Teresa McQueen

Mutant FLT3: A Direct Target of Sorafenib in Acute Myelogenous Leukemia

BACKGROUND Internal tandem duplication (ITD) mutations in the juxtamembrane domain-coding sequence of the Fms-like tyrosine kinase 3 (FLT3) gene have been identified in 30% of acute myeloid leukemia (AML) patients and are associated with a poor prognosis. The kinase inhibitor sorafenib induces growth arrest and apoptosis at much lower concentrations in AML cell lines that harbor FLT3-ITD mutations than in AML cell lines with wild-type FLT3. METHODS The antileukemic activity of sorafenib was investigated in isogenic murine Ba/F3 AML cell lines that expressed mutant (ITD, D835G, and D835Y) or wild-type human FLT3, in primary human AML cells, and in a mouse leukemia xenograft model. Effects of sorafenib on apoptosis and signaling in AML cell lines were investigated by flow cytometry and immunoblot analysis, respectively, and the in vivo effects were determined by monitoring the survival of leukemia xenograft-bearing mice treated with sorafenib (groups of 15 mice). In a phase 1 clinical trial, 16 patients with refractory or relapsed AML were treated with sorafenib on different dose schedules. We determined their FLT3 mutation status by a polymerase chain reaction assay and analyzed clinical responses by standard criteria. All statistical tests were two-sided. RESULTS Sorafenib was 1000- to 3000-fold more effective in inducing growth arrest and apoptosis in Ba/F3 cells with FLT3-ITD or D835G mutations than in Ba/F3 cells with FLT3-D835Y mutant or wild-type FLT3 and inhibited the phosphorylation of tyrosine residues in ITD mutant but not wild-type FLT3 protein. In a mouse model, sorafenib decreased the leukemia burden and prolonged survival (median survival in the sorafenib-treated group vs the vehicle-treated group = 36.5 vs 16 days, difference = 20.5 days, 95% confidence interval = 20.3 to 21.3 days; P = .0018). Sorafenib reduced the percentage of leukemia blasts in the peripheral blood and the bone marrow of AML patients with FLT3-ITD (median percentages before and after sorafenib: 81% vs 7.5% [P = .016] and 75.5% vs 34% [P = .05], respectively) but not in patients without this mutation. CONCLUSION Sorafenib may have therapeutic efficacy in AML patients whose cells harbor FLT3-ITD mutations.

The Synthetic Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid Induces Caspase-Dependent and -Independent Apoptosis in Acute Myelogenous Leukemia

In acute myeloid leukemia (AML), resistance to chemotherapy is associated with defects in both the extrinsic and intrinsic pathways of apoptosis. Novel agents that activate endogenous apoptosis-inducing mechanisms directly may be potentially useful to overcome chemoresistance in AML. We examined the mechanisms of apoptosis induction by the novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) in AML cells. CDDO-induced apoptosis was associated with the loss of mitochondrial inner transmembrane potential, caspases activation, the translocation of apoptosis-inducing factor to the nucleus, and DNA fragmentation in AML cells. Apoptosis was equally evident in cells deficient in caspase-9 or caspase-8 after exposure to CDDO, suggesting caspase-independent cell death. The use of small interfering RNA to reduce the expression of apoptosis-inducing factor partially inhibited CDDO-induced apoptosis in AML cells. Cells overexpressing Bcl-2 were markedly resistant to CDDO-induced apoptosis. Moreover, CDDO promoted the release of cytochrome c from isolated mitochondria, suggesting that CDDO targets the mitochondria directly to trigger the intrinsic pathway of cell death in intact cells. Together, these results suggest that CDDO functions by activating the intrinsic pathway of apoptosis and initiates caspase-dependent and independent cell death. The direct modulation of mitochondrial-mediated, caspase-independent apoptosis by CDDO may be advantageous for overcoming chemoresistance in AML.

