Human Acute Myelogenous Leukemia Cell Lines Research Articles

Suppression of mTOR Signaling Attenuates Apoptotic Effects Induced By the NEDD8-Activating Enzyme Inhibitor Pevonedistat (MLN4924) in Acute Myeloid Leukemia Cells

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Therefore, novel therapies for AML are needed. Recently, the NEDD8 conjugation pathway and the mammalian target of rapamycin (mTOR) signaling pathway have emerged as important regulatory pathways for cancer therapy. Since some components of the mTORC1/2 pathways such as Deptor are neddylation targets, we postulated that combined exposure to NEDD8-activating enzyme (NAE) inhibitor pevonedistat and the dual mTORC1/2 inhibitor sapanisertib (TAK-228) might have additive or synergistic effects in AML.To test pevonedistat and sapanisertib effects on AML cells, we used the agents alone or in combination followed by assessment of proliferation, apoptosis and signal transduction in human AML cell lines as well as in primary cells. By AnnexinV/7AAD flow cytometry analysis, we found that pevonedistat significantly induced AML cell death and apoptosis in a time- and dose-dependent manner. Western blot showed that poly (ADP-ribose) polymerase (PARP) and caspases 3/8/9 were cleaved, indicating that pevonedistat-treated cells were actively undergoing apoptosis. Among the six AML cell lines (HL-60, KG1a, Molm-13, MV4-11, THP-1 and U937) tested, MV4-11 was the most sensitive cell line (IC50 <25nM) and KG1a was the least sensitive cell line (IC50 >1000nM) after 48 hours of exposure. The average IC50 on primary AML blast cells (5 primary samples) was 400nM at 48 hours. Western blot analysis showed that the neddylated cullin levels were markedly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. We also found that pevonedistat induced morphologic changes and increased CD11b expression in Molm-13 and MV4-11 cells, indicating cell differentiation.The effects of pevonedistat on AML cells could be attenuated by pre- or co-treatment with the mTORC1/2 inhibitor sapanisertib. We found that sapanisertib alone induced limited cell death in most AML cell lines and primary AML patient blasts. The results from XTT assay and flow cytometry analysis indicated that sapanisertib alone significantly decreased cell size, arrested cells in G0-G1 phase and reduced protein content of AML cells but did not induce robust apoptosis. This suggested that through inactivating mTOR signal pathway, sapanisertib suppressed cell activity and induced a dormant/quiescent status and thereby attenuated the apoptotic effects of pevonedistat. With concurrent pevonedistat and sapanisertib exposure, the expression of apoptotic mediators such as cleaved PARP and caspases 3 were decreased compare to pevonedistat treatment alone (See Figure).To confirm the role of mTOR inhibition on pevonedistat-induced AML apoptosis, we targeted mTORC1/2 by knocking down mTOR expression with small hairpin RNAs (shRNA). Downregulation of mTOR expression mirrored the effects of sapanisertib. mTOR inhibition by shRNAs resulted in decreased AML cell size and arrest of cells in G0-G1 phase. In addition, pevonedistat-induced cell death was significantly impeded in the cells infected with mTOR shRNA lentivirus compared to scrambled shRNA (p<0.01). Therefore, we validated that mTOR signal inactivation protects AML cells from NAE inhibitor pevonedistat-induced apoptosis using both pharmaceutical and genetic approaches. To further study the mechanism behind mTOR inhibition inducing resistance to pevonedistat, we tested rapamycin, an mTORC1 inhibitor. Concurrent exposure to rapamycin did not significantly alter the effects of pevonedistat and did not change cell size and cell cycle status. In addition, rapamycin did not inhibit the phosphorylation of 4E-BP1, suggesting that mTORC1 inhibition alone may be insufficient to inhibit the effects of pevonedistat, and suggesting a role for mTORC2.Taken together, our data demonstrate that the NEDDylation pathway inhibitor pevonedistat has strong potential as a therapeutic agent for AML therapy via apoptosis induction. However, when AML cells are arrested in G0-G1 with reduced proliferation rates by dual mTORC1/2-inhibition, the ability of pevonedistat to induce apoptosis is suppressed. Whether this is due solely to the cytostatic effects of dual mTOR inhibitors or also involves other mechanisms remains uncertain. While both sapanisertib and pevonedistat have clear anti-leukemia effects, their concurrent use may inhibit NAE inhibitor-induced apoptosis. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

A novel dipeptide type inhibitor of the Wnt/β-catenin pathway suppresses proliferation of acute myelogenous leukemia cells

The Wnt/β-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/β-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated “31” that was found to be an inhibitor of the Wnt/β-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/β-catenin signaling pathway.

