Cell-type-specific innate immune response to oncolytic Newcastle disease virus.

Virotherapy of cancer exploits the potential of naturally occurring and engineered oncolytic viruses to selectively replicate in and cause cytotoxicity to tumor cells without affecting healthy normal cells. The tumor selectivity of Newcastle disease virus (NDV), a member of the family Paramyxoviridae, depends on the differential type I interferon (IFN) response. Further understanding of the key mechanisms and immune effector molecules involved will aid in augmenting the oncolytic properties of NDV. Here we report on the infection kinetics and innate immune responses to a recombinant LaSota strain of NDV (rLaSota eGFP) in human tumor and normal cells. We observed varying replicative fit and cytotoxicity of rLaSota eGFP depending on the tumor cell type, with severely restricted replication in normal cells. The absence of retinoic acid-inducible gene I (RIG-I), a cytosolic RNA sensor, determined sensitivity to NDV. Productive NDV infection with a moderate IFN-α induction in human multiple myeloma cells suggested a role for IFN-independent mechanisms or lack of type I IFN reinforcement by RIG-I. Proinflammatory cytokines and chemokines were altered differentially in infected normal and tumor cells. Our results suggest that tumor selectivity is dependent on variations in the cellular antiviral response to infection with NDV and RIG-I expression.

ATP analog enhances the actions of a heat shock protein 90 inhibitor in multiple myeloma cells.

Heat shock protein (HSP) 90 regulates client oncoprotein maturation. The chaperone function of HSP90 is blocked by 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), although it results in transcription and translation of antiapoptotic HSP proteins. Using three myeloma cell lines, we tested whether inhibition of transcription/translation of HSP or client proteins will enhance 17-AAG-mediated cytotoxicity. 8-Chloro-adenosine (8-Cl-Ado), currently in clinical trials, inhibits bioenergy production, mRNA transcription, and protein translation and was combined with 17-AAG. 17-AAG treatment resulted in HSP transcript and protein level elevation. In the combination, 8-Cl-Ado did not abrogate HSP mRNA and protein induction. HSP90 requires ATP to stabilize client proteins; hence, expression of signal transducer and activator of transcription 3 (STAT3), Raf-1, and Akt was analyzed. 17-AAG alone resulted in <10% change in STAT3, Raf-1, and Akt protein levels, whereas no change was observed for 4E-BP1. In contrast, the combination treatment resulted in a >50% decrease in client protein levels and marked hypophosphorylation of 4E-BP1. 8-Cl-Ado alone resulted in a <30% decrease of client proteins and 4E-BP1 hypophosphorylation. 8-Cl-Ado combined with 17-AAG resulted in more than additive cytotoxicity. In conclusion, 8-Cl-Ado, which targets transcription, translation, and cellular bioenergy, enhanced 17-AAG-mediated cytotoxicity in myeloma cells.

Transcription inhibition of heat shock proteins: a strategy for combination of 17-allylamino-17-demethoxygeldanamycin and actinomycin d.

The heat shock protein (HSP) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical trials because of its unique mechanism of action and antitumor activity. However, 17-AAG triggers the transcription and elevation of antiapoptotic HSP90, HSP70, and HSP27, which lead to chemoresistance in tumor cells. We hypothesized that inhibiting HSP90, HSP70, and HSP27 transcription may enhance 17-AAG-induced cell death in multiple myeloma cell lines. Actinomycin D (Act D), a clinically used agent and transcription inhibitor, was combined with 17-AAG. The concentrations for 17-AAG and Act D were selected based on the target actions and plasma levels during therapy. Inducible and constitutive HSP27, HSP70, and HSP90 mRNA and protein levels were measured by real-time reverse transcription-PCR and immunoblot assays. Compared with no treatment, Act D alone decreased HSP mRNA levels in MM.1S and RPMI-8226 cell lines. Combining Act D with 17-AAG did not attenuate 17-AAG-mediated increases in transcript levels of inducible HSP70; however, constitutive HSP mRNA levels were decreased. In contrast to its effect on mRNA levels, Act D was able to abrogate 17-AAG-mediated increases in all HSP protein levels. The cytotoxicity of combined Act D and 17-AAG was assessed. Treatment with Act D alone caused <40% cell death, whereas the combination of 17-AAG and Act D resulted in an increase of cell death in both multiple myeloma cell lines. In conclusion, these results indicate that 17-AAG-mediated induction of HSP70 and HSP27 expression can be attenuated by Act D and therefore can potentially improve the clinical treatment of multiple myeloma.

