Ajoy K. Samanta

Janus Kinase 2: A Critical Target in Chronic Myelogenous Leukemia

The Bcr-Abl tyrosine kinase is the causative factor in most chronic myelogenous leukemia (CML) patients. We have shown that Bcr-Abl is associated with a cluster of signaling proteins, including Janus kinase (Jak) 2, growth factor receptor binding protein 2-associated binder (Gab) 2, Akt, and glycogen synthase kinase (GSK)-3beta. Treatment of CML cell lines and mouse Bcr-Abl+ 32D cells with either Jak2 short interfering RNA or Jak2 kinase inhibitor AG490 inhibited pTyr Gab2 and pSer Akt formation, inhibited the activation of nuclear factor-kappaB, and caused the activation of GSK-3beta, leading to the reduction of c-Myc. Importantly, BaF3 cells expressing T315I and E255K imatinib-resistant mutants of Bcr-Abl underwent apoptosis on exposure to AG490 yet were resistant to imatinib. Similar to wild-type Bcr-Abl+ cells, inhibition of Jak2 by Ag490 treatment resulted in decrease of pSer Akt and c-Myc in imatinib-resistant cells. These results identify Jak2 as a potentially important therapeutic target for CML.

Jak2 inhibition deactivates Lyn kinase through the SET-PP2A-SHP1 pathway, causing apoptosis in drug-resistant cells from chronic myelogenous leukemia patients

Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) become drug resistant by mutations within the kinase domain of Bcr–Abl, and by other changes that cause progression to advanced stage (blast crisis) and increased expression of the Lyn tyrosine kinase, the regulation of which is not understood yet. In Bcr–Abl+ cells inhibition of Jak2, a downstream target of Bcr–Abl, by either Jak2 inhibitors or Jak2-specific short interfering RNA (siRNA) reduced the level of the SET protein, and increased PP2A Ser/Thr phosphatase and Shp1 tyrosine phosphatase activities, which led to decreased levels of activated Lyn. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn kinase compared with Jak2 inhibition alone. In contrast, inhibition of either PP2A or Shp1 combined with Jak2 inhibition interfered with the loss of Lyn kinase activation more so than Jak2 inhibition alone, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase caused by Jak2 inhibition. Inhibition of Jak2 induced apoptosis and reduced colony formation in IM-sensitive and -resistant Bcr–Abl mutant cell lines. Jak2 inhibition also induced apoptosis in CML cells from blast crisis patients but not in normal hematopoietic cells. These results indicate that Lyn is downstream of Jak2, and Jak2 maintains activated Lyn kinase in CML through the SET–PP2A–Shp1 pathway.

Overexpression of MEKK3 Confers Resistance to Apoptosis through Activation of NFκB

Many cancers have constitutively activated NFκB, the elevation of which contributes to cancer cell resistance to chemotherapeutic agent-induced apoptosis. Although mitogen-activated protein kinase/extracellular-regulated kinase kinase kinase-3 (MEKK3) has been shown to participate in the activation of NFκB, its relations to apoptosis and cancer are unclear. In this study, we established cell model systems to examine whether stable expression of MEKK3 could lead to increased NFκB activity and confer resistance to apoptosis. In addition, we investigated in breast and ovarian cancers whether MEKK3 expression may be altered and correlated with aberrant NFκB activity. We show that stable cell lines overexpressing MEKK3 not only had elevated levels of NFκB binding activity but also were more responsive to cytokine stimulation. These stable cells showed 2–4-fold higher basal expression of Bcl-2 and xIAP than the parental cells. Consistent with this increased expression of cell survival genes, MEKK3 stable cells showed reduced activation of caspases 3 and 8 and poly(ADP-ribose) polymerase cleavage and dramatically increased resistance to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin, daunorubicin, camptothecin, and paclitaxel. Intriguingly, analysis of human breast and ovarian cancers showed that a significant fraction of these samples have elevated MEKK3 protein levels with corresponding increases in NFκB binding activities. Thus, our results established that elevated expression of MEKK3 appears to be a frequent occurrence in breast and ovarian cancers and that overexpression of MEKK3 in cells leads to increased NFκB activity and increased expression of cell survival factors and ultimately contributes to their resistance to apoptosis. As such, MEKK3 may serve as a therapeutic target to control cancer cell resistance to cytokine- or drug-induced apoptosis.

Janus kinase 2 regulates Bcr-Abl signaling in chronic myeloid leukemia.

Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr–Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr–Abl+ cells reduced levels of the Bcr–Abl protein and also the phosphorylation of Tyr177 of Bcr–Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr–Abl+ cells with Jak2 inhibitors for 4–6 h but not with IM also reduced Bcr–Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr–Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr–Abl in immune complexes but did not reduce levels of Bcr–Abl, suggesting that the reduction of Bcr–Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr–Abl stability and oncogenic signaling in CML cells are under the control of Jak2.

Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML)

Bcr-Abl is the predominant therapeutic target in chronic myeloid leukemia (CML), and tyrosine kinase inhibitors (TKIs) that inhibit Bcr-Abl have been successful in treating CML. With progression of CML disease especially in blast crisis stage, cells from CML patients become resistant to imatinib mesylate (IM) and other TKIs, resulting in relapse. Because Bcr-Abl is known to drive multiple signaling pathways, the study of the regulation of stability of Bcr-Abl in IM-resistant CML cells is a critical issue as a possible therapeutic strategy. Here, we report that a new dual-kinase chemical inhibitor, ON044580, induced apoptosis of Bcr-Abl+ IM-sensitive, IM-resistant cells, including the gatekeeper Bcr-Abl mutant, T315I, and also cells from blast crisis patients. In addition, IM-resistant K562-R cells, cells from blast crisis CML patients, and all IM-resistant cell lines tested had reduced ability to form colonies in soft agar in the presence of 0.5 µM ON044580. In in vitro kinase assays, ON044580 inhibited the recombinant Jak2 and Abl kinase activities when the respective Jak2 and Abl peptides were used as substrates. Incubation of the Bcr-Abl+ cells with ON044580 rapidly reduced the levels of the Bcr-Abl protein and also reduced the expression of HSP90 and its client protein levels. Lysates of Bcr-Abl+ cell lines were found to contain a large signaling network complex composed of Bcr-Abl, Jak2, HSP90, and its client proteins as detected by a gel filtration column chromatography, which was rapidly disrupted by ON044580. Therefore, targeting Jak2 and Bcr-Abl kinases is an effective way to destabilize Bcr-Abl and its network complex, which leads to the onset of apoptosis in IM-sensitive and IM-resistant Bcr-Abl+ cells. This inhibitory strategy has potential to manage all types of drug-resistant CML cells, especially at the terminal blast crisis stage of CML, where TKIs are not clinically useful.

BCR-ABL oncogenic transformation of NIH 3T3 fibroblasts requires the IL-3 receptor

Oncogenic transformation of hematopoietic cells by the Bcr-Abl oncoprotein directly involves the activation Jak2 tyrosine kinase and the Stat5 transcription factor. Both proteins are normally linked to the interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor receptors for growth and survival. Since fibroblastic cells are not targets of BCR-ABL-induced oncogenesis, we determined whether forced expression of the IL-3 receptor would allow oncogenic transformation of NIH 3T3 fibroblasts known to be resistant to transformation by BCR-ABL. NIH 3T3 cells transduced with the human IL-3 receptor α and β chains were highly susceptible to oncogenic transformation by expression of BCR-ABL. Forced expression of both receptor chains but not either one alone allowed efficient foci formation of NIH 3T3 cells expressing BCR-ABL (triple positive cells), and these cells formed colonies in soft agar, whereas BCR-ABL+ NIH 3T3 cells lacking IL-3 receptor expression did not. Signaling studies indicate that the BCR-ABL/IL-3 receptor+ NIH 3T3 cells utilize the Gab2/PI-3 kinase pathway activated by Jak2, and the Stat5 pathway activated separately by Bcr-Abl, whereas BCR-ABL+ NIH 3T3 cells lacking the IL-3 receptor do not utilize the Jak2 pathway, but still maintain activation of Stat5. The Bcr-Abl kinase inhibitor imatinib mesylate (1 μM) and two Jak2 kinase inhibitors strongly inhibited agar colony formation and the activation of Gab2 caused by Jak2. All of these findings indicate that Bcr-Abl oncoprotein requires the IL-3 receptor/Jak2/Stat5 pathways for oncogenic transformation of NIH 3T3 fibroblasts.

MEKK3 expression correlates with nuclear factor κ B activity and with expression of antiapoptotic genes in serous ovarian carcinoma

Constitutively activated nuclear factor κ B (NFκB) contributes to the development of cancer by regulating the expression of genes involved in cell survival, metastasis, and angiogenesis. The authors have demonstrated that MEKK3 plays a critical role in cytokine‐mediated NFκB activation, and that stable expression of MEKK3 in cultured cells leads to increased NFκB activity.