Evaluation of apoptosis induction by concomitant inhibition of MEK, mTOR, and Bcl-2 in human acute myelogenous leukemia cells.

Aberrant activation of multiple signaling pathways is common in acute myelogenous leukemia (AML) cells, which can be linked to a poor prognosis for patients with this disease. Previous research with mTOR or MEK inhibitors revealed cytostatic, rather than cytotoxic, effects in in vitro and in vivo AML models. We evaluated the combination effect of the mTOR inhibitor AZD8055 and the MEK inhibitor selumetinib on human AML cell lines and primary AML samples. This combination demonstrated synergistic proapoptotic effects in AML cells with high basal activation of MEK and mTOR. We next incorporated the BH3 mimetic ABT-737 into this combination regimen to block Bcl-2, which further enhanced the apoptogenic effect of MEK/mTOR inhibition. The combination treatment also had a striking proapoptotic effect in CD33(+)/CD34(+) AML progenitor cells from primary AML samples with NRAS mutations. Mechanistically, upregulation of the proapoptotic protein Bim, accompanied by the downregulation of the antiapoptotic protein Mcl-1 (mainly via protein degradation), seemed to play critical roles in enhancing the combination drug effect. Furthermore, the modulation of survivin, Bax, Puma, and X-chromosome-linked inhibitor of apoptosis protein (XIAP) expression suggested a role for mitochondria-mediated apoptosis in the cytotoxicity of the drug combination. Consequently, the concomitant blockade of prosurvival MEK/mTOR signaling and the deactivation of Bcl-2 could provide a mechanism-based integrated therapeutic strategy for the eradication of AML cells.

Preclinical Characterization of AMG 330, a CD3/CD33-Bispecific T-Cell–Engaging Antibody with Potential for Treatment of Acute Myelogenous Leukemia

There is high demand for novel therapeutic options for patients with acute myelogenous leukemia (AML). One possible approach is the bispecific T-cell-engaging (BiTE, a registered trademark of Amgen) antibody AMG 330 with dual specificity for CD3 and the sialic acid-binding lectin CD33 (SIGLEC-3), which is frequently expressed on the surface of AML blasts and leukemic stem cells. AMG 330 binds with low nanomolar affinity to CD33 and CD3ε of both human and cynomolgus monkey origin. Eleven human AML cell lines expressing between 14,400 and 56,700 CD33 molecules per cell were all potently lysed with EC(50) values ranging between 0.4 pmol/L and 3 pmol/L (18-149 pg/mL) by previously resting, AMG 330-redirected T cells. Complete lysis was achieved after 40 hours of incubation. In the presence of AML cells, AMG 330 specifically induced expression of CD69 and CD25 as well as release of IFN-γ, TNF, interleukin (IL)-2, IL-10, and IL-6. Ex vivo, AMG 330 mediated autologous depletion of CD33-positive cells from cynomolgous monkey bone marrow aspirates. Soluble CD33 at concentrations found in bone marrow of patients with AML did not significantly affect activities of AMG 330. Neoexpression of CD33 on newly activated T cells was negligible as it was limited to 6% of T cells in only three out of ten human donors tested. Daily intravenous administration with as low as 0.002 mg/kg AMG 330 significantly prolonged survival of immunodeficient mice adoptively transferred with human MOLM-13 AML cells and human T cells. AMG 330 warrants further development as a potential therapy for AML.

