Acute Myelogenous Leukemia Cell Lines Research Articles

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.

Identification of Wee1 as a novel therapeutic target for mutant RAS-driven acute leukemia and other malignancies.

Direct targeting of rat sarcoma (RAS), which is frequently mutated, has proven to be challenging, and inhibition of individual downstream RAS mediators has resulted in limited clinical efficacy. We designed a chemical screen to identify compounds capable of potentiating mammalian target of rapamycin (mTOR) inhibition in mutant RAS-positive leukemia, and identified a Wee1 inhibitor. Synergy was observed in both mutant neuroblastoma RAS viral oncogene homolog (NRAS)- and mutant kirsten RAS viral oncogene homolog (KRAS)-positive acute myelogenous leukemia (AML) cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-binding protein 1 and s6 kinase, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 knockdown, as well as partial reversal of drug combination-induced apoptosis by a cyclin-dependent kinase 1 (CDK1) inhibitor. Importantly, we also extended our findings to other mutant RAS-expressing malignancies, including mutant NRAS-positive melanoma, and mutant KRAS-positive colorectal cancer, pancreatic cancer and lung cancer. We observed favorable responses with combined Wee1/mTOR inhibition in human cancer cell lines from multiple malignancies, and inhibition of tumor growth in in vivo models of mutant KRAS lung cancer and leukemia. The present study introduces for the first time Wee1 inhibition combined with mTOR inhibition as a novel therapeutic strategy for the selective treatment of mutant RAS-positive leukemia and other mutant RAS-expressing malignancies.

Importance of Glutamine Metabolism in Leukemia Cells by Energy Production Through TCA Cycle and by Redox Homeostasis

Some cancer cells depend on glutamine despite of pronounced glycolysis. We examined the glutamine metabolism in leukemia cells, and found that HL-60 cells most depended on glutamine in the 4 acute myelogenous leukemia (AML) cell lines examined: growth of HL-60 cells was most suppressed by glutamine deprivation and by inhibition of glutaminolysis, which was rescued by tricarboxylic acid (TCA) cycle intermediate, oxaloacetic acid. Glutamine is also involved in antioxidant defense function by increasing glutathione. Glutamine deprivation suppressed the glutathione content and elevated reactive oxygen species most evidently in HL-60 cells. Glutamine metabolism might be a therapeutic target in some leukemia.

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.

WTAP is a novel oncogenic protein in acute myeloid leukemia

WTAP is a novel oncogenic protein in acute myeloid leukemia

Developing aptamer probes for acute myelogenous leukemia detection and surface protein biomarker discovery

BackgroundThe majority of patients with acute myelogenous leukemia (AML) still die of their disease. In order to improve survival rates in AML patients, new strategies are necessary to discover biomarkers for the detection and targeted therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface.MethodsThe NB4 AML cell line was used as the target cell population for selecting single stranded DNA aptamers. After determining the affinity of selected aptamers to leukocytes, the aptamers were used to phenotype human bone marrow leukocytes and AML cells in clinical specimens. Then a biotin-labelled aptamer was used to enrich and identify its target surface protein.ResultsThree new aptamers were characterized from the selected aptamer pools (JH6, JH19, and K19). All of them can selectively recognize myeloid cells with Kd in the low nanomole range (2.77 to 12.37 nM). The target of the biotin-labelled K19 aptamer probe was identified as Siglec-5, a surface membrane protein in low abundance whose expression can serve as a biomarker of granulocytic maturation and be used to phenotype AML. More importantly, Siglec-5 expression can be used to detect low concentrations of AML cells in human bone marrow specimens, and functions as a potential target for leukemic therapy.ConclusionsWe have demonstrated a pipeline approach for developing single stranded DNA aptamer probes, phenotyping AML cells in clinical specimens, and then identifying the aptamer-recognized target protein. The developed aptamer probes and identified Siglec-5 protein may potentially be used for leukemic cell detection and therapy in our future clinical practice.

Inhibition of Wee1 Sensitizes Cancer Cells to Antimetabolite Chemotherapeutics In Vitro and In Vivo, Independent of p53 Functionality

Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia.

