miRNA-21 regulates arsenic-induced anti-leukemia activity in myelogenous cell lines

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules involved in modulation of cellular sensitivity to anti-cancer drugs. miRNA-21 (miR-21), one of the most prominent miRNAs in the genesis and progression of many human cancers, has been rarely characterized in myelogenous leukemia. Arsenic trioxide (ATO) was successfully used in the treatment of acute promyelocytic leukemia (APL) etc. However, cytotoxicity or insensitivity is a major concern in the successful treatment of leukemia. Here, we used a specific precursor miRNA-21 (pre-miR-21) or anti-miRNA-21 oligonucleotide (AMO-miR-21) to study sensitivity of HL60 and K562 cells to ATO. Cell viability and cell cycle were evaluated by MTT assay and PI assay using flow cytometry, respectively. Levels of miR-21 and its target PDCD4 were quantified by real-time PCR and/or western blot. AMO-miR-21 or ATO alone led to growth inhibition, apoptosis and G1 phase arrest of cell cycle. Apoptotic cells were confirmed morphologically with Hoechst staining. Moreover, there was somewhat synergistic effect of AMO-miR-21 and ATO in growth inhibition and apoptosis promotion. Meanwhile, enforced pre-miR-21 expression increased resistance to ATO, nevertheless not affecting cell growth alone. Dual-luciferase reporter vector containing two tandem PDCD4 3′ UTR validated that PDCD4 was directly up-regulated by miR-21. Therefore, miRNA-21 by targeting PDCD4 may play a functional role in modulating ATO-induced cell death, and strategy using AMO-miR-21 and its combination with ATO may be useful as a myelogenous leukemia therapy.     

Altered gene expression in busulfan-resistant human myeloid leukemia

Busulfan (Bu) resistance is a major obstacle to hematopoietic stem cell transplantation (HSCT) of patients with chronic or acute myelogenous leukemia (CML or AML). We used gene expression analysis to identify cellular factors underlying Bu resistance. Two Bu-resistant leukemia cell lines were established, characterized and analyzed for differentially expressed genes. The CML B5/Bu250(6) cells are 4.5-fold more resistant to Bu than their parental B5 cells. The AML KBM3/Bu250(6) cells are 4.0-fold more Bu-resistant than KBM3 parental cells. Both resistant sublines evade Bu-mediated G2-arrest and apoptosis with altered regulations of CHK2 and CDC2 proteins, constitutively up-regulated anti-apoptotic genes (BCL-X(L), BCL2, BCL2L10, BAG3 and IAP2/BIRC3) and down-regulated pro-apoptotic genes (BIK, BNIP3, and LTBR). Bu-induced apoptosis is partly mediated by activation of caspases; use of the inhibitor Z-VAD-FMK completely abrogated PARP1 cleavage and reduced apoptosis by approximately 50%. Furthermore, Bu resistance in these cells may be attributed in part to up-regulation of HSP90 protein and activation of STAT3. The inhibition of HSP90 with geldanamycin attenuated phosphorylated STAT3 and made B5/Bu250(6) and KBM3/Bu250(6) more Bu-sensitive. The analysis of cells derived from patients classified as either clinically resistant or sensitive to high-dose Bu-based chemotherapy indicated alterations in gene expression that were analogous to those observed in the in vitro model cell lines, confirming the potential clinical relevance of this model for Bu resistance.     

Proteasome inhibitors abolish cell death downstream of caspase activation during anti-microtubule drug-induced apoptosis in leukemia cells

PURPOSE: Anti-microtubule drugs and proteasome inhibitors are currently among the most intensively studied anti-tumor agents, however little is known about their pharmacological interactions at the cellular level. MATERIALS AND METHODS: The human promyelocytic leukemia cell line, HL-60, was exposed to nocodazole or etoposide in combination with proteasome or caspase inhibitors. Apoptotic cell death was detected by flow cytometry as sub-G1 population. Caspase and proteasome activities were monitored by the fluorogenic substrates Ac-DEVD-AMC and Suc-LLVY-AMC, respectively, in cell lysate. Heat shock protein 70 (HSP70) expression was determined by Western blotting. RESULTS: Nocodazole, a microtubule inhibitor, induced caspase-dependent apoptosis in the HL-60 cell line. At sub-cytotoxic concentrations, proteasome inhibitors, including MG-132 or clasto-beta-lactone, decreased nocodazole-induced apoptotic DNA fragmentation without affecting the induction of caspase-3 activity. In contrast, MG-132 decreased both DNA fragmentation and caspase activation induced by etoposide, a topoisomerase-II inhibitor. HSP70 had previously been found to inhibit apoptosis independently from caspase activation. In this study, MG-132 up-regulated HSP70 protein expression, both in the presence or absence of nocodazole. CONCLUSION: Proteasome inhibitors decreased anti-microtubule agent-induced apoptotic DNA fragmentation downstream of caspase-3 activation, possibly due to increased HSP70 expression. The results indicate that combination treatment with these novel anti-tumor agents in leukemia requires careful evaluation of their molecular interaction at the level of apoptosis induction.     

