ALBERT

Description: More than 98% of patients with polycythemia vera (PV) carry the activating JAK2 mutation V617F, which may play a role in the pathogenesis of PV by promoting hematopoetic cell proliferation and survival. PV is characterized by an increased red blood cell mass and is generally accompanied by increased myeloid and megakaryocytic production. Current therapy consists of supportive care measures including aspirin, phlebotomy, interferon and hydroxyurea to reduce the risk of complications of the disease such as thrombotic events. Such palliative therapy is often unsatisfactory due to side effects and the persistent susceptibility of the patients to development of thrombosis, and the failure to prevent transformation to myelofibrosis or acute leukemia. XL019 potently and selectively inhibits JAK2 (Ki of 2 nM); relative to other JAK family kinases (JAK1 Ki of 134 nM, JAK3 Ki of 195 nM and TYK2 Ki of 344 nM). Cellular selectivity of XL019 was confirmed in primary human cell assays; while EPO-stimulated pSTAT5 in primary erythroid cells was quite sensitive to XL019 treatment (IC50 of 64 nM), stimulation of T-cells with IL-2 or B-cells with IL-4 or IL-6 gave pSTAT IC50s of 〉800 nM in each case. Potent effects on JAK2-STAT signaling were also demonstrated in single-dose pharmacodynamic studies in xenograft tumors, including HEL92.1.7, CFPAC-1 and DU 145. XL019 is being evaluated in a Phase 1 dose escalation study in subjects with PV who have failed or are intolerant of standard therapies defined as follows: symptomatic splenomegaly in spite of current therapy, thrombocytosis with a platelet count 〉450K/μl, or non-controlled constitutional symptoms such as pruritus, night sweats, or weight loss. The primary objectives of this study are to determine the safety and tolerability of XL019 when administered orally either once daily, or every Monday, Wednesday, and Friday in 28-day cycles (starting doses 10 mg QD and 25 mg QMWF). Secondary objectives include determination of the pharmacokinetics and pharmacodynamics of XL019, and to evaluate clinical response using the following endpoints: hematologic response; time to hematologic response; duration of hematologic response; phlebotomy independence; change in spleen and/or liver size; change in bone marrow morphology; impact on quality of life (QOL); and impact on functional exercise capacity. Pharmacodynamic assessments for this study include JAK2 V617F allele burden, erythropoietin-independent colony formation, changes in cytokine levels and JAK2 signaling pathways in peripheral blood mononuclear cells, and changes in bone marrow histology. Our initial findings regarding the tolerability, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of XL019 in PV patients will be presented.

Description: Since the identification of the JAK family of intracellular tyrosine kinases and their downstream signaling partners, the STATs (Signal Transducers and Activators of Transcription), evidence has accumulated that activation of JAK signaling pathways can contribute to proliferation and survival of cancer cells. A direct link to oncogenesis was made when an activated form of JAK2 (JAK2 V617F) was identified in the majority of patients with myeloproliferative disorders (MPDs) including approximately half of patients with myelofibrosis (MF). Introduction of this JAK2 mutation into the bone marrow of mice induced erythrocytosis [Wernig et al. (2006) Blood107:4274] supporting JAK2 as a relevant target for treatment of myeloproliferative disorders. We have identified a novel small molecule inhibitor of JAK2, XL019, which may prove to have utility in treating MPDs. XL019 is a potent and reversible inhibitor of the JAK2 enzyme, with a Ki of 2 nM, and shows excellent selectivity (minimum of 50-fold selectivity against 〉120 protein kinases tested including other JAK family members). XL019 downregulates STAT signaling in cell lines expressing both wild type and activated forms of JAK2. IC50s for inhibition of STAT5 phosphorylation by XL019 ranged from 623 nM (HEL92.1.7) to 3398 nM (KG-1) in tumor cell lines. XL019 showed increased potency in primary human erythroid cells in vitro, where the IC50 for inhibition of EPO-stimulated phospho-STAT5 was 64 nM. XL019 inhibits proliferation in cell lines harboring activated or overexpressed JAK2, including certain lines derived from patients with Hodgkin’s Lymphoma (L-1236, 928 nM IC50), AML (MV4-11, 992 nM IC50), essential thrombocythemia (SET-2, 386 nM IC50), and erythroleukemia (HEL92.1.7, 6777 nM IC50). Preclinical single-dose pharmacodynamic studies demonstrate a potent effect of XL019 on JAK-STAT signaling in HEL92.1.7, CFPAC-1 and DU 145 xenograft tumors. Twice daily dosing (bid) led to substantial tumor growth inhibition in the DU145 and HEL models (maximum tumor growth inhibition of 86% and 60%, respectively), accompanied by increases in tumor cell apoptosis (4 – 4.4 fold increase) and decreases in tumor microvasculature (44% reduction in DU 145 xenograft tumors). Based on these encouraging data, XL019 is being evaluated in subjects with primary or post PV/ET MF in a Phase I dose escalation study, which has completed enrollment of the first cohort. The primary objectives of this study are to determine the safety and tolerability of XL019 when administered orally once daily for 21 days in 28 day cycles. Secondary objectives include determination of the pharmacokinetics and pharmacodynamics of XL019, and to evaluate response using the International Working Group for MF consensus response criteria. We have developed a flow-based mechanistic assay for JAK2 activity, which measures GM-CSF-stimulated phospho-STAT5 in peripheral granulocytes in whole blood samples. Additional pharmacodynamic assessments for this study include JAK2 V617F allele burden, erythropoietin-independent colony formation, cytokine levels, and changes in bone marrow histology. Our initial findings regarding the pharmacokinetics, pharmacodynamics, tolerability and efficacy of XL019 in MF patients will be presented.

