil colonies were obtained by plating PBMCs from healthy controls or HES patients in methylcellulose, in the presence of IL-3, IL-5, and GM-CSF. Effects of IM and TG101348 on eosinophil colony growth were studied in parallel experiments. Results: Consistent with prior observations, the HMC-1 cell line was resistant to inhibition by IM (IC50>10 mM), but was moderately sensitive to dasatinib (Table). In contrast, TG101348 inhibited HMC-1 growth at nanomolar concentrations, similar to its effect on HEL cells that harbor the JAK2V617F mutation. TG101348 inhibited HMC-1 growth 53-fold and 4-fold more potently than IM and dasatinib, respectively (Figure). Sorafenib, a multikinase inhibitor that reportedly has activity against KIT kinase (enzyme IC50=68 nM), had only a limited effect on HMC-1 growth (IC50=4000 nM). In contrast to HMC-1, EOL-1 growth was inhibited by IM, dasatinib, sorafenib, and TG101348 at picomolar concentrations (Table). The T674I mutation (in the context of FIP1L1-PDGFRA) corresponds to the T315I mutation in BCR-ABL – it occurs infrequently in CEL and is usually found in the context of transformation to acute leukemia. Growth of Ba/F3 cells harboring FIP1L1-PDFGRA-T674I has been shown to be inhibited by sorafenib (confirmed in this report; Table) and nilotinib, at nanomolar concentrations. TG101348 is less potent at inhibiting growth of these cells, with cellular IC50 of ~2 mM – pharmacokinetic data from the ongoing Phase I study of TG101348 shows that this concentration is achievable in plasma with once daily dosing. Drug Cellular IC50 HMC-1 (nM) EOL-1 (pM) Ba/F3 T674I (nM) HEL (nM) Imatinib 18800 4 10700 - Dasatinib 1300 <1 30900 - Sorafenib 4000 2 16 - Jak Inhibitor I 926 30 4300 ? TG101348 355 <1 1950 300 Figure Figure Conclusions: The JAK2-selective inhibitor TG101348 is a potent inhibitor of KITD816V and FIP1L1-PDGFRA, warranting clinical trials using this drug in SM and FIP1L1- PDGFRA-positive CEL. Data regarding TG101348 effects on signaling intermediates in leukemic cell lines and on eosinophil colony growth will be presented at the meeting – data from the latter experiments may indicate a therapeutic role for TG10134 in the treatment of patients with HES.

Abstract Background: The recent discovery of JAK2V617F, as an activating mutation in the majority of patients with myeloproliferative neoplasms, has spurred development of JAK2-selective small molecule inhibitors for the treatment of these diseases. TG101348 is a potent (enzyme IC50=3 nM) and selective (35- and 334-fold selectivity over JAK1 and JAK3, respectively) orally bioavailable JAK2 inhibitor that is effective in treating a murine model of Polycythemia vera (PV) (Cancer Cell. 2008 13:311). TG101348 selectively inhibits growth of hematopoietic colonies harboring V617F, MPLW515K, or JAK2 exon 12 mutations (Leukemia. 2008 Mar 20, Epub). Methods: A Phase I dose-escalation study of TG101348 is ongoing for patients with high- or intermediate-risk (with symptomatic disease) primary myelofibrosis (PMF) and post-PV or post-essential thrombocythemia (ET) myelofibrosis (Cancer.2007109:2083). TG101348 was administered orally once daily in 28-day cycles in cohorts of at least 3 patients. Additional eligibility criteria included absolute neutrophil count of ≥1 × 109/L and platelet count of ≥50 × 109/L, but a JAK2 mutation was not required. Primary objectives were determination of safety and tolerability, as well as pharmacokinetic behavior of TG101348. The secondary objectives were evaluation of treatment response (Blood.2006108:1497), pharmacodynamic activity, and drug effect on V617F allele burden and plasma cytokine levels. The spleen response was documented as change in palpable spleen tip from the costal margin. For patients achieving less than a complete remission after 3 cycles of treatment at their initial dose, step-wise escalation was permitted at each cycle if a cohort had been shown to have tolerated that dose. Results: Fifteen patients (9 males; median age=66 years; range=53 to 79 years) have been enrolled in the study to date – 10 with PMF (8 V617F+) and 5 with post-PV MF (all V617F+). The median follow-up to date is 13 weeks (range 1–28). Treatment was discontinued in 1 patient, in cohort 1, on the 2nd day of dosing after it was discovered that the patient had baseline QTcF prolongation (Grade 1). In the remaining 14 patients, no dose-limiting toxicities or other significant non-hematological