Dual targeting of HSC70 and HSP72 inhibits HSP90 function and induces tumor-specific apoptosis

Heat-shock protein 70 (HSP70) isoforms contribute to tumorigenesis through their well-documented antiapoptotic activity and via their role as cochaperones for the HSP90 molecular chaperone. HSP70 expression is induced following treatment with HSP90 inhibitors, which may attenuate the cell death effects of this class of inhibitor. Here we show that silencing either heat-shock cognate 70 (HSC70) or HSP72 expression in human cancer cell lines has no effect on HSP90 activity or cell proliferation. However, simultaneously reducing the expression of both of these isoforms induces proteasome-dependent degradation of HSP90 client proteins, G1 cell-cycle arrest, and extensive tumor-specific apoptosis. Importantly, simultaneous silencing of HSP70 isoforms in nontumorigenic cell lines does not result in comparable growth arrest or induction of apoptosis, indicating a potential therapeutic window.     

Src family kinases promote AML cell survival through activation of signal transducers and activators of transcription (STAT)

We investigated the role of Src family kinases (SFKs) in the regulation of STAT activation in myeloid leukemia cells. Two of 6 AML cell lines displayed constitutive STAT5 activation, whereas four cell lines had constitutive SFK activity. Treatment with the SFK inhibitors suppressed STAT5 activation and decreased viability. Akt phosphorylation and Mcl-1 expression decreased after SFK inhibition accompanied by apoptosis induction. In primary AML specimens, SFK inhibitors suppressed proliferation in 5 of 14 specimens. These data indicate that Src-STAT5 and Src-Akt pathways are integral survival signal pathways in AML cells. Src inhibition may represent a novel treatment strategy for investigation in AML.     

Heat-shock protein 90 inhibitors in cancer therapy: 17AAG and beyond

Heat-shock protein 90 (HSP90) has diverse functions in mammalian cells. It acts as molecular chaperone, together with several co-chaperone molecules (such as Hop, Hip, p23, cdc37, Aha, and immunophilins). HSP90 binds to its client proteins (such as steroid receptors, AKT, Bcr-Abl, Apaf-1, survivin, cyclin dependent kinases which are involved in signal transduction that regulate cell cycle, survival, and death, and promote their proper protein folding, assembly, and transportation across different cellular compartments. Failure of Hsp90 chaperone activity leads to misfolding of client proteins, which leads to ubiquitination and proteasome degradation, and this deregulating cellular homeostasis. Since tumor cells frequently overexpress the active form of HSP90, which is more susceptible to inhibition by small molecules such as geldanamycin and its analogs, HSP90 became an attractive target for cancer therapy. This paper will review the recent advances in HSP90-biology and will discuss the emerging role of the HSP90 inhibitors such as 17-allylamino-17 demethoxy-geldanamycin and other HSP-90-directed small molecules in cancer therapy.     

MS-275, a novel histone deacetylase inhibitor with selectivity against HDAC1, induces degradation of FLT3 via inhibition of chaperone function of heat shock protein 90 in AML cells

This study explored the effect of MS-275, a novel histone deacetylase inhibitor (HDACI), against a variety of human leukemia cells with defined genetic alterations. MS-275 profoundly induced growth arrest of acute myelogenous leukemia (AML) MOLM13 and biphenotypic leukemia MV4-11 cells, which possess internal tandem duplication mutation in the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD), with IC50s less than 1 microM, as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay on day two of culture. Exposure of these cells to MS-275 decreased levels of total, as well as, phosphorylated forms of FLT3, resulting in inactivation of its downstream signal pathways, including Akt, ERK, and STAT5. Further studies found that MS-275 induced acetylation of heat shock protein 90 (HSP90) in conjunction with ubiquitination of FLT3, leading to degradation of FLT3 proteins in these cells. This was blunted by treatment with the proteasome inhibitor bortezomib, confirming that FLT was degraded via ubiquitin/proteasome pathway. Moreover, we found that further inhibition of MEK/ERK signaling potentiated the action of MS-275 in leukemia cells. Taken together, MS-275 may be useful for treatment of individuals with leukemia possessing activating mutation of FLT3 gene.     