Preclinical and Early Clinical Evaluation of the Oral AKT Inhibitor, MK-2206, for the Treatment of Acute Myelogenous Leukemia

Recent studies suggested that AKT activation might confer poor prognosis in acute myelogenous leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We, therefore, explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206. Experimental Methods: We first studied the effects of MK-2206 in human AML cell lines and primary AML specimens in vitro. Subsequently, we conducted a phase II trial of MK-2206 (200 mg weekly) in adults requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse phase protein array (RPPA). In preclinical studies, MK-2206 dose-dependently inhibited growth and induced apoptosis in AML cell lines and primary AML blasts. We then treated 19 patients with MK-2206 but, among 18 evaluable participants, observed only 1 (95% confidence interval, 0%-17%) response (complete remission with incomplete platelet count recovery), leading to early study termination. The most common grade 3/4 drug-related toxicity was a pruritic rash in 6 of 18 patients. Nevertheless, despite the use of MK-2206 at maximum tolerated doses, RPPA analyses indicated only modest decreases in Ser473 AKT (median 28%; range, 12%-45%) and limited inhibition of downstream targets. Although preclinical activity of MK-2206 can be demonstrated, this inhibitor has insufficient clinical antileukemia activity when given alone at tolerated doses, and alternative approaches to block AKT signaling should be explored.

Enhancing Ceramide Cytotoxicity in Acute Myelogenous Leukemia

Abstract 4905 Background:While understanding of Acute Myelogenous Leukemia (AML) pathogenesis has advanced greatly in recent years, drug discovery and development have added little to therapy. Ceramide and other sphingolipids are of therapeutic interest for many neoplasms (Jiang Y, DiVittore NA, Kaiser JM, et al. Combinatorial therapies improve the therapeutic efficacy of nanoliposomal ceramide for pancreatic cancer. Cancer Biol Ther. Oct 1 2011;12(7):574–585). Cellular ceramide accumulation favors a pro-apoptotic state, while accumulation of sphingosine-1-phosphate promotes survival. We have targeted the ceramide balance of AML cells in vitro with varying concentrations of liposomal formulations of C6 ceramide (Lip-C6), the sphingosine kinase-1 inhibitor safingol (Lip-Saf), and tamoxifen (Lip-Tam) to determine potential synergistic anti-leukemic efficacy. Safingol is a sphingosine kinase inhibitor currently in phase 1 trials. In addition, Tamoxifen can reverse drug resistance of many cancer cell types (Chapman JV, Gouaze-Andersson V, Messner MC, et al. Metabolism of short-chain ceramide by human cancer cells–implications for therapeutic approaches. Biochem Pharmacol. Aug 1 2010;80(3):308–315.). This has been shown to be due to the ability of Tamoxifen to block the activities of glucosylceramide synthase (GCS) and p-glycoprotein (P-GP), which coordinate to detoxify ceramide at the Golgi membrane. In the present study, liposomal drug formulations were chosen to enhance drug delivery and prevent premature drug metabolism. Methods:The cell lines C1498, HL-60, HL-60/VCR, GFPp210, Wehi-3B, K562, U937, and KG-1 were used in this study. Drugs were synthesized into liposomal formulations by the Penn State Hershey Drug Discovery and Delivery Core laboratory. Cellular viability was measured after treatment with Lip-C6, Lip-Saf, Lip-Tam, or a combination for 48 hours. Synergy and dose-response curves were modeled using CalcuSyn software. Apoptosis and cell proliferation were assessed using flow cytometry after treatment of drug for 24 hours at the calculated IC50 from MTS assays. Autophagy was also measured in C1498 cells to confirm an established safingol cell-death mechanism. Primary human AML collected by our lab from consenting, patients was assessed in methylcellulose for blast clonogenicity. Results and Conclusions:Several cell lines showed a favorable change in the IC50 of the drugs when used in combination, indicating a possible synergistic anti-leukemic mechanism of action. When Lip-C6 was combined with Lip-Saf in a varying ratios, synergistic growth inhibition was observed in the human AML cell lines HL-60, HL-60/VCR, and KG-1 (Figure 1). Interestingly, Lip-Tam caused complete cell population death at concentrations less than 15 μM in the Wehi-3B, K562, GFPp210, and C1498 lines. When cells were treated with Lip-C6 and Lip-Tam in a 1:1 combination, complete cell population killing was noted at concentrations of less than 10 μM in every cell line tested. Additionally, flow cytometric data confirmed findings of other investigators suggesting that Lip-Saf caused enhanced autophagy. Therefore, the observed synergistic leukemia cell death is likely due in part to the novel combination of an autophagy-inducer with an apoptosis-inducer. Clonogenic data has shown that combination of Lip-C6and Lip-Saf cooperate to inhibit formation of blast colonies from human AML, indicating a potential use in lessening leukemia burden (Figure 1).In conclusion, novel ceramide-centered drug combinations promote improved cell death of leukemia cell lines via accumulation of ceramide and inhibition of ceramide metabolic pathways. This study acts as a persuasive proof-of-concept of the effect of inhibiting a single ceramide degradation pathway within cells. By inhibiting sphingosine kinase-1 or the GCS activity of P-GP in these cell lines, it becomes apparent that the cellular ceramide balance shifts to favor a pro-apoptotic state. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Unbiased Analyses of Signaling Through Leukemia Associated MicroRNA