Panobinostat, An Oral Pan-Histone Deacetylase (HDAC) Inhibitor Activates a Microrna Signature That Targets Rad51 To Attenuate Homologous DNA Repair and Sensitize AML Cells To Sapacitabine

Acute myelogenous leukemia (AML) is characterized by multiple genetic and epigenetic abnormalities including a profound dysregulation of microRNA expression. Specific expression signatures of miRNA are associated with disease behavior and clinical outcome.Effective clinical treatment of AML has largely depended on a class of antimetabolites – the nucleoside analogs. Of these, sapacitabine is a nucleoside analog prodrug that is in development for the therapy of AML. It is converted to its active metabolite 2-C-cyano-2-deoxy-1-β(-D-arabino-pentafuranosyl) cytosine (CNDAC), which interferes with DNA synthesis by initially causing a single stranded DNA break that is converted into a double strand break in the subsequent replicative cycle. Such double strand breaks are primarily repaired by the homologous recombination repair (HR) pathway. Consequently, efficient HR may offer a potential resistance mechanism to therapy with sapacitabine.Panobinostat and vorinostat are pan HDAC inhibitors (HDACi) that modulate gene expression, induce apoptosis, and are clinically active against specific leukemias and lymphomas. In pilot experiments, we identified that exposure of AML cell lines to a combination of panobinostat or vorinostat and sapacitabine had a greater than additive effect on cell viability. Loss of cell survival was preceded by a rapid and marked decline in the levels of Rad51, a protein that is critical in repairing CNDAC-induced damage via the HR repair pathway, both in AML cell lines and primary tumor cells that were exposed to either panobinostat or vorinostat. Therefore, we hypothesized that exposure of AML cells to HDACi would activate the expression of a set microRNAs, that would target Rad51. Loss of Rad51 protein, would in turn impede the successful completion of HR rendering the cells incapable of repairing the damage caused by CNDAC and thus sensitizing them to the nucleoside analog.Our preliminary data using microRNA arrays indicated that exposure of AML cells to panobinostat resulted in the induction of nine microRNA that putatively target Rad51. We then ectopically expressed three of the nine Rad51- directed miRNA and found that miR-182 efficiently targeted the Rad51 protein. Using real time-PCR we found that the levels of miR-182 were significantly lower in primary AML cells when compared to hematopoietic cells from normal donors. Using chromatin immunoprecipitation (ChIP) assays we identified that HDAC1 and HDAC2 were recruited to the miR182 promoter in AML cell lines as well as in primary AML cells, which was probably linked to loss of gene expression. Correspondingly, HDAC inhibition led to the accumulation of activating chromatin modifications at the miR-182 promoter, which was followed by the induction of miR-182 levels in both AML cells lines and primary AML cells. To determine whether HDAC inhibition targets Rad51 via miR-182, we expressed antagomiRs that interfere with the binding of miR-182 to Rad51, and found that the levels of Rad51 were preserved in cells expressing anti-miR182 before being exposed to panobinostat. Finally, we identified that exposure to panobinostat suppressed ongoing HR as measured by the absence of Rad51 foci in cells exposed to this agent, whereas cells exposed to CNDAC alone exhibited robust Rad51 foci formation, indicating an active HR pathway.In conclusion, HDAC inhibition results in the induction of miR-182 which targets Rad51 and attenuates HR to sensitize cells to sapacitabine in AML. This therapeutic strategy may be effective in circumventing potential resistance mechanisms to nucleoside analog therapy in AML. Disclosures:Ewald:Novartis: Employment.