Arsenic trioxide (ATO) and MEK1 inhibition synergize to induce apoptosis in acute promyelocytic leukemia cells.

Recent studies suggest that components of the prosurvival signal transduction pathways involving the Ras-mitogen-activated protein kinase (MAPK) can confer an aggressive, apoptosis-resistant phenotype to leukemia cells. In this study, we report that acute promyelocytic leukemia (APL) cells exploit the Ras-MAPK activation pathway to phosphorylate at Ser112 and to inactivate the proapoptotic protein Bad, delaying arsenic trioxide (ATO)-induced apoptosis. Both in APL cell line NB4 and in APL primary blasts, the inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) and Bad phosphorylation by MEK1 inhibitors enhanced apoptosis in ATO-treated cells. We isolated an arsenic-resistant NB4 subline (NB4-As(R)), which showed stronger ERK1/2 activity (2.7-fold increase) and Bad phosphorylation (2.4-fold increase) compared to parental NB4 cells in response to ATO treatment. Upon ATO exposure, both NB4 and NB4-As(R) cell lines doubled protein levels of the death antagonist Bcl-xL, but the amount of free Bcl-xL that did not heterodimerize with Bad was 1.8-fold greater in NB4-As(R) than in the parental line. MEK1 inhibitors dephosphorylated Bad and inhibited the ATO-induced increase of Bcl-xL, overcoming ATO resistance in NB4-As(R). These results may provide a rationale to develop combined or sequential MEK1 inhibitors plus ATO therapy in this clinical setting.     

A potential role for HSP90 inhibitors in the treatment of JAK2 mutant-positive diseases as demonstrated using quantitative flow cytometry

The V617F mutation of the JAK2 tyrosine kinase is found in a majority of patients with myeloproliferative disorders. Flow cytometry assays for quantitation of phosphorylated and total protein for JAK2, STAT5, and heat shock proteins (HSPs) were developed to facilitate the study of the JAK/STAT pathway. A cell line homozygous for V617F (HEL) was treated with inhibitors of JAK2 tyrosine kinase activity and the HSP90 inhibitor 17-AAG. 17-AAG reduced HSP90 levels, but increased HSP70 levels. Phospho-STAT5, total STAT5, and total AKT levels were also reduced by17-AAG treatment. Further, phospho-JAK2, total JAK2, and cell viability were reduced to a greater extent by 17-AAG than by the pan-JAK kinase family inhibitor JKII or the JAK2-specific inhibitor AG490, and these inhibitors failed to synergize with 17-AAG. Flow-cytometry-based assays for JAK/STAT signaling pathway and HSPs are likely to have broad clinical utility for monitoring patients with abnormalities in the JAK2 pathway.     

Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response,chaperone regulation and apoptosis in myeloma cells

Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways.     

Expression analysis of PCNA gene in chronic myelogenous leukemia--combined application of siRNA silencing and expression arrays

Imatinib metylase is the first choice treatment for BCR/ABL positive chronic myelogenous leukemia (CML). However, as some CML patients develop resistance to imatinib therapy, there is a significant interest in development of alternative treatment strategies, such as identifying targets other than BCR/ABL that may participate in CML. Previously, we demonstrated strong PCNA up-regulation in CML patients. To further study its role in CML pathogenesis, we performed silencing of PCNA expression followed by array experiments. PCNA inhibition led to down-regulation of CDK1, CDK4, PLK1, ERK3, JNK1, STAT5, and several inhibitors of apoptosis (DAXX, Mdm2, survivin). The following genes were up-regulated: CDK inhibitors p21 and p19-INK4D, pro-apoptotic FAST kinase, fibronectin, etc. However, as PCNA affects cell growth in naturally proliferating cells as well as in cancerous cells, it seems to act a secondary role relating to proliferation activity of leukemic cells.     