Targeting MET transcription as a therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is an incurable indolent malignancy with an average lifespan of 3 years, underscoring the need for new therapies. Studies have shown that the receptor MET and its ligand hepatocyte growth factor play an important role in proliferation, migration, adhesion, and survival of MM cells. Hence, an effective way to decrease MET receptor may act as a viable therapeutic option. Since MET mRNA and protein have short half-lives, we hypothesized that transcription inhibitor will reduce MET transcript and protein levels and this will lead to cell death. Pharmacological (flavopiridol) and molecular (shRNA) transcription inhibitor were used to impede formation of MET transcripts. The diminution of global RNA synthesis with flavopiridol was related to phosphorylation status of Ser residues (r (2) = 0.90 and 0.92 for Ser2 and Ser5) on the C-terminal-domain of RNA polymerase II. This was accompanied with a time-dependent decrease in MET transcript, which reached to less than 30% (1 microM) and 10% (3 microM) by 24 h. This decline in transcript level was directly associated with a reduction in MET protein level (r (2) = 0.82) and resulted in cell death. Assessment of MET in MM survival was done by using shRNA targeted towards MET. When cells were infected with shRNA viral construct, there was increased cell death with a decline in MET transcript and protein. Taken together, our study demonstrates that MET plays a critical role in the survival and removal or lowering of MET by flavopiridol or shRNA results in the demise of MM cells.

HSP70 inhibition reverses cell adhesion mediated and acquired drug resistance in multiple myeloma.

Heat shock proteins (HSPs) are a super family of highly conserved molecular chaperone proteins, which are induced in response to stress. HSP70 has been demonstrated to inhibit apoptosis induced by a number of chemotherapeutic agents. Previous investigations have suggested the development of drug resistance in multiple myeloma (MM) cells after adhesion to stroma. This study used MM cell lines and primary plasma cells to determine if HSP70 had a role in development of chemo resistance. Adhesion of MM cells to either bone marrow stromal cells or fibronectin (FN) enhanced HSP70 expression. Inhibition of the HSP70 expression decreased 8226 cell adhesion to stroma or FN and induced more apoptosis in FN-adhered 8226 cells than in suspension cultures at 24 h. Further, HSP70 inhibitors enhanced melphalan-induced apoptosis and reversed melphalan-induced cell adhesion-mediated drug resistance (CAM-DR) phenotype. In addition, compared to parental cells, KNK-437, a heat shock factor inhibitor caused more apoptosis in melphalan-resistant 8226/LR5 cells and sensitized them to melphalan. Primary CD138 positive cells showed high expression of HSPA4 mRNA, and KNK-437 caused apoptosis in these cells. In conclusion, our data suggest inhibition of HSP70, reduced adhesion and caused apoptosis of both acquired and de novo drug resistant MM cells.

Bone marrow stroma confers resistance to Apo2 ligand/TRAIL in multiple myeloma in part by regulating c-FLIP.

Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients' stroma-conditioned medium, we determined that soluble factor(s) produced by stroma-myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.

The growth factor fusion construct containing B-lymphocyte stimulator (BLyS) and the toxin rGel induces apoptosis specifically in BAFF-R-positive CLL cells.