The pp90rsk-CREB Signaling Pathway Regulates Apoptosis In Acute Myelogenous Leukemia

CREB (cAMP Response-Element Binding Protein) is a nuclear transcription factor critical for hematopoietic cell proliferation, differentiation, and survival. We previously demonstrated that 60% of patients with Acute Myelogenous Leukemia (AML) overexpress CREB in leukemic blasts, and CREB overexpression in these patients was associated with an increased risk of relapse and decreased event-free survival. Previous studies have suggested that CREB may play an important role in the regulation of apoptosis in a wide variety of cancers. Specifically, CREB has been shown to up-regulate members the anti-apoptotic protein family such as Bcl-2, Bcl-XL and Mcl-1, leading to chemotherapy resistance in vitro. CREB-mediated resistance to apoptosis may underlie the increased rate of relapse and poor survival of AML patients with CREB overexpression. Thus, we hypothesized that targeted inhibition of CREB in AML cells would promote AML cell apoptosis. To test this hypothesis, we developed a small-molecule inhibitor of CREB function, XX-650-23. This molecule disrupts the interaction between CREB and its binding partner CBP (CREB-Binding Protein), which is required for full activation of CREB-mediated gene transcription. Treatment of primary AML patient bone marrow samples with XX-650-23 induced apoptosis and cell death at a dose of 2 uM. The degree of apoptosis varied with the expression level of CREB in primary AML cells tested. Higher CREB levels correlated with higher sensitivity to XX-650-23. In non-leukemic primary patient bone marrow samples, CREB levels were very low, and XX-650-23 did not induce apoptosis in these cells. AML cell lines (KG-1 and HL-60) also underwent apoptosis following CREB inhibition, in proportion to CREB expression level. CREB knockdown or overexpression in KG-1 cells decreased and increased susceptibility to apoptosis, respectively. Mechanistically, the onset of apoptosis in AML cells occurred simultaneously with down-regulation of Bcl-2, a validated CREB-regulated gene. Inhibition of Bcl-2 function using the specific Bcl-2 inhibitor ABT-737 (100 nM) induced apoptosis similar to XX-650-23, indicating that Bcl-2 inhibition alone is sufficient to cause apoptosis. Thus, targeted inhibition of CREB results in Bcl-2 downregulation and is sufficient to induce apoptosis in AML cells. Proteomic analysis using Mass Cytometry-Time of Flight (CyTOF) revealed that one compensatory cellular response to CREB inhibition is increased phosphorylation of CREB. This phosphorylation decreased in the presence of BI-D1870, a specific inhibitor of the pp90RSK kinase (RSK), but not by pharmacologic inhibition of the p38 or ERK kinases, using SB202190 or U0126, respectively. We therefore examined the role of pp90RSK in the regulation of apoptosis in AML cells. Pharmacologic inhibition of RSK independently lead to AML cell apoptosis (BI-D1870, IC50=3.3 uM), in part due to blockade of CREB phosphorylation. In summary, our data provide the first evidence that inhibition of CREB, or its chief activator RSK, is sufficient to induce apoptosis in AML cells. Current work focuses on defining CREB target genes mediating XX-650-23 response using chromatin-immunoprecipitation with massively parallel DNA sequencing (ChIP-Seq), and defining the RSK kinome in AML cells using 2-dimensional gel phosphoprotein profiling. These studies will more fully define the role of the RSK-CREB signaling axis in AML proliferation, survival, and apoptosis. Disclosures:No relevant conflicts of interest to declare.

Hypoxia Regulates Exosomal Microrna Content, Trafficking and Function Of Key Elements In The AML Microenvironment