Activation of Integrin-Linked Kinase Is a Critical Prosurvival Pathway Induced in Leukemic Cells by Bone Marrow–Derived Stromal Cells

Integrin-linked kinase (ILK) directly interacts with beta integrins and phosphorylates Akt in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. In this study, we examined the functional role of ILK activation in leukemic and bone marrow stromal cells on their direct contact. Coculture of leukemic NB4 cells with bone marrow-derived stromal mesenchymal stem cells (MSC) resulted in robust activation of multiple signaling pathways, including ILK/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducers and activators of transcription 3 (STAT3), and Notch1/Hes. Blockade of PI3K or ILK signaling with pharmacologic inhibitors LY294002 or QLT0267 specifically inhibited stroma-induced phosphorylation of Akt and glycogen synthase kinase 3beta, suppressed STAT3 and ERK1/2 activation, and decreased Notch1 and Hes1 expression in leukemic cells. This resulted in induction of apoptosis in both leukemic cell lines and in primary acute myelogenous leukemia samples that was not abrogated by MSC coculture. In turn, leukemic cells growing in direct contact with bone marrow stromal elements induce activation of Akt, ERK1/2, and STAT3 signaling in MSC, accompanied by significant increase in Hes1 and Bcl-2 proteins, which were all suppressed by QLT0267 and LY294002. In summary, our results indicate reciprocal activation of ILK/Akt in both leukemic and bone marrow stromal cells. We propose that ILK/Akt is a proximal signaling pathway critical for survival of leukemic cells within the bone marrow microenvironment. Hence, disruption of these interactions by ILK inhibitors represents a potential novel therapeutic strategy to eradicate leukemia in the bone marrow microenvironment by simultaneous targeting of both leukemic cells and activated bone marrow stromal cells.

Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway

Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenib-induced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.

The Warburg Effect in Leukemia-Stroma Cocultures Is Mediated by Mitochondrial Uncoupling Associated with Uncoupling Protein 2 Activation

In 1956, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect (i.e., the inhibition of anaerobic fermentation by oxygen). Since then, permanent defects in oxygen consumption that could explain the dependence of cancer cells on aerobic glycolysis have not been identified. Here, we show that under normoxic conditions exposure of leukemia cells to bone marrow-derived mesenchymal stromal cells (MSC) promotes accumulation of lactate in the culture medium and reduces mitochondrial membrane potential (DeltaPsiM) in both cell types. Notably, the consumption of glucose was not altered in cocultures, suggesting that the accumulation of lactate was the result of reduced pyruvate metabolism. Interestingly, the decrease in DeltaPsiM was mediated by mitochondrial uncoupling in leukemia cells and was accompanied by increased expression of uncoupling protein 2 (UCP2). HL60 cells fail to increase UCP2 expression, are not uncoupled after coculture, and do not exhibit increased aerobic glycolysis, whereas small interfering RNA-mediated suppression of UCP2 in OCI-AML3 cells reversed mitochondrial uncoupling and aerobic glycolysis elicited by MSC. Taken together, these data suggest that microenvironment activation of highly conserved mammalian UCPs may facilitate the Warburg effect in the absence of permanent respiratory impairment.

Inhibition by all-trans-retinoic acid of tumor necrosis factor and nitric oxide production by peritoneal macrophages.

Besides their growth‐inhibiting and differentiation‐inducing properties, retinoids have been shown to exert immunomodulatory and anti‐inflammatory functions by mechanisms that are not well understood. Tumor necrosis factor‐α (TNF), a cytokine produced by mononuclear phagocytes, has been shown to be an important mediator of endotoxin‐induced septic shock, cachexia, bone resorption, and inflammation. Nitric oxide may also have a role in septic shock, hypotension, and vasodilatation. In this study, we examined the effects of retinoids on the production of TNF and nitric oxide by murine peritoneal macrophages. Of the various retinoids studied, all‐trans‐retinoic acid (RA) was most potent; it almost completely inhibited the production of TNF by macrophages activated with endotoxin and interferon‐γ. The inhibitory effect was dependent on the dose and duration of RA exposure to macrophages. RA also blocked phorbol ester‐induced TNF production in a macrophage cell line (RAW 264.7). Besides TNF, the retinoid suppressed the production of nitric oxide from activated peritoneal macrophages. The importance of these results in relation to controlling various harmful effects of cytokines released by activated macrophages is discussed. J. Leukoc. Biol. 55: 336–342; 1994.

Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia.