Immunogenic apoptosis in human acute myeloid leukemia (AML): primary human AML cells expose calreticulin and release heat shock protein (HSP) 70 and HSP90 during apoptosis

Several previous studies have demonstrated that both conventional cytotoxic drugs as well as targeted therapeutics can induce apoptosis in primary human acute myelogenous leukemia (AML) cells. However, the apoptotic phenotype of dying AML cells has been less extensively characterized. Even though specific antileukemic immune reactivity is important in AML, especially for allotransplanted patients, it has not been investigated whether dying primary human AML cells show phenotypic characteristics consistent with immunogenic apoptosis [calreticulin exposure, heat shock protein (HSP) release]. We therefore investigated whether in vitro cultured primary human acute myeloid leukemia (AML) cells show calreticulin exposure and HSP70/HSP90 release during spontaneous (stress-induced) apoptosis when cultured in medium alone and when cultured in the presence of antileukemic drugs. Both surface exposure of calreticulin and release of HSP70 and HSP90 was detected but showed a wide variation between patients. This variation was also maintained when the AML cells were cultured in the presence of cytotoxic drugs (cytarabine, daunorubicin, mitomycin), all-trans retinoic acid (ATRA) and valproic acid. Finally, AML cells collected during in vivo ATRA therapy showed increased calreticulin exposure during spontaneous in vitro apoptosis, suggesting that in vivo pharmacotherapy can modulate the apoptotic phenotype. To conclude, apoptotic AML cells can show phenotypic characteristics consistent with immunogenic apoptosis, but there is a wide variation between patients and the level of calreticulin exposure/HSP release seems to depend on individual patient characteristics rather than the apoptosis-inducing agent.     

HSP90 inhibitor AUY922 abrogates up‐regulation of RTKs by mTOR inhibitor AZD8055 and potentiates its antiproliferative activity in human breast cancer

mTOR inhibition led to activation of upstream receptor tyrosine kinases (RTKs) and AKT, which may attenuate the efficacy of mTOR kinase inhibitors. We sought to discover efficient drug combination with mTOR inhibitors by elucidating the survival feedback loops induced by mTOR inhibition in breast cancer. The feedback signaling upon treatment of mTOR inhibitor AZD8055 was determined and the combinatorial activity of AZD8055 and HSP90 inhibitor AUY922 in cell signaling and proliferation were detected. Treatment of breast cancer T47D cells with AZD8055 induced activation of AKT and phosphatidylinositol 3‐kinase (PI3K), which was accompanied with increase in expression of multiple upstream proteins including EGFR, HER2, HER3 and IRS‐1. Different RTKs were revealed to be responsible for the reactivation of AKT by AZD8055 in different breast cancer cell lines. Down‐regulation of these proteins differentially enhanced the antiproliferative activity of AZD8055. AZD8055 and AUY922 displayed synergistic effect against a panel of human breast cancer cells irrespective their genotype, which was associated with enhanced cell cycle arrest and inhibition of DNA synthesis. AUY922 destabilized multiple tested tyrosine kinases and abrogated activation of AKT induced by AZD8055. AZD8055 also inhibited up‐regulation of HSP70 and HSP27 upon AUY922 treatment. Cotreatment of these two drugs demonstrated synergistic activity against triple negative MDA‐MB‐468 xenograft without enhanced toxicity. The combination of AZD8055 and AUY922 demonstrated synergistic activity against various types of breast cancer and established a mechanistic rationale for a combination approach using catalytic mTOR kinase inhibitor and HSP90 inhibitor in the treatment of breast cancer.     

Targeting Hsp90 by 17-AAG in leukemia cells: mechanisms for synergistic and antagonistic drug combinations with arsenic trioxide and Ara-C.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new anticancer agent currently in clinical trials. The ability of 17-AAG to abrogate the function of heat-shock protein Hsp90 and modulate cellular sensitivity to anticancer agents has prompted recent research to use this compound in drug combination therapy. Here we report that 17-AAG has striking opposite effects on the activity of arsenic trioxide (ATO) and ara-C. Combination of 17-AAG with ATO exhibited a synergistic effect in leukemia cells, whereas coincubation of 17-AAG and ara-C showed antagonistic activity. Mechanistic studies revealed that ATO exerted cytotoxic action by reactive oxygen species generation, and activated Akt survival pathway. 17-AAG abrogated Akt activation and enhanced the activity of ATO. In contrast, treatment of leukemia cells with 17-AAG caused a G1 arrest, a decrease in DNA synthesis and reduced ara-C incorporation into DNA, leading to antagonism. The ability of 17-AAG to enhance the antileukemia activity of ATO was further demonstrated in primary leukemia cells isolated from patients with acute myeloid leukemia and chronic lymphocytic leukemia, including cells from refractory patients. Our data suggest that combination of 17-AAG and ATO may be an effective therapeutic regimen. Caution should be exercised in using 17-AAG together with ara-C, as their combination effects are schedule dependent.     