The cytokine B lymphocyte stimulator (BLyS) mediates its effect through cell-surface receptors BAFF-R, TACI, and BCMA. BLyS receptors are expressed only on B cells and not present in other normal cells including normal T lymphocytes. Chronic lymphocytic leukemia (CLL) is a B-cell disease and CLL lymphocytes express BLyS receptors. Gelonin, a type 1 ribosome-inactivating toxin, lacks cell membrane binding domain and hence is nontoxic to intact cells. We generated a construct of recombinant gelonin (rGel) fused to BLyS to specifically target quiescent B-CLL lymphocytes. The construct rGel/BLyS specifically binds and internalizes through BAFF-R into CD19(+) B-CLL lymphocytes and induces apoptosis at nanomolar concentrations. In contrast, rGel alone was not able to internalize into these leukemic lymphocytes. Mechanistically, the rGel/BLyS construct inhibits protein synthesis with an IC(50) of less than 3 nM compared with more than 5000 nM for rGel toxin alone. This rGel/BLyS-mediated decrease in protein synthesis was associated with a decline in short-lived proteins such as MCL-1 and XIAP, the 2 survival proteins in B-CLL. There was a strong relationship between a decrease in these proteins and the cleavage of PARP, a hallmark feature of apoptosis. Taken together, these data suggest that the rGel/BLyS fusion toxin may have potential therapeutic efficacy for B-CLL patients.

Forodesine, an inhibitor of purine nucleoside phosphorylase, induces apoptosis in chronic lymphocytic leukemia cells.

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine, a potent inhibitor of PNP, was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK), we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at Ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data, a phase 2 clinical trial of forodesine has been initiated for CLL patients.

Identification of compounds that enhance the anti-lymphoma activity of rituximab using flow cytometric high-content screening.

In this report, we describe a new flow cytometry technique termed flow cytometric high-content screening (FC-HCS) which involves semi-automated processing and analysis of multiparameter flow cytometry samples. As a first test of the FC-HCS technique, we used it to screen a 2000-compound library, called the National Cancer Institute (NCI) Diversity Set, to identify agents that would enhance the anti-lymphoma activity of the therapeutic monoclonal antibody rituximab. FC-HCS identified 15 compounds from the Diversity Set that significantly enhanced the ability of rituximab to inhibit cell cycle progression and induce apoptosis in lymphoma cells. The validity of the screening results was confirmed for several compounds using additional assays of cell proliferation, apoptosis and cell growth. The FC-HCS technique was relatively simple and reliable and could process up to 1000 samples/day on a single flow cytometer. The FC-HCS technique may be useful for a variety of applications including drug discovery, immunologic monitoring of patients, functional genomics studies and tissue engineering efforts.

Arsenic trioxide inhibits translation of mRNA of bcr-abl, resulting in attenuation of Bcr-Abl levels and apoptosis of human leukemia cells.

Present studies demonstrate that treatment with arsenic trioxide (AT) lowered ectopically expressed or endogenous levels of Bcr-Abl protein, as well as induced apoptosis of Bcr-Abl-expressing cultured and primary chronic myeloid leukemia cells, including those refractory to imatinib mesylate. Treatment with AT neither affected bcr-abl mRNA transcript levels nor promoted the proteasomal degradation of Bcr-Abl. Importantly, in [(35)S]methionine-labeled leukemia cells, exposure to AT rapidly lowered the levels of the newly synthesized Bcr-Abl, indicating inhibition of bcr-abl mRNA translation. Treatment with AT rapidly inhibited the activity of 3-phosphoinositide-dependent protein kinase-1, as well as of p70 S6 kinase-1. p70 S6 kinase-1 is known to be a positive regulator of the translation of a group of mRNAs that possesses a long and highly structured 5'-untranslated region (UTR) containing a tract of oligopyrimidines (TOP). Because bcr-abl mRNA was discovered to possess a long and highly structured 5'-UTR containing a 12-pyrimidine TOP sequence in its 5'-UTR, we determined the effect of AT in Jurkat cells with ectopic expression of a 5'-UTR-deleted mutant of the bcr-abl gene, i.e., Jurkat/Bcr-Abl (5'UTR-) cells. Treatment with AT neither lowered the levels of the 5'-UTR-deleted mutant of Bcr-Abl nor induced apoptosis of Jurkat/Bcr-Abl (5'UTR-) cells. Taken together, these findings demonstrate a novel mechanism by which AT down-regulates Bcr-Abl levels and induces apoptosis of Bcr-Abl-positive chronic myelogenous leukemia cells.