adverse events have been observed to date. The pharmacokinetic parameters of TG101348 in plasma were well characterized following single daily oral doses of 30 mg, 60 mg, 120 mg or 240 mg. Dose-proportional pharmacokinetics were observed following single and multiple doses of TG101348. Treatment response data are as follows: Cohort 1 (starting dose=30 mg; 3 patients; median follow-up=26 weeks): 2 of the 3 patients had improvement in constitutional symptoms including fatigue, night sweats and pruritus. No other benefits were noted. These patients have now been dose escalated to either 120 or 240 mg/day. Cohort 2 (starting dose=60 mg; 3 patients; median follow-up=18 weeks): 1 patient had a 40% decrease in spleen size (18 to 10 cm). All 3 patients have now been dose escalated to 120 or 240 mg/day. Cohort 3 (starting dose=120 mg; 3 patients; median follow-up=13 weeks): 2 of the 3 patients reported improvement in constitutional symptoms including fatigue, night sweats, and pruritus. 1 patient had a 25% decrease in spleen size (28 to 21 cm). 2 patients have been dose escalated to 240 mg/day. Cohort 4 (starting dose=240 mg; 3 patients; median follow-up=8 weeks): 1 patient developed Grade 3 anemia (baseline Grade 1) at the end of the first cycle. 1 patient reported decreased fatigue. All 3 patients have experienced a 20 to 53% reduction in spleen size; baseline measurements were 10, 13, and 17 cm. Cohort 5 (starting dose=360 mg; 2 patients; median follow-up=1 week). Both patients had decreased splenomegaly within 1 week of therapy, including 1 with disappearance of palpable splenomegaly from a baseline of 20 cm; this patient also reported significant improvement in constitutional symptoms. Conclusions: Preliminary findings indicate that TG101348 is well tolerated in patients with myelofibrosis. Although it is too early to make valid conclusions, a decrease in spleen size has been noted at higher dose levels. Similarly, improvement in constitutional symptoms has been reported by some patients. Updated results on current and future patients, including data on pharmacokinetics, pharmacodynamics, and changes in V617F allele burden and plasma cytokine levels will be presented at the meeting.

Abstract Abstract 459 Background: A Phase I study of TG101348, a potent, oral JAK2-selective inhibitor in patients with high- or intermediate-risk primary, post-polycythemia vera (PV) and post-essential thrombocythemia (ET) myelofibrosis (MF) recently completed enrollment. Data from the dose escalation cohort (n=28; 30–800 mg administered as a single daily dose) showed dose-linear plasma exposure, with mean elimination T1/2 at steady state ranging from 16 to 34 hours. The dose-limiting toxicity was reversible asymptomatic grade 3 or 4 amylasemia/lipasemia; the maximum tolerated dose was 680 mg. We now present updated results from an extension study in which subjects continued treatment beyond six 28-day cycles. Result: In the entire study, 59 subjects (median age 64 years) were treated. Forty four patients had PMF, 12 post-PV MF, and 3 post-ET MF; 86% were JAK2V617F-positive. Median palpable spleen size was 18 cm and 21 (36%) patients were transfusion-dependent at study enrollment. Forty three (73%) subjects continued treatment on the extension study; the median (range) cumulative exposure to TG101348 was 380 days (170-767); the number of cycles of treatment completed ranged from 7–29, and the proportion of subjects who completed 12, 18, and 24 cycles of treatment was 72%, 28%, and 14%, respectively. The median (range) treatment dose at data cutoff was 440 mg/day (120 to 680 mg/day; n = 35). Toxicity: TG101348 was well tolerated. Non-hematologic, at least possibly related adverse events included nausea (69%; Grade 3=3%), diarrhea (64%; Grade 3=10%), and vomiting (58%; Grade 3=3%), that were dose-dependent; the symptoms were self-limited or controlled with symptomatic treatment and/or dose reduction. Also observed were asymptomatic increases in serum lipase (27%), AST/ALT (27%/25%), creatinine (24%), and alkaline phosphatase (17%). Grade 3/4 hematological adverse events possibly related to TG101348 included treatment-emergent anemia (35% of patients who were not transfusion dependent at baseline), thrombocytopenia (24%), and neutropenia (10%). Spleen: During cycles 1–6, 42% of patients achieved Clinical Improvement (CI) per the International Working Group for Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) criteria