Abstract 2373MicroRNA (miRNA or miR) expression profiling of primary human acute myelogenous leukemia (AML) biopsies and human AML cell lines has identified miRNA expression patterns associated with cytogenetic subtypes of AML. Elevated expression of miR-196b is associated with 11q23 translocations (MLL), whereas elevated expression of miR-126 is associated with AML1/CBFβ rearrangements in AML. Moreover, miR-196b blocks normal granulopoiesis and miR-126 inhibits erythropoiesis. To understand how these miRNAs function in normal and abnormal-malignant hematopoiesis, we sought to identify the target mRNAs they regulate. Currently miRNA target identification relies mostly on computational prediction algorithms with high error rates, or indirect methods such as microarray expression analyses of mRNAs altered upon overexpression or inhibition of the miRNA of interest. Here we applied a novel unbiased method to identify direct miR-196b and miR-126 target mRNAs by capture of miRNA-bound mRNA complexes from human fibroblasts and human AML cell lines. mRNAs that were bound to the miRNA of interest were applied to microarray analyses, and those that were present in at least 2-fold or greater quantities over those associated with a negative control C. elegans miRNA were identified as putative targets.We identified 651 potential mRNA targets of miR-196b and 402 potential targets of miR-126. We validated this technique in several ways. First, we showed that captured mRNAs were significantly enriched for miRNA seed sequences by comparison with computer algorithm predicted targets (244/651 for miR-196b and 42/402 for miR-126), and then remaining mRNA 3’UTR sequences were examined using miRNA binding site prediction software. We also confirmed the microarray findings by qRT-PCR analyses of the captured mRNAs from multiple independent experiments. Bioinformatic gene network analyses revealed that the 651 miR-196b targets were significantly enriched for participation in cell cycle checkpoint control, kinetochore/centromere interaction during mitosis, and apoptosis. Additionally, some of the miR-196b candidate targets are know to be deregulated in bone marrow failure (myelodysplastic syndrome or MDS, hemolytic anemia, and thrombocytopenia) or have roles in granulocyte activation. Bioinformatic network analyses of the 402 miR-126 targets revealed significant enrichment for genes involved in mitotic spindle assembly, kinetochore/mitotic spindle interaction during mitosis, Rho-mediated cytoskeletal dynamics, and DNA damage repair. 199 targets were found in common between miR-196b and miR-126. Notably, cell cycle (G2/M checkpoint, and negative cell cycle regulators) was one of the most significantly enriched biological processes in the 199 shared target genes (p<0.001). Moreover, some of the miR-196b/miR-126 common targets participate in translational control complexes dysregulated in bone marrow failure (MDS), as well as transcriptional circuits deregulated in abnormal myeloid differentiation.In sum, we optimized and implemented an innovative microRNA-mRNA target capture approach that resulted in identification of hundreds of previously unknown miRNA targets in human cells. These recent discoveries of putative miR-196b and miR-126 targets suggest novel mechanisms of miRNA gene regulation in normal and defective hematopoiesis and leukemogenesis. The identification of these miRNA targets may also help explain their distinct expression patterns in cytogenetic subtypes of AML. Disclosures:No relevant conflicts of interest to declare.

AEE788 is a vascular endothelial growth factor receptor tyrosine kinase inhibitor with antiproliferative and proapoptotic effects in acute myeloid leukemia