Histone deacetylase inhibitor LAQ824 both lowers expression and promotes proteasomal degradation of Bcr-Abl and induces apoptosis of imatinib mesylate-sensitive or -refractory chronic myelogenous leukemia-blast crisis cells.

Treatment with LAQ824 (Novartis Pharmaceutical, Inc.), a cinnamyl hydroxamic acid analogue inhibitor of histone deacetylases, depleted the mRNA and protein expression of Bcr-Abl in human chronic myeloid leukemia blast crisis (CML-BC) cells. Exposure to LAQ824 induced the expression of the cell cycle-dependent kinase inhibitors p21 and p27 and caused cell cycle G(1)-phase accumulation and apoptosis of CML-BC cells. LAQ824 also induced acetylation of heat shock protein 90. This inhibited the chaperone association of Bcr-Abl with heat shock protein 90, thereby promoting the proteasomal degradation of Bcr-Abl. Cotreatment with LAQ824 increased imatinib mesylate-induced apoptosis of CML-BC cells. Additionally, LAQ824 down-regulated the levels of mutant Bcr-Abl possessing the T315I point mutation, as well as induced apoptosis of imatinib-refractory primary CML-BC cells. Therefore, LAQ824 may be a promising therapeutic agent in the treatment of imatinib-sensitive or -refractory human leukemia.

Molecular determinants of epothilone B derivative (BMS 247550) and Apo-2L/TRAIL-induced apoptosis of human ovarian cancer cells.

OBJECTIVE: We determined the cytotoxic effects BMS 247550 (Epo B), a derivative of epothilone B, on cisplatinum- or paclitaxel-sensitive or -resistant human ovarian cancer cells. Additionally, we determined the effect of Epo B on Apo-2L/TRAIL-induced apoptosis of ovarian cancer cells. METHODS: Epo B-induced cytotoxic and cell cycle effects were evaluated by the MTT assay and flow cytometry, respectively. Epo B-induced apoptosis was assessed by immunoblot analyses of the processing and proteolytic activity of caspases, flow cytometric measurement of annexin V staining, and the TUNEL assay. The effects of Epo B and/or Apo-2L/TRAIL on the protein expressions of the death receptors DR4 and DR5 as well as of XIAP and survivin were determined by immunoblot analyses. RESULTS: In the cell cycle-synchronized ovarian cancer cells, Epo B induced tubulin polymerization and mitotic arrest, followed by apoptosis. This was associated with the cytosolic accumulation of cytochrome (cyt) c and Smac/DIABLO as well as PARP cleavage activity of caspase-3. Epo B was able to exert cytotoxic effects against cisplatinum- and paclitaxel-resistant ovarian cancer cells. Epo B increased the expressions of DR4 and DR5, as well as augmented Apo-2L/TRAIL-induced processing of caspase-8 and Bid. This was associated with more caspase-3 activity, a decline in the intracellular levels of XIAP, cIAP, and survivin, and apoptosis of ovarian cancer cells. CONCLUSIONS: These data support the in vivo testing of Epo B against cisplatinum- and paclitaxel-resistant ovarian cancers, and suggest that a pretreatment with Epo B may sensitize human ovarian cancers to the cytotoxic effects of Apo-2L/TRAIL.

Regulation of 17-AAG-induced apoptosis: role of Bcl-2, Bcl-XL, and Bax downstream of 17-AAG-mediated down-regulation of Akt, Raf-1, and Src kinases.