Description: JAK2 V617F has been identified as a constitutive activating mutation in approximately half of patients with myelofibrosis (MF). MF, a myeloproliferative disorder comprised of primary myelofibrosis and the clinically indistinguishable entities of post-polycythemia vera or post essential thrombocythemia MF, has been reported to have a median survival of 4 years [Dupriez et al. (1996) Blood88:1013–18]. No effective therapies exist for patients with MF. XL019 is a potent, highly selective and reversible inhibitor of JAK2 which may have utility in treating MF, by ameliorating hepato-splenomegaly, constitutional symptoms, and progressive anemia. The objectives of this phase 1 study include safety evaluation, preliminary assessments of efficacy using International Working Group (IWG) response criteria for MF, and evaluation of pharmacokinetic and pharmacodynamic endpoints. Pharmacodynamic evaluations include quantitative PCR for peripheral blood JAK2 V617F allele burden and erythropoietin-independent colony formation. In addition, plasma and fixed blood samples are being collected to evaluate changes in protein biomarkers and JAK2 signaling pathways. To date, XL019 has been studied in 21 patients over multiple dose levels ranging from doses of 25 mg to 300 mg using different schedules of administration (3 weeks on, 1 week off; QD; and QMWF). Median age was 64 years (range, 47–87 years) and 16 patients (76%) carried the JAK2V617F mutation. Additionally, one patient had a MPLW515F mutation in the absence of a JAK2 mutation. No treatment-related hematologic adverse events (i.e. thrombocytopenia, anemia, neutropenia) have been observed to date. Reversible low-grade peripheral neuropathy (PNP) was observed in 7/9 patients treated at daily doses of ≥100 mg (Grade 1: 5 patients; Grade 2: 2 patients). XL019 doses below 100 mg using 2 different dosing schedules are currently being evaluated. To date, XL019 has resulted in reductions in splenomegaly and leukocytosis, stabilization of hemoglobin counts, improvements in blast counts, and resolution or improvement in generalized constitutional symptoms. The median spleen size in 15 patients measured below the costal margin by palpation was 14cm (range, 3–26cm). Three of 15 patients with palpable splenomegaly at baseline were JAK2 V617F mutation negative and did not experience spleen size reduction. Twelve of 12 (100%) evaluable patients with an activating mutation (JAK2 V617F: 11 patients; MPLW515F: 1 patient) experienced reduction in spleen size and 5 (42%) had a ≥50% decline from baseline. Ten of 11 patients with JAK2V617F activating mutations and baseline constitutional symptoms, reported improvements in generalized constitutional symptoms which include pruritus and fatigue. No significant non-hematologic or hematologic toxicity has been observed at the current dose level. On 25 mg dosing schedules, no signs of PNP have been observed with a follow-up period of up to 4 months. Overall, XL019 has demonstrated encouraging clinical activity and is generally well tolerated.

Description: Approximately 50% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) lack activating mutations in JAK2. Among these patients, ~10% harbor an activating mutation in the thrombopoietin receptor, MPLW515L. We have reported that expression of MPLW515L in a murine bone marrow transplant model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis, that are not observed in the JAK2V617F model. These observations provide an opportunity to assess the efficacy of small molecule JAK2 inhibitors on a myeloproliferative disease (MPD) induced by MPLW515L in vivo, and to determine whether such inhibitors attenuate thrombocytosis. We have tested EXEL-8232 for efficacy in suppression of thrombocytosis in vivo, and for its ability to attenuate JAK2V617F-negative MPD mediated by MPLW515L. EXEL-8232 is a potent small molecule inhibitor of JAK2 and is structurally similar to XL019, a compound currently in clinical trials for MF and polycythemia vera. EXEL-8232 is selective for JAK2 with a biochemical IC50 of 2 nM, and abolished constitutive phosphorylation of JAK2 and STAT5, as well as cytokine-independent growth, of Ba/F3 cells in vitro. After disease was established 12 days post-bone marrow transplantation, EXEL-8232 was administered for 28 days q12h by oral gavage at doses of 30mg/kg or 100mg/kg respectively. Animals treated with 100mg/kg normalized high platelet counts in excess of2 million/ml and normalized leukocytosis from a median of 134,000/ml in vehicle treated controls. Furthermore, drug treatment eliminated extramedullary hematopoiesis in the spleen, as well as bone marrow fibrosis. Of note, EXEL-8232 had no impact on erythrocytosis in diseased animals or in wild type controls, and wild type animals treated with either dosage of 30mg/kg or 100mg/kg did not develop thrombocytopenia. Consistent with these clinical responses, the surrogate endpoints for response to treatment included a reduction of genomic disease burden in the 100mg/kg treated arm (p