Aberrant activation of tyrosine kinase receptors is frequently observed in acute myelogenous leukemia (AML). Moreover, activating mutations of the fms-like tyrosine kinase 3 (FLT3) receptor can be found in approximately 30% of patients, thereby representing one of the most frequent single genetic alterations in AML. AEE788, a novel dual receptor tyrosine kinase inhibitor of endothelial growth factor and vascular endothelial growth factor (VEGF), is being studied in several solid tumors with remarkable success. It is not known, however, about the efficacy of this inhibitor in the treatment of AML. Therefore, we investigated the effect of AEE788 in the treatment of three human AML cell lines and seven AML patient samples. Cell survival in THP-1, MOLM-13, and MV4-11 cell lines (the two last harboring the FLT3/internal tandem duplication mutation) and AML blasts incubated with 0.5 to 15 microM AEE788 were quantified. We also studied the activation of VEGF/VEGF receptors loop, FLT3, and their downstream effectors (Akt, extracellular signal-regulated kinase, signal transducers and activators of transcription 5, and nuclear factor-kappaB). Our data showed that AEE788 was a tyrosine kinase inhibitor of FLT3 activity and had antiproliferative and proapoptotic activity in AML-derived cell lines and AML blasts that presented phosphorylation of the FLT3 receptor. Consistently, in these cells AEE788 abrogated VEGF/VEGF receptors activation and the survival signaling pathways studied. Taken together, the activity of AEE788 might represent a promising new option of targeting FLT3 for the treatment of AML.

A Therapeutic Approach for Acute Leukemias: Glycolysis Inhibition Revisited

Background: Recent evidence suggests that myeloid and lymphoid leukemia cells are dependent on aerobic glycolysis for ATP production, known as the Warburg effect. Early glycolysis inhibitors were effective but only at high mM concentrations. The novel third generation glycolysis inhibitor, 3-bromo-2-oxopropionate-1-propyl ester (3-BrOP), has shown efficacy in models of neuroblastoma, glioblastoma, colon carcinoma, and lymphoma in the mcM range and has improved oral bioavailability and biodistribution. We proposed that potent inhibition of glycolysis would lead to ATP depletion and induce growth arrest and cell death in acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) cells. Objective: To determine the feasibility and effect of 3-BrOP on human AML, precursor-B, and T cell leukemia cell lines and patient samples.Design/Methods: A panel of fifteen human pre-B ALL, T-ALL, and AML cell lines were treated with 3-BrOP to determine IC50s via cell counts, cell cycle, and Alamarblue. In addition, western blots and ATP assays were performed to determine caspase activation and ATP depletion.Results: All cell lines demonstrate growth arrest, decreased alamar blue metabolism and significant apoptosis when treated with 3-BrOP. The five human precursor-B ALL cell lines had IC50s between 10–30mcM, while the T ALL cell lines had IC50s between 15–30mcM. The human AML cell line panel had IC50s between 20–40mcM. In addition, patient samples representing ALL (IC50 15 mcM) and AML (IC50 30mcM) were sensitive to 3-BrOP mediated cell death. Of note, caspase-3 cleavage was detected by 2 hours of exposure, and was nearly complete by 6 hours in both pre-B and T-ALL suggesting a potent an early pro-apoptotic signal. Overall, cell cycle analysis at 48 hours of treatment, showed the majority of cells had died (83–97%, p<0.001). The remaining cells were apoptotic with >80% of Nalm6 (pre-B ALL) and 72% of U937 (AML) cells having sub-G0 DNA content (p<0.001). As expected, exposure to 3-BrOP reduced intracellular ATP concentrations in a dose and time dependent manner. In Nalm6 cells (pre-B ALL), by 48 hours of exposure, the ATP levels were 50% of control at a dose of 15 mcM. At a dose of 22.5 mcM, the ATP level was 13% of control, and by 30 mcM there was no detectable ATP. Importantly, at higher doses (>30mcM) ATP levels were reduced significantly within 24 hours. These effects on ATP levels correlate with dose and time dependent viability.Conclusions: These data demonstrate that a broad range of acute leukemia cell lines and patient samples appear to be dependent on glycolysis for ATP production and survival. The novel 3rd generation glycolysis inhibitor, 3-BrOP, induces cell death across all subtypes of human acute leukemia cell lines. Glycolysis inhibition via 3-BrOP represents a novel and clinically feasible therapeutic approach for acute leukemias and warrants further pre-clinical evaluation.

