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Janus Kinase 2 (JAK2) is a cytokine receptor associated tyrosine kinase. Cytokine stimulation results in JAK2 activation and tyrosine phosphorylation of the cytokine receptor. Cytosolic SH2 domain containing proteins, such as the signal transducer and activator of transcription 3 (STAT3) are recruited to phospho-tyrosine residues on the activated cytokine receptor, and phosphorylated by JAK2 to form stable dimers, followed by their translocation to the nucleus where they function as transcription factors. Deregulation of the JAK-STAT pathway is seen in several epithelial tumors and many hematological malignancies. Recent studies demonstrate that an activating mutation in the pseudokinase domain of JAK2 (V617F) underlies hematological disorders like polycythemia vera. Therefore, inhibition of JAK2 may have therapeutic significance in many cancers. The tryphostin AG490 is the most widely studied inhibitor of JAK2, which inhibits tumor cell growth and increases sensitivity to apoptotic stimuli in vitro. However, in vivo studies with AG490 have been less promising due to its poor pharmacology and requirement for high concentrations to achieve significant anti-tumor activity. To identify a more effective inhibitor of the JAK2-STAT pathway, we screened over 300 analogues of AG490 for their ability to inhibit IL-6 dependent activation of STAT3. A lead compound, Degrasyn (WP1130), was identified from this screen that inhibited IL-6 mediated STAT3 activation at low microM concentrations. Preliminary studies using in vitro kinase assays revealed that Degrasyn is a weak JAK2 kinase inhibitor despite being a strong suppressor of STAT3 activation, suggesting a different mechanism of inhibition of the JAK2-STAT pathway. We show that Degrasyn inhibits STAT3 phosphorylation by inducing the down-regulation of JAK2 protein without affecting STAT3. The loss of JAK2 protein via Degrasyn is a rapid and irreversible process. A decrease in JAK2 protein levels is observed as early as 30 minutes after treatment with near complete loss of JAK2 after 2 hours of Degrasyn incubation. While other tyrosine kinases are not affected, both wild type and mutant (V617F) forms of JAK2 are equally down-regulated by Degrasyn. Loss of JAK2 protein by Degrasyn is not blocked by inhibition of calpain or serine/threonine proteases or by inhibition of the proteosomal or lysosomal pathway. Real-Time PCR analysis of JAK2 transcript levels after Degrasyn treatment showed no significant change, suggesting a direct effect of Degrasyn on the JAK2 protein itself. Recent studies suggest that Degrasyn alters the cytoplasmic compartmentalization of JAK2, sequestering the kinase in an insoluble fraction. In vivo studies show that Degrasyn has significant anti-tumor effects against models of leukemia and lymphoma. These results suggest that Degrasyn induces JAK2 degradation by a unique mechanism and may be useful in treating tumors and diseases where the JAK2 kinase plays a pivotal role.

BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.

BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of chromosomal translocation in chronic myelogenous leukemia (CML). The tyrosine kinase activity of BCR-ABL activates signaling cascades that induce cytokine independence and transformation of myeloid progenitor cells. Targeted inhibition of this kinase with specific inhibitors (imatinib or BMS-354825) is a very effective therapy for some CML patients but resistance to these agents (through point mutations and other mechanisms) leads to advanced disease with very few therapeutic options. An alternate therapeutic strategy is to reduce BCR-ABL expression or its critical downstream signaling elements important for transformation. We examined BCR-ABL signaling elements and gene expression changes that occur in CML cells following kinase inhibition by imatinib in newly established imatinib sensitive and resistant cells to identify critical signaling elements involved in CML cell death. Imatinib rapidly and progressively suppressed c-myc expression in imatinib sensitive but not resistant cells prior to the onset of apoptosis. These results suggested that c-myc expression was regulated by BCR-ABL signaling and may play a role in CML tumorigenicity. To confirm a role for c-myc in CML cell growth and/or survival, c-myc expression was specifically down-regulated by siRNA using a novel electroporation instrument (AMAXA) that permits high level gene transfer with limited toxicity in CML cell lines. Jak2 siRNA was used as a control. c-myc, but not Jak2 siRNA, suppressed c-myc expression and cell growth and survival in both imatinib sensitive and resistant CML cells, suggesting that targeted suppression of c-myc may have therapeutic activity against both kinase inhibitor sensitive and resistant CML cells. Since the tyrphostin AG490 was previously shown to inhibit c-myc expression in CML cells through its inhibitory effects on Jak2, we screened a series of > 200 AG490 derivatives for their ability to rapidly reduce c-myc expression in hematological malignancies. After several rounds of testing we synthesized an agent (WP-1066) capable of rapid c-myc downregulation (beginning 1–5 min after treatment with 1–2 microM) but poor Jak2 kinase inhibitory activity (IC50 > 100 microM). These results suggested a more direct effect of WP-1066 on c-myc protein expression than AG490 and mechanistic studies suggest that WP-1066 reduces c-myc protein stability but does not affect c-myc gene expression. In BCR-ABL expressing cells WP-1066 rapidly reduced c-myc protein levels in CML cells and inhibited the growth and survival of cell lines or patient specimens expressing wild-type or mutant forms of BCR-ABL that effect tyrosine kinase inhibitory activity (T315I in BV-173R cells). Equal concentrations of imatinib or WP-1066 reduced BCR-ABL activation and downstream signaling (Stat5 phosphorylation) in CML cells. However, WP-1066 differed from imatinib in its ability to downregulate BCR-ABL protein expression without affects on c-abl or Stat5 expression. Similar results were obtained in clinical specimens taken from patients with BCR-ABL point mutations that mediate imatinib (or BMS-354825) resistance. Nude mouse studies demonstrated that WP-1066 reduced the growth of K562 tumors to an extent similar to that of imatinib. Together these results suggest that WP-1066 downregulates BCR-ABL and c-myc expression, induces apoptosis in CML cells expressing wild-type or mutant BCR-ABL and may have therapeutic activity in imatinib (or BMS-354825) resistant CML tumors.