17-allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone function of heat shock protein-90 (Hsp-90) and promotes the proteasomal degradation of its misfolded client proteins. Here, we demonstrate that treatment of the human acute myeloid leukemia HL-60 cells with 17-AAG attenuates the intracellular levels of a number of Hsp-90 client proteins, including Akt, c-Raf-1, and c-Src. Also, 17-AAG induced the mitochondrial release and cytosolic accumulation of cytochrome c (cyt c) and second mitochondria-derived activator of caspases (Smac)/DIABLO, resulting in the activation of caspase-9 and caspase-3 and apoptosis. Treatment with 17-AAG triggered the B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax) conformational change associated with apoptosis, while Bax-deficient cells were resistant to 17-AAG-induced apoptosis. In addition, in HL-60/Bcl-2 and HL-60/Bcl-xL cells, which ectopically express Bcl-2 and Bcl-xL respectively, 17-AAG-induced Bax conformational change, cytosolic accumulation of cyt c and Smac/DIABLO, and apoptosis were markedly inhibited. Although the rate of 17-AAG-mediated decline in Akt, c-Raf-1, and c-Src levels was blunted, the total decline was not compromised in HL-60/Bcl-2 and HL-60/Bcl-xL cells. Cotreatment with HA14-1, a nonpeptidic ligand that can bind and inhibit the antiapoptotic activity of Bcl-2, significantly overcame the resistance to 17-AAG-induced apoptosis in HL-60/Bcl-2 cells. Together, these findings indicate that although 17-AAG treatment causes the levels of a number of survival-signaling protein kinases to decline, the downstream engagement of the mitochondrial pathway of apoptosis is regulated by the activity of the Bcl-2 family of proteins. Also, neutralizing the antiapoptotic effect of Bcl-2 would further enhance the antileukemia activity of 17-AAG.

Flavopiridol down-regulates antiapoptotic proteins and sensitizes human breast cancer cells to epothilone B-induced apoptosis.

The molecular mechanisms underlying the cell cycle growth-inhibitory and apoptotic effects of flavopiridol (FP) were determined in human breast cancer cells. Treatment with FP caused accumulation in the G(1) phase of the cell cycle and induced apoptosis of SKBR-3 and MB-468 cells. This was associated with down-regulation of the levels of cyclins D1 and B1, as well as with inhibition of cyclin-dependent kinase (cdk) 1, cdk2, and cdk4. FP-induced apoptosis was accompanied by a conformational change and mitochondrial localization of Bax. This resulted in the accumulations of cytochrome c, Smac, and Omi/HtrA2 in the cytosol and induced the poly(ADP-ribose) polymerase cleavage activity of caspase-3. Treatment with FP also attenuated the mRNA and protein levels of XIAP, cIAP-2, Mcl-1, Bcl-x(L), and survivin. In MB-468 cells with overexpression of Bcl-2 (468/Bcl-2), FP-induced Bax conformational change and apoptosis were inhibited, whereas the FP-mediated decline in the levels of IAP proteins, Mcl-11 and Bcl-x(L) remained unaltered. The effects of cotreatment with FP and the nontaxane tubulin-polymerizing agent epothilone (Epo) B were also determined in MB-468 cells. Sequential treatment with Epo B followed by FP induced significantly more apoptosis of MB-468 cells than treatment with the reverse sequence of FP followed by Epo B or treatment with either agent alone (P < 0.05). Treatment with Epo B followed by FP induced more Bax conformational change and was associated with a greater decline in the levels of XIAP, cIAP-2, Mcl-1, and Bcl-x(L). However, MB-468/Bcl-2 cells remained relatively resistant to Epo B followed by FP. Taken together, these findings suggest that the superior sequence-dependent anti-breast cancer activity of Epo B followed by FP may be due to FP-induced Bax conformational change and down-regulation of the antiapoptotic IAP, Bcl-x(L), and Mcl-1 proteins, but this treatment may not overcome the resistance to apoptosis of breast cancer cells conferred by overexpression of Bcl-2.

Cotreatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) enhances imatinib-induced apoptosis of Bcr-Abl-positive human acute leukemia cells.

Here we demonstrate that treatment with SAHA (suberoylanilide hydroxamic acid), a known inhibitor of histone deacetylases (HDACs), alone induced p21 and/or p27 expressions but decreased the mRNA and protein levels of Bcr-Abl, which was associated with apoptosis of Bcr-Abl-expressing K562 and LAMA-84 cells. Cotreatment with SAHA and imatinib (Gleevec) caused more down-regulation of the levels and auto-tyrosine phosphorylation of Bcr-Abl and apoptosis of these cell types, as compared with treatment with either agent alone (P <.05). This finding was also associated with a greater decline in the levels of phospho-AKT and Bcl-x(L). Significantly, treatment with SAHA also down-regulated Bcr-Abl levels and induced apoptosis of CD34(+) leukemia blast progenitor cells derived from patients who had developed progressive blast crisis (BC) of chronic myelocytic leukemia (CML) while receiving therapy with imatinib. Taken together, these findings indicate that cotreatment with SAHA enhances the cytotoxic effects of imatinib and may have activity against imatinib-refractory CML-BC.

Novel targeted therapies for Bcr-Abl positive acute leukemias: beyond STI571.

In the pathophysiology of CML, the constitutive activity of the Bcr-Abl tyrosine kinase (TK) is, most likely, the sole molecular abnormality of the chronic phase. It also remains a critical molecular determinant of malignant behavior of the leukemic progenitors in the accelerated and blastic phase of CML. Therefore, downregulation of the levels and activity of Bcr-Abl is clearly the lynchpin of a rational therapeutic strategy against all phases of CML. Support for this has only been strengthened by the observations that resistance to imatinib mesylate (imatinib) commonly involves a breakthrough and the persistent activity of Bcr-Abl TK. This is due to either mutations that inhibit imatinib action on Bcr-Abl TK or amplification of the bcr-abl gene. Recent studies have demonstrated that other small molecule tyrosine kinase inhibitors that also inhibit Bcr-Abl TK may be highly active in inducing differentiation and apoptosis of CML progenitors, regardless of their sensitivity to imatinib. Small molecule inhibitors that downregulate the levels of Bcr-Abl by inhibiting its translation, e.g., arsenic trioxide, or promoting its proteasomal degradation, e.g., geldanamycin analogues, have also been identified. Finally the identification of other potent survival and antiapoptotic signaling pathways in imatinib-resistant CML progenitors indicates that inhibitors of these pathways will eventually be treatment strategies for advanced phases of CML.

Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC(50) of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Mechanisms of resistance to imatinib mesylate in Bcr-Abl-positive leukemias.

The constitutive activity of the Bcr-Abl tyrosine kinase plays a critical role in the molecular pathogenesis of not only the chronic but also the accelerated and blastic phases of chronic myelogenous leukemia. Therefore, Bcr-Abl tyrosine kinase is a rational therapeutic target in all phases of chronic myelogenous leukemia. Although imatinib mesylate (STI571, Gleevec, Novartis, Basal, Switzerland) produces high rates of complete clinical and cytogenetic responses in the chronic phase, resistance is universal and clinical relapse develops rapidly in the advanced phases of chronic myelogenous leukemia. This resistance has been shown to be caused by specific ATP binding site mutations or amplification of Bcr-Abl gene, resulting in a Bcr-Abl tyrosine kinase that is resistant to further inhibition by imatinib. Alternative (Bcr-Abl-independent) mechanisms driving the growth and survival of the malignant clone may also be responsible for imatinib resistance. Novel tyrosine kinase inhibitors that also target Bcr-Abl tyrosine kinase, or agents that downregulate Bcr-Abl levels regardless of its wild-type or mutant status, may need to be developed clinically for the future therapy of imatinib-resistant chronic myelogenous leukemia.