Comparison of antitumor effects of multitargeted tyrosine kinase inhibitors in acute myelogenous leukemia

We compared the antitumor activities of the multitargeted tyrosine kinase inhibitors imatinib, sorafenib, and sunitinib to determine which inhibitor is best suited to be used for the treatment of acute myelogenous leukemia (AML). In nine human AML cell lines, sorafenib and sunitinib were more potent inhibitors of cellular proliferation than imatinib (IC50, 0.27 to >40, 0.002-9.1, and 0.007-13 micromol/L for imatinib, sorafenib, and sunitinib, respectively). Sorafenib and sunitinib were potent inhibitors of cells with fms-like tyrosine kinase 3 internal tandem duplication (IC50, 2 and 7 nmol/L) and c-KIT N822K mutations (IC50, 23 and 40 nmol/L). In four cell lines (MV4-11, Kasumi-1, KG-1, and U937) that spanned a range of drug sensitivities, sorafenib and sunitinib had similar activity in apoptosis and cell cycle assays, except that sunitinib did not promote apoptosis in U937 cells. Both drugs inhibited mitogen-activated protein kinase signaling but had no effect on AKT signaling in most of the cell lines tested. Sorafenib was substantially more bound than sunitinib in human plasma (unbound fraction, 0.59% versus 8.4%) and cell culture medium (unbound fraction, 1.3% versus 39%), indicating that sorafenib was more potent than sunitinib and that unbound sorafenib concentrations with activity against most AML cell lines are achievable in vivo. There was more intracellular accumulation of sorafenib than of sunitinib and imatinib in AML cells. Between 1 and 10 micromol/L, sorafenib inhibited the proliferation of six of nine primary AML blast samples by > or =50%. Our results highlight the pharmacologic features of sorafenib that may provide it an advantage in the treatment of AML.

SSBP2, a candidate tumor suppressor gene, induces growth arrest and differentiation of myeloid leukemia cells

Acute myelogenous leukemia (AML) is the most common leukemia in adults with clonal proliferation of myeloid stem cells. Two or more cooperating mechanisms, namely block in differentiation, enhanced proliferation and resistance to programmed cell death, underlie this neoplastic transformation. Nonrandom, complete and partial deletions of chromosome 5 are common anomalies in AML. Using positional cloning strategies, we characterized an evolutionarily conserved candidate myeloid leukemia suppressor gene encoding sequence-specific single-stranded DNA binding protein 2 (SSBP2) from chromosome 5q13.3, a locus that is frequently deleted in AML. Recent studies in Drosophila and Xenopus demonstrate a pivotal role for SSBPs in embryonic differentiation. In mammals, SSBP2 is one of three highly related and ubiquitously expressed genes. Here, we identify frequent loss of SSBP2 protein expression in human AML cell lines using highly specific antibodies. Furthermore, inducible expression of SSBP2 in the AML cell line U937 leads to loss of clonogenicity, G1 arrest and partial differentiation. Remarkably, inducible expression of SSBP2 is accompanied by downregulation of C-MYC expression. Our findings are consistent with human SSBP2 being a novel regulator of hematopoietic growth and differentiation, whose loss confers a block in differentiation advantage to myeloid leukemic cells.

Expression of Cyclooxygenase-2 (COX-2) in Human Leukemias and Hematopoietic Cells.

COX-2 has been implicated in the development of many epithelial cancers, as well as in tumor angiogenesis. COX-2 inhibitors have been shown to have anti-tumor activity in experimental cancer. Little information exists, however, on the expression or role of COX-2 in hematologic malignancies. We have use a variety of immunochemical assays to document expression of COX-2 in human and murine leukemias and hematopoietic cells. The factor-dependent murine cell lines FDCP1 and 32D expressed COX-2 when growing continuously in the presence of IL-3; expression declined markedly when growth factor was removed. FDCP1 cells constitutively expressing bcl-2, pim-1, or bcr-abl had markedly elevated levels of COX-2, and continued to express this enzyme even after removal of growth factor. To assess COX-2 expression in human hematopoietic cells we developed a flow cytometry assay using a FITC-labelled anti-COX-2 MoAb (Cayman). Cells were washed once in serum-free medium, fixed briefly in 1% paraformaldehyde, permeabilized with PBS/0.2% Triton X100, then stained with antibody. Negative control samples were processed similarly but stained with antibody that had been preincubated with immunizing peptide. Specific COX-2 staining was interpreted as the difference between the histograms from blocked versus unblocked anti-COX-2 antibody, as determined by Kolmogorov-Smirnoff analysis. In buffy coat preparations from normal donors, we found constitutive expression of COX-2 in lymphocytes (both B-cells and T-cells). In contrast little or no COX-2 was detected in unstimulated neutrophils or monocytes. In human acute myelogenous leukemia (AML) cell lines we found COX-2 expression to be universal and easily detected. In several cell lines we confirmed the results of our flow cytometry assay with immunoblotting. We further examined 25 cryopreserved samples of human acute leukemia blasts obtained from peripheral blood. COX-2 expression was variable, but universal. Levels generally were less than those seen in immortalized cell lines, and did not correlate with blasts morphology (AML, ALL, APL, AMoL, CML-BT). To determine if COX-2 inhibitors could play a role in the treatment of acute leukemias, we performed cytotoxicity assays using the COX-2 specific inhibitors, celecoxib and NS398. Survival and growth of human AML cell lines were inhibited by both agents. These data demonstrate that 1) a variety of oncogenes can induce expression of COX-2 in hematopoietic cells, 2) clinical human acute leukemias uniformly express COX-2 in circulating blasts, and 3) COX-2 inhibitors are cytotoxic for human leukemia cells. Combination therapies for acute leukemias may evaluate the incorporation of COX-2 inhibitors for added cytotoxic effects or angiogenesis inhibition.