Clinical implications of c-Kit mutations in acute myelogenous leukemia

c-Kit is a receptor tyrosine kinase (RTK) with a pivotal role in melanogenesis, gametogenesis, and hematopoiesis. Aberrantly activated RTK and related downstream signaling partners were identified as key elements in the molecular pathogenesis of several malignancies. This finding culminated in a two-class model integrating constitutive activating and maturation arrest-inducing mutations as key elements for the pathogenesis of acute myelogenous leukemia (AML). c-Kit is expressed by myeloblasts in about 60% to 80% of patients, and the most frequently observed activating RTK mutations in AML (next to FLT3) are mutations or internal tandem duplications in c-Kit, with an overall incidence of 17%. The identification of small-molecule tyrosine kinase inhibitors capable of blocking key kinase switches introduced a paradigm change in the treatment of diseases like gastrointestinal stromal tumors and chronic myelogenous leukemia. Despite encouraging preclinical data, it appears that a complex clonal disease like AML will probably benefit from a synergistic approach of targeted drugs used (at least for now) in combination with conventional chemotherapy.     

FLT3 expressing leukemias are selectively sensitive to inhibitors of the molecular chaperone heat shock protein 90 through destabilization of signal transduction-associated kinases.

PURPOSE: We conducted studies to evaluate the hypothesis that FLT3 is a client of heat shock protein (Hsp) 90 and inhibitors of Hsp90 may be useful for therapy of leukemia. EXPERIMENTAL DESIGN: The effects of the Hsp90-inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on cell growth, expression of signal transduction kinases, apoptosis, FLT3 phosphorylation and interaction with Hsp90 was determined in FLT3(+) human leukemias. RESULTS: We found that FLT3 is included in a multiprotein complex that includes Hsp90 and p23. 17-AAG inhibited FLT3 phosphorylation and interaction with Hsp90. FLT3(+) leukemias were significantly more sensitive to the Hsp90 inhibitors 17-AAG and Herbimycin A in cell growth assays than FLT3-negative leukemias. Cells transfected with FLT3 became sensitive to 17-AAG. Cell cycle inhibition and apoptosis were induced by 17-AAG. Cells with constitutive expression of FLT3, as a result of internal tandem duplication, were the most sensitive; cells with wild-type FLT3 were intermediate in sensitivity, and FLT3-negative cells were the least sensitive. 17-AAG resulted in reduced cellular mass of FLT3, RAF, and AKT. The mass of another Hsp, Hsp70, was increased. The expression level of MLL-AF4 fusion protein was not reduced by 17-AAG in human leukemia cells. CONCLUSIONS: FLT3(+) leukemias are sensitive to 17-AAG and Herbimycin A. 17-AAG inhibits leukemia cells with either FLT3-internal tandem duplication or wild-type FLT3, in part through destabilization of client kinases including FLT3, RAF, and AKT. 17-AAG is potentially useful for therapy of FLT3-expressing leukemias, including the mixed lineage leukemia fusion gene leukemias.     

Multifaceted approach to the treatment of bcr-abl-positive leukemias

Bcr-Abl-positive leukemias include chronic myelogenous leukemia (CML), both myeloid and lymphoid blast-phase CML, and some cases of acute lymphoblastic leukemia. The chimeric bcr-abl gene codes for a tyrosine kinase that is constitutively activated in the leukemic cells and plays the central role in leukemogenesis. Hematologic malignancies, including Bcr-Abl-positive leukemias, also frequently have overactivity of the Ras signaling pathway, leading to abnormal transduction of growth and survival signals. New and investigational therapeutic options that target these specific molecular defects of leukemic cells include the tyrosine kinase inhibitor imatinib mesylate (STI571) and farnesyltransferase inhibitors (R115777, SCH66336), which block localization of Ras proteins to the cell membrane. While single-agent therapy with these new agents may produce hematologic and cytogenetic remissions in patients with Bcr-Abl-positive leukemias, molecular remissions are less common, and resistance may develop. Therefore, the development of a multifaceted therapeutic approach to these leukemias is of great interest. Arsenic trioxide (ATO), which has significant activity in patients with relapsed and refractory acute promyelocytic leukemia, is a potential addition to the therapeutic arsenal. While some of the molecular activities of ATO are specific to acute promyelocytic leukemia, arsenicals also have a broad variety of antineoplastic properties that may be useful in combination therapy with agents that target specific molecular defects of Bcr-Abl-positive leukemias.