Small, non-coding micro RNA (miRNA) are recognized for their potent regulatory capacity. Several recent studies indicate the prognostic value of miRNA profiling in acute myelogenous leukemia (AML), although a more mechanistic understanding of the role miRNA play in AML biology is still lacking. We recently demonstrated that patient-derived AML blasts release exosomes (nanometer-size, extracellular vesicles) that traffic a non-random subset of miRNA to stromal bystander cells, eliciting changes in transcriptional activity and growth factor secretion (Huan et al., Cancer Res. 2013). Here we hypothesized that exosome miRNA provide a candidate mechanism for the adaptation of the bone marrow to a specialized leukemic niche. As oxygen levels in the bone marrow are substantially lower than those commonly used in tissue culture, we undertook a systematic study of miRNA incorporation and exosome trafficking in AML under physiological oxygen conditions. In carefully calibrated tissue culture conditions we initially observed an up to 7-fold net increase in exosome number released by Molm14 (Flt3-ITD+ AML cell line) leukemia cells at 1% O2versus 21% O2. Nanoparticle tracking analysis and RNA bioanalyzer tracings suggested that the decreased O2 did not alter vesicle composition, average RNA amount per exosome, or global RNA profiles. Further emphasizing the critical nature of appropriate compartmental oxygenation in exosome trafficking, both murine and human stromal cells demonstrated increased uptake of Molm14 exosomes under hypoxia. Low-oxygen conditions alter transcriptional profiles, phenotypic behavior and drug resistance in AML. Therefore, we next evaluated the miRNA expression of leukemic cells and their incorporation in exosomes at 1% versus 21% O2, utilizing the Affymetrix microarray platform containing >5,000 human (hsa) miRNA probesets, followed by select qRT-PCR validation. Array experiments showed broad differences between cellular and exosomal miRNA and revealed that certain miRNA were selectively regulated in an oxygen-responsive manner. For example, hematopoiesis relevant hsa-miR-124, -146a, and -155 increased an average of 4.6-, 5.5-, and 4.9-fold, respectively, in exosomes from hypoxia-conditioned cells. Intriguingly, several known, non-AML specific, hypoxia-responsive miRNA substantially increased in cells cultured at 1% O2 (e.g. miR-210 by 33-fold), but changed less than 2-fold in exosomes. Several recent reports show that leukemia cells actively convert the bone marrow microenvironment and contribute to the erosion of hematopoiesis by modulating hematopoietic-stromal interactions, in part via decrease in SDF1a, SCF, and Angpt1. We investigated the ability of AML-derived exosomes to regulate these transcripts, and found a 50% decrease in SCF and over 90% decrease in Angpt1 in murine stromal cells after in vitro exposure to leukemia exosomes, again with relatively greater differences for exosomes from hypoxia-conditioned AML cells. These experiments were complemented by observations of altered clonogenicity (CFU-C) of murine lin-negative hematopoietic cells after AML exosome exposure, whereby hypoxia conditioning prompted a decline in colony count to 46% from vesicle-free media baseline, compared with 31% decrease at 21 % O2. Exosomes equilibrate across biological fluids and can be recovered from serum. To translate our observations to an in vivo setting, we developed a xenograft model using Molm14 cells in immune-deficient NSG mice. Early after grafting animals, exosomes could be reproducibly isolated from as little as 20 microL serum and candidate miRNA (hsa-miR-146, -150, 155, 210) were amplified, allowing us to quantitatively track leukemia progression via a unique miRNA signature even before circulating leukemia cells were detectable in the peripheral blood. A comparison of leukemic animals to NSG controls bearing cord blood MNC grafts revealed that changes in circulating miRNA were disease specific and resembled those in the hypoxia setting in vitro. In sum, our work demonstrates that physiologic oxygen levels not only increase AML exosome trafficking between cells, but selectively alter the miRNA profile contained therein. These changes produce phenotypic alterations in stromal and hematopoietic bystander cells that correlate with the functional conversion of the bone marrow to a leukemic niche. Disclosures:No relevant conflicts of interest to declare.

Acquired multidrug resistance in human K562/ADM cells is associated with enhanced autophagy

Autophagy, as a necessary process for survival in mammalian cells deprived of nutrients or growth factors, will be activated in many tumor cells while treated with chemotherapeutic drugs, but the role of autophagy in acquired multidrug resistance of human acute myelogenous leukemia to adriamycin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental acute myelogenous leukemia cell line K562 and resistant sub cell line, K562/ADM, which were obtained by treating adriamycin with increasing concentrations. K562/ADM and K562 cells were exposed to PBS culture medium for 3 hours, then the stress-induced autophagy was measured. Real-time quantitative RT-PCR revealed the expression of LC3 mRNA was higher in K562/ADM than in K562 cells. LC3-II, as an autophagosomal marker, was more abundant in K562/ADM than in K562 cells measured by Western blotting. To determine the effect of 3-MA, a known specific inhibitor of autophagy, on overcoming acquired multidrug resistance induced by adriamycin, the MTT assay and flow cytometry were performed. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of adriamycin in acquired resistant cells (K562/ADM). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in multidrug resistance of K562/ADM cells induced by adriamycin.