Constitutive activation of STAT transcription factors in acute myelogenous leukemia

Hematopoietic growth factors (HGF) are essential for proliferation and differentiation of hematopoietic precursors and activate a distinct set of JAK-STAT (Janus kinases-signal transducers and activators of transcription) proteins. Previous results from our group have shown a strong expression of JAK-STAT proteins in primary acute myelogenous leukemia (AML) blasts and AML cell lines. Here, we asked whether a constitutive activation of the JAK-STAT pathway might be involved in the pathogenesis of AML. We could demonstrate a constitutive activation of STAT1, 3 and 5 by immunoprecipitation of the tyrosine phosphorylated proteins in different human AML cell lines. Three patterns of STAT activation were found: (I) activation of only STAT1, (II) activation of STAT1 in combination with STAT3, and (III) activation of STAT1, 3 and 5. Furthermore, STAT1 and 3 formed stable heterodimers only in cell lines with constitutive STAT3 activation. In all cell lines analyzed, tyrosine phosphorylation of the four known Janus kinases could not be detected, although JAK1 was stably associated with STAT3. To further analyze whether a constitutive activation of tyrosine kinases might contribute to the autonomous growth of AML blasts, inhibitor studies were performed. The tyrphostin AG490, an inhibitor of the JAK-STAT pathway, but not A1, an inactive tyrphostin induced a time- and dose-dependent growth arrest without overt morphological signs of differentiation in AML cell lines. Our results show that STAT transcription factors are constitutively activated in human AML cell lines and might contribute to the autonomous proliferation of AML blasts. Inhibition of this pathway might be of interest for the establishment of more specific antileukemic strategies.

Constitutive activation of STAT transcription factors in acute myelogenous leukemia

Abstract: Hematopoietic growth factors (HGF) are essential for proliferation and differentiation of hematopoietic precursors and activate a distinct set of JAK-STAT (Janus kinases-signal transducers and activators of transcription) proteins. Previous results from our group have shown a strong expression of JAK-STAT proteins in primary acute myelogenous leukemia (AML) blasts and AML cell lines. Here, we asked whether a constitutive activation of the JAK-STAT pathway might be involved in the pathogenesis of AML. We could demonstrate a constitutive activation of STAT1, 3 and 5 by immunoprecipitation of the tyrosine phosphorylated proteins in different human AML cell lines. Three patterns of STAT activation were found: (I) activation of only STAT1, (II) activation of STAT1 in combination with STAT3, and (III) activation of STAT1, 3 and 5. Furthermore, STAT1 and 3 formed stable heterodimers only in cell lines with constitutive STAT3 activation. In all cell lines analyzed, tyrosine phosphorylation of the four known Janus kinases could not be detected, although JAK1 was stably associated with STAT3. To further analyze whether a constitutive activation of tyrosine kinases might contribute to the autonomous growth of AML blasts, inhibitor studies were performed. The tyrphostin AG490, an inhibitor of the JAK-STAT pathway, but not A1, an inactive tyrphostin induced a time- and dose-dependent growth arrest without overt morphological signs of differentiation in AML cell lines. Our results show that STAT transcription factors are constitutively activated in human AML cell lines and might contribute to the autonomous proliferation of AML blasts. Inhibition of this pathway might be of interest for the establishment of more specific antileukemic strategies.