for spleen response. The CI rate for the 43 subjects who continued treatment beyond 6 cycles was 65%, and the median time (range) to CI was 140 days (77-802). After 12 cycles, 36 (95%) of the 38 evaluable patients had some degree of spleen size reduction; 32 (85%), 26 (68%), and 10 (26%) of subjects had a minimum 25%, 50%, and 100% decrease in palpable splenomegaly. JAK2V617F allele burden: Of the 48 JAK2V617F mutation-positive subjects, 23 (48%) had a baseline mutant allele burden of >20%. Of these, 20 subjects with a baseline median allele burden (range) of 62% (23% to 100%) completed at least 6 cycles of treatment. Sixteen subjects (80%) exhibited a median 62% (range 6% to 96%) decrease in JAK2V617F burden, including 9 (45%) with a ≥50% decrease; 4 subjects (20%) had increases (range 18% to 58%). Eighteen subjects (78%) completed 12 cycles of treatment; 13 (72%) had a median 50% decrease (range 29% to 82%), including 7 (39%) subjects with a ≥50% decrease; 5 subjects (28%) had no change or increases (range 0% to 22%) in JAK2V617F. Constitutional symptoms: For patients with data at baseline and cycle 6, early satiety, fatigue, night sweats, cough, and pruritus were reported by 56%, 47%, 24%, 22%, and 19% of subjects, respectively. Improvement (complete resolution) rates observed at cycle 6 were: 75% (40%), 65% (29%), 89% (89%), 88% (75%), and 100% (57%) for the aforementioned symptoms, respectively. Leukocytosis/Thrombocytosis: Leukocytosis was present at baseline in 33 subjects; following 6 and 12 cycles of therapy, 57% (n=28) and 56% (n=25) achieved normal WBC counts. Thrombocytosis was noted at baseline for 10 (17%) subjects; following 6 and 12 cycles of therapy, 90% (n=10) and 88% (n=8) achieved normal platelet counts. Conclusion: The current study provides proof-of-concept that selective JAK2 inhibition has anti-myeloproliferative/anti-clonal activity in myelofibrosis. The majority of subjects treated with TG101348 for more than 6 cycles experienced a rapid, significant, and durable decrease in splenomegaly, normalization of leukocyte/platelet counts, and improvement of disease-related symptoms, all within the context significantly decreased JAK2V617F burden. Disclosures: Off Label Use: There is no FDA approved drug for Myelofibrosis.All the drugs discussed will be off-label. Stone:Novartis: Consultancy. Silverman:TargeGen: Consultancy. Shorr:TargeGen: Employment, Equity Ownership.

Abstract Abstract 755 Background: TG101348 is a potent, orally bioavailable, JAK2-selective small molecule inhibitor, that is currently being evaluated in a Phase I study for the treatment of myelofibrosis. Data from the dose escalation cohort (n=28; 30-800mg administered as a single daily dose) showed dose-linear plasma exposure, with mean elimination T1/2 at steady state ranging from 20 to 52 hours. The dose-limiting toxicity was asymptomatic grade 3 or 4 amylasemia/lipasemia that was reversible, and the maximum tolerated dose (MTD) was 680mg. The most frequent non-hematological toxicities were mild nausea, vomiting, and/or diarrhea that were easily controlled or resolved spontaneously. Grade 3/4 neutropenia and thrombocytopenia were observed in 14% and 25% of patients, respectively. TG101348 had activity in reducing spleen size, leukocyte count, and JAK2V617F (VF) allele burden. Here, we present updated results with a focus on data from the dose confirmation cohort who initiated treatment at a dose of 680mg/day. Results: Fifty nine patients (median age=66 years; range 43-86) have been treated – 28 in the dose escalation phase, and 31 in the dose confirmation phase. Overall, 44 patients had PMF, 12 post-PV MF, and 3 post-ET MF; 86% were VF-positive. Median palpable spleen size was 18cm and 22 patients were RBC transfusion-requiring at study enrollment. After a median follow-up of 12 weeks (range <1-76), 18 (31%) patients have discontinued treatment due to toxicity (n=7; thrombocytopenia=3, neutropenia=1), comorbidities (n=5), withdrawal of consent (n=4), or non-compliance/lack of response (1 each). The remaining 41 patients are currently at the following dose levels: 680mg (n=14), 520-600mg (n=16), 360-440mg (n=10), and 240mg (n=1). The cumulative drug exposure to date is 362 patient-months; exposure at or above MTD (≥680mg) is 154 patient-months. Forty patients (68%) started treatment at ≥680mg. Toxicity: TG101348 is well tolerated. Of the patients who started at ≥680mg, Gr3/4 