The TPO/c-MPL Pathway in the Bone Marrow may Protect Leukemia Cells from Chemotherapy in AML Patients

Accumulating evidence indicates that the interaction of human LSCs (leukemic stem cells) with the hematopoietic microenvironment, mediated by the thrombopoietin (TPO)/c-MPL pathway, may be an underlying mechanism for resistance to cell cycle-dependent cytotoxic chemotherapy. However, the role of TPO/c-MPL signaling in AML (acute myelogenous leukemia) chemotherapy resistance hasn't been fully understood. The c-MPL and TPO levels in different AML samples were measured by flow cytometry and ELISA. We also assessed the TPO levels in the osteoblasts derived from bone mesenchymal stem cells (BMSCs). The survival rate of an AML cell line that had been co-cultured with different BMSC-derived osteoblasts was measured to determine the IC50 of an AML chemotherapy drug daunorubicin (DNR). The levels of TPO/c-MPL in the initial and relapse AML patients were significantly higher than that in the control (P < 0.05). The osteoblasts derived from AML patients' BMSCs secreted more TPO than the osteoblasts derived from normal control BMSCs (P < 0.05). A strong positive correlation between the TPO level and c-MPL expression was found in the bone marrow mononuclear cells of the relapse AML patients. More importantly, the IC50 of DNR in the HEL + AML-derived osteoblasts was the highest among all co-culture systems. High level of TPO/c-MPL signaling may protect LSCs from chemotherapy in AML. The effects of inhibition of the TPO/c-MPL pathway on enhancing the chemotherapy sensitivity of AML cells, and on their downstream effector molecules that direct the interactions between patient-derived blasts and leukemia repopulating cells need to be further studied.

Phosphatidylserine index as a marker of the procoagulant phenotype of acute myelogenous leukemia cells

Patients with acute myelogenous leukemia (AML) are at risk for thrombotic complications. Risk to develop thrombosis is closely tied to leukemia subtype, and studies have shown an association between leukocytosis and thrombosis in AML M3. We evaluated the relative roles of cell count and the surface expression of tissue factor (TF) and phosphatidylserine (PS) in the procoagulant phenotype of AML cell lines. The TF-positive AML M3 cell lines, NB4 and HL60, and AML M2 cell line, AML14, exhibited both extrinsic tenase and prothrombinase activity in a purified system and promoted experimental thrombus formation. In contrast, the TF-negative AML cell line, HEL, exhibited only prothrombinase activity and did not affect the rate of occlusive thrombus formation. In plasma, NB4, HL60 and AML14 shortened clotting times in a cell-count, PS- and TF-dependent manner. Exposure of cultured NB4, HL60, and AML14 cells to the chemotherapeutic agent daunorubicin increased their extrinsic tenase activity and PS expression. Clot initiation time inversely correlated with logarithm of PS index, defined as the product of multiplying leukocyte count with cell surface PS exposure. We propose that leukemia cell PS index may serve as a biomarker for procoagulant activity.

The application of Heck reaction in the synthesis of guaianolide sesquiterpene lactones derivatives selectively inhibiting resistant acute leukemic cells

A series of guaianolide-type sesquiterpene lactones derivatives with arylation of α-methylene-γ-lactone moiety was synthesized using Heck reactions, and was evaluated for their activities against acute myelogenous leukemia (AML) cell line HL-60 and doxorubicin-resistant cell line HL-60/A. Although all compounds were significantly less active against HL-60 than the parent molecules, surprisingly, compounds 3a, 4c–4e, 5e, and 8d exhibited high potency against doxorubicin-resistant cell line HL-60/A (IC50=6.2–19μM), and their activities against HL-60/A were comparable to that of their parent molecules. In view of their novel activities against HL-60/A, compound 5e with inhibitory activity against HL-60/A (IC50=6.2±0.5μM) was selected for study its preliminary mechanism. The result reveals that compound 5e can obviously induce apoptosis.