Topoisomerase II Levels and Drug Sensitivity in Adult Acute Myelogenous Leukemia

AbstractThe topoisomerase (topo) II-directed agents etoposide, daunorubicin (DNR), and amsacrine (m-AMSA) are widely used in the treatment of acute myelogenous leukemia (AML). In the present study, multiple aspects of topo II-me-diated drug action were examined in marrows from adult AML patients. Colony-forming assays revealed that the dose of etoposide, DNR, or m-AMSA required to diminish leukemic colony formation by 90% (LD90) varied over a greater than 20-fold range between different pretreatment marrows. Measurement of nuclear DNR accumulation in the absence and presence of quinidine revealed evidence of P-glycoprotein (Pgp) function in 8 of 82 samples at diagnosis and 5 of 36 samples at first relapse, but the largest quinidine-induced increment in DNR accumulation (<2-fold) was too small to explain the variations in drug sensitivity. Restriction enzyme-based assays and sequencing of partial topo IIα and topo IIβ cDNAs from the most highly resistant specimens failed to demonstrate topo II gene mutations that could account for resistance. Western blotting of marrow samples containing greater than 80% blasts revealed that the content of the two topo II isoenzymes varied over a greater than 20-fold range, but did not correlate with drug sensitivity in vitro or in vivo. In addition, levels of topo IIα and topo IIβ in 46 of 47 clinical samples were lower than in human AML cell lines. Immunoperoxidase staining showed that these low topo II levels were accompanied by marked cell-to-cell heterogeneity, with topo Ha being abundant in some blasts and diminished or absent from others. There was a trend toward increasing percentages of topo IIα-positive cells in pretreatment marrows that contained more S-phase cells. Consistent with this observation, treatment of patients with granulocyte-macrophage colony-stimulating factor for 3 days before chemotherapy resulted in increases in topo IIα-positive cells concomitant with increases in the number of cells traversing the cell cycle. These observations have implications for the regulation of topo II in AML, for the use of topo II-directed chemotherapy, and for future attempts to relate drug sensitivity to topo II levels in clinical material.

Topoisomerase II levels and drug sensitivity in adult acute myelogenous leukemia

The topoisomerase (topo) II-directed agents etoposide, daunorubicin (DNR), and amsacrine (m-AMSA) are widely used in the treatment of acute myelogenous leukemia (AML). In the present study, multiple aspects of topo II-mediated drug action were examined in marrows from adult AML patients. Colony-forming assays revealed that the dose of etoposide, DNR, or m-AMSA required to diminish leukemic colony formation by 90% (LD90) varied over a greater than 20-fold range between different pretreatment marrows. Measurement of nuclear DNR accumulation in the absence and presence of quinidine revealed evidence of P-glycoprotein (Pgp) function in 8 of 82 samples at diagnosis and 5 of 36 samples at first relapse, but the largest quinidine-induced increment in DNR accumulation (&lt; 2-fold) was too small to explain the variations in drug sensitivity. Restriction enzyme-based assays and sequencing of partial topo II alpha and topo II beta cDNAs from the most highly resistant specimens failed to demonstrate topo II gene mutations that could account for resistance. Western blotting of marrow samples containing greater than 80% blasts revealed that the content of the two topo II isoenzymes varied over a greater than 20-fold range, but did not correlate with drug sensitivity in vitro or in vivo. In addition, levels of topo II alpha and topo II beta in 46 of 47 clinical samples were lower than in human AML cell lines. Immunoperoxidase staining showed that these low topo II levels were accompanied by marked cell-to- cell heterogeneity, with topo II alpha being abundant in some blasts and diminished or absent from others. There was a trend toward increasing percentages of topo II alpha-positive cells in pretreatment marrows that contained more S-phase cells. Consistent with this observation, treatment of patients with granulocyte-macrophage colony- stimulating factor for 3 days before chemotherapy resulted in increases in topo II alpha-positive cells concomitant with increases in the number of cells traversing the cell cycle. These observations have implications for the regulation of topo II in AML, for the use of topo II-directed chemotherapy, and for future attempts to relate drug sensitivity to topo II levels in clinical material.