We investigated the constitutive activation of the MEK/ERK pathway in acute myelogenous leukemia (AML) via a flow cytometric technique to quantitate expression of phosphorylated ERK (p-ERK). A total of 42 AML samples (16 newly diagnosed, 26 relapsed/refractory) were analyzed. Normal bone marrow CD34+ cells (n=10) had little or no expression of p-ERK, while G-CSF-mobilized CD34+ cells exhibited enhanced p-ERK levels. Markedly elevated p-ERK levels were found in 83.3% of the AML samples, with no differences observed between the newly diagnosed and relapsed/refractory samples. Treatment with a MEK inhibitor resulted in significantly decreased p-ERK levels in both the newly diagnosed and relapsed/refractory samples, which was associated with growth arrest, but not apoptosis induction. In summary, we defined conditions for the analysis of MAPK signaling in primary AML samples. Normal CD34+ cells expressed very low levels of p-ERK, and increased p-ERK levels were found in normal G-CSF-stimulated circulating CD34+ cells. Constitutively high p-ERK levels observed in the majority of AML samples suggest deregulation of this pathway that appears to be independent of disease status. The ability of ERK inhibition to promote growth arrest rather than apoptosis suggests that clinical trials of MEK/ERK inhibitors may be more effective when combined with chemotherapy.

Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia

XIAP is a member of the inhibitors-of-apoptosis family of proteins, which inhibit caspases and block cell death, with prognostic importance in AML. Here we demonstrate that cytokines regulate the expression of XIAP in leukemic cell lines and primary AML blasts. Inhibition of phosphatidylinositol-3 kinase (PI3K) with LY294002 and of the mitogen-activated protein kinase (MAPK) cascade by PD98059 resulted in decreased XIAP levels (34±8.7 and 23±5.7%, respectively). We then generated OCI-AML3 cells with constitutively phosphorylated Akt (p473-Akt) by retroviral gene transfer. Neither these nor Akt inhibitor-treated OCI-AML3 cells showed changes in XIAP levels, suggesting that XIAP expression is regulated by PI3K downstream effectors other than Akt. The induction of XIAP expression by cytokines through PI3K/MAPK pathways is consistent with its role in cell survival. Exposure of leukemic cells to chemotherapeutic agents decreased XIAP protein levels by caspase-dependent XIAP cleavage. Targeting XIAP by XIAP antisense oligonucleotide resulted in downregulation of XIAP, activation of caspases and cell death, and sensitized HL-60 cells to Ara-C. Our results suggest that XIAP is regulated by cytokines through PI3K, and to a lesser degree through MAPK pathways. Selective downregulation of XIAP expression might be of therapeutic benefit to leukemic patients.

The novel triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid inhibits metastatic murine breast tumor growth through inactivation of STAT3 signaling

We and others have reported that C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) effectively inhibits the growth of multiple cancer cell types. Our previous studies indicated that prolonged CDDO-Me treatment inactivated extracellular signal-regulated kinase signaling in acute myelogenous leukemia cells. Whether treatment with CDDO-Me has an earlier effect on other proteins that are important for either signal transduction or oncogenesis is unknown. Constitutively activated signal transducer and activator of transcription 3 (STAT3) is frequently found in human breast cancer samples. Constitutively activated STAT3 was shown to up-regulate c-Myc in several types of cancer and has a feedback effect on Src and Akt. To examine the effects of CDDO-Me on STAT3 signaling in breast cancer, we used the murine 4T1 breast tumor model, which is largely resistant to chemotherapy. In vitro, after treatment of 4T1 cells with 500 nmol/L CDDO-Me for 2 h, we found (a) inactivation of STAT3, (b) inactivation of Src and Akt, (c) 4-fold reduction of c-Myc mRNA levels, (d) accumulation of cells in G(2)-M cell cycle phase, (e) abrogation of invasive growth of 4T1 cells, and (f) lack of apoptosis induction. In in vivo studies, CDDO-Me completely eliminated 4T1 breast cancer growth and lung metastases induced by 4T1 cells in mice when treatment started 1 day after tumor implantation and significantly inhibited tumor growth when started after 5 days. In vivo studies also indicated that splenic mature dendritic cells were restored after CDDO-Me treatment. In summary, these data suggest that CDDO-Me may have therapeutic potential in breast cancer therapy, in part, through inactivation of STAT3.

MEK Inhibition Enhances ABT-737-Induced Leukemia Cell Apoptosis via Prevention of ERK activated MCL-1 induction and Modulation of MCL-1/BIM Complex

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-XL, ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.