neutropenia was observed in 15/0% and Gr3/4 thrombocytopenia in 20/10%. Twenty four (60%) patients did not require RBC transfusions at baseline (median Hgb=9.6g/dL; range 7.4-13.1); of these, 42% and 8% of patients developed Gr3 and Gr4 anemia, respectively. The majority of patients who started at ≥680mg developed mild nausea (1 Gr3), vomiting (1 Gr3), and/or diarrhea (3 Gr3) that were self-limited or easily controlled. Other non-hematological toxicities included Gr1/2 transaminitis (38%), Gr1/2 serum creatinine elevation (38%), and asymptomatic hyperlipasemia (33%). Efficacy: Thirty three patients who started at ≥680mg have completed at least 3 cycles of treatment; at 3 months, reduction in palpable spleen size (baseline median=18cms; range 6-32) was at least 50% in 22 (67%) patients; the spleen became non-palpable in 9 (27%) patients. All 21 patients with leukocytosis at baseline (WBC range 11 to 203 ×109/L) who started at ≥680mg have experienced a marked reduction in their WBC count (range 4 to 90); 70% had a normal WBC count at their last follow-up visit. Overall, 48 of the 51 VF-positive patients completed at least 1 cycle and were evaluable for response in VF allele burden; at last available follow-up, the median decrease in granulocyte mutant allele burden was 48%; 21 (44%) patients have had a ≥50% reduction, and in the group who started treatment at ≥680mg, 48% have had a ≥50% reduction. Of those evaluable, there was clinically significant benefit or resolution of constitutional symptoms, including early satiety, fatigue, cough, pruritus, and night sweats. Conclusions: TG101348 continues to be well tolerated in patients with myelofibrosis. Spleen and leukocyte responses are frequent, observed early, and produce substantial clinical benefit for patients. These responses are associated with significant decrease in VF allele burden and point to activity of TG101348 against the malignant clone in myelofibrosis. Disclosures: Pardanani: TargeGen: Research Funding; Cytopia: Research Funding. Off Label Use: Data from ongoing clinical trial will be presented. Gotlib:TargeGen: Research Funding. Jamieson:Merck: Research Funding; Pfizer: Research Funding; Wintherix: Consultancy; TargeGen: Research Funding; Celgene: Research Funding. Cortes:Targegen: Research Funding. Stone:Cephalon: ad hoc advisory board. Silverman:TargeGen: Consultancy. Shorr:TargeGen: Employment, Equity Ownership. Gilliland:Merck: Employment. Tefferi:TargeGen: Research Funding.

Purpose Myelofibrosis is a myeloid malignancy associated with anemia, splenomegaly, and constitutional symptoms. Patients frequently harbor JAK-STAT activating mutations that are sensitive to TG101348, a selective small-molecule Janus kinase 2 (JAK2) inhibitor. Patients and Methods In a multicenter phase I trial, oral TG101348 was administered once a day to patients with high- or intermediate-risk primary or post–polycythemia vera/essential thrombocythemia myelofibrosis. Results Fifty-nine patients were treated, including 28 in the dose-escalation phase. The maximum-tolerated dose was 680 mg/d, and dose-limiting toxicity was a reversible and asymptomatic increase in the serum amylase level. Forty-three patients (73%) continued treatment beyond six cycles; the median cumulative exposure to TG101348 was 380 days. Adverse events included nausea, vomiting, diarrhea, anemia, and thrombocytopenia; corresponding grades 3 to 4 incidence rates were 3%, 3%, 10%, 35%, and 24%. TG101348 treatment had modest effect on serum cytokine levels, but greater than half of the patients with early satiety, night sweats, fatigue, pruritus, and cough achieved rapid and durable improvement in these symptoms. By six and 12 cycles of treatment, 39% and 47% of patients, respectively, had achieved a spleen response per International Working Group criteria. The majority of patients with leukocytosis or thrombocytosis at baseline (n = 28 and n = 10, respectively) achieved normalization of blood counts after six (57% and 90%, respectively) and 12 (56% and 88%, respectively) cycles. A significant decrease in JAK2 V617F allele burden was observed at 6 months in mutation-positive patients (n = 51; P = .04), particularly in the subgroup with allele burden greater than 20% (n = 23; P < .01); the decrease was durable at 12 months. Conclusion TG101348 is well tolerated and produces significant reduction in disease burden and durable clinical benefit in patients with myelofibrosis.