Description: Introduction: Cabozantinib (cabo) is an inhibitor of tyrosine kinases including MET, VEGFR2, RET, and the TAM family kinases TYRO3, AXL, and MER. Cabo has shown clinical activity in patients with castration-resistant prostate cancer and other solid tumors with bone metastases. Multiple myeloma (MM) is the second most common hematologic malignancy, and represents ~2% of all cancer deaths. MM is a monoclonal B-cell (plasma cell) neoplasia with clinical hallmarks of multiple osteolytic lesions causing bone pain, pathologic fractures, and hypercalcemia. Circulating levels of HGF and VEGF are upregulated in MM patients, and regulation of plasma cell-osteoblast communication by the HGF-MET signaling pathway has been implicated in the development of lytic bone disease in these patients. Thus, the primary objectives of this research were to A): determine the activity of cabo on bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model (study 1), and B): investigate the impact of cabo on overall survival of these mice when dosed alone or in combination with bortezomib (btz) (study 2). Methods: Female C57BL/KaLwRij mice were allocated to treatment groups (n=15-16 per group) with equivalent average body weights. Four experimental groups were utilized in each of 2 studies: a vehicle control group, and groups receiving single agent btz (0.5 mg/kg ip twice a week) or cabo (10 mg/kg, PO QD). Study 1 also included a higher dose cabo group (30 mg/kg, PO QD), and study 2 included a combination group: btz (0.5 mg/kg ip twice a week) plus cabo (10 mg/kg, PO QD). In study 2, each single agent group also received the vehicle from the alternate single-agent group via the appropriate route and schedule. On day 0, animals were inoculated with 5TGM1 mouse myeloma cells by IV administration. Dosing began on day 1 and continued daily until euthanasia at day 35 (study 1) or day 70 (study 2). Body weights were determined twice a week and blood samples were collected on days -1, 15, 22, and 34 for analysis of paraprotein (IgG2b) and TRACP 5b. In study 1 the development of osteolytic lesions was detected by radiography at the end of the study. Some animals were euthanized before the end of the experiment due to paraplegia. Animals euthanized within four days of the end of the experiment in study 1 were included in the analyses. Results: In study 1, btz reduced serum IgG2b levels, and decreased the frequency of soft tissue lesions, but did not show bone protective properties. Cabo exhibited bone protective effects: mean and total area of osteolytic lesions were reduced at the 30 mg/kg dose, and serum TRACP 5b values and osteoclast counts at the tumor-bone interface were reduced at both the 10 and 30 mg/kg doses. Relative bone area did not differ from control according to histomorphometry. The rise in serum IgG2b started earlier than vehicle control in both cabo-treated groups, but a significant difference was not observed in relative IgG2b at sacrifice. Cabo dose dependently increased the necrotic tumor area in bone, indicating the possibility that the rise in IgG2b may have been due to lysis of plasma cells. Both doses of cabo decreased the frequency of soft tissue lesions. In study 2, the median survival times were 36 d (vehicle), 43 d (btz), 48 d (cabo), and 55 d (cabo+btz). The prolongation of OS compared to vehicle was statistically significant for the cabo group but not for the btz group. Prolongation of OS in the combination group was significant compared to btz alone, but not when compared to cabo alone. Conclusions: Cabo showed both bone-protective and anti-tumor effects in this murine model of MM. In addition, statistically-significant prolongation of overall survival was observed with the combination of cabo+btz compared to btz alone, and with single-agent cabo compared to vehicle. Based on these results, further investigation of cabozantinib alone or in combination with other agents in multiple myeloma is warranted. Disclosures Aftab: Exelixis: Employment, Equity Ownership. Suominen:Pharmatest: Employment; Exelixis: Research Funding. Clary:Exelixis: Employment, Equity Ownership. Käkönen:Pharmatest: Employment; Exelixis: Research Funding. Fagerlund:Pharmatest: Employment; Exelixis: Research Funding. Alhoniemi:Pharmatest: Employment; Exelixis: Research Funding. Rissanen:Pharmatest: Employment; Exelixis: Research Funding. Halleen:Pharmatest: Employment; Exelixis: Research Funding.