Oroxylin A has therapeutic potential in acute myelogenous leukemia by dual effects targeting PPARγ and RXRα

Oroxylin A (OA) is a flavonoid derived from a Chinese herb that has previously been reported to inhibit the proliferation of several cancer cell lines. It is found that OA significantly inhibited the growth of myeloid leukemia cell lines and as xenografts in immunodeficient mice and primary blasts from acute myelogenous leukemia (AML) patients. Furthermore, OA-induced cell cycle arrest and differentiation were observed in OA-treated AML cell lines. OA-induced increase of CD11b/CD14 expression was reversed by GW9662, a specific PPARγ inhibitor, or transient transfection with PPARγ siRNA. Docking study showed OA bound to ligand-binding domain of PPARγ via forming hydrogen bonds with Arg288 and Leu340 sites. Results of fluorescence polarization-based ligand assay verified PPARγ-binding activity of OA, and in OA-treated cells, intranuclear accumulation and increased binding activity of PPARγ to PPRE were detected. We also found that GW9662 attenuated OA-induced upregulation of C/EBPβ, an important regulator of leukemic differentiation, and p21, which is a potent inhibitor of CDKs that can inhibit phosphorylation of Rb by cyclin D1-CDK4 complexes. Moreover, our results showed that OA displayed synergistic effects with all-trans retinoic acid and VD3 in part related to reduction of intranuclear phosphorylated RXRα that has been reported to block nuclear receptor/RXRα heterodimer transcriptional activity. This reduction of phosphorylated RXRα was associated with inhibition of the specific upstream MAP kinase ERK1/2. We suggest that OA may provide a novel complement to AML treatment by its dual effects of augmenting PPARγ activity and sensitizing nuclear receptors to specific ligands.

Farnesyltransferase inhibitor tipifarnib inhibits Rheb prenylation and stabilizes Bax in acute myelogenous leukemia cells

Although farnesyltransferase inhibitors have shown promising activity in relapsed lymphoma and sporadic activity in acute myelogenous leukemia, their mechanism of cytotoxicity is incompletely understood, making development of predictive biomarkers difficult. In the present study, we examined the action of tipifarnib in human acute myelogenous leukemia cell lines and clinical samples. In contrast to the Ras/MEK/ERK pathway-mediated Bim upregulation that is responsible for tipifarnib-induced killing of malignant lymphoid cells, inhibition of Rheb-induced mTOR signaling followed by dose-dependent upregulation of Bax and Puma occurred in acute myelogenous leukemia cell lines undergoing tipifarnib-induced apoptosis. Similar Bax and Puma upregulation occurred in serial bone marrow samples harvested from a subset of acute myelogenous leukemia patients during tipifarnib treatment. Expression of FTI-resistant Rheb M184L, like knockdown of Bax or Puma, diminished tipifarnib-induced killing. Further analysis demonstrated that increased Bax and Puma levels reflect protein stabilization rather than increased gene expression. In U937 cells selected for tipifarnib resistance, neither inhibition of signaling downstream of Rheb nor Bax and Puma stabilization occurred. Collectively, these results not only identify a pathway downstream from Rheb that contributes to tipifarnib cytotoxicity in human acute myelogenous leukemia cells, but also demonstrate that FTI-induced killing of lymphoid versus myeloid cells reflects distinct biochemical mechanisms downstream of different farnesylated substrates. (ClinicalTrials.gov identifier NCT00602771).

Synthesis of Micheliolide Derivatives and Their Activities against AML Progenitor Cells

Micheliolide (MCL) derivatives with etherification or esterification of the hydroxyl group at the C4 position were synthesized and evaluated for their activities against different acute myelogenous leukemia (AML) cell lines. These derivatives demonstrated comparable activities against AML cell lines HL-60 and doxorubicin resistant cell line HL-60/A. As to multi-drug resistant AML progenitor cells KG-1a, MCL and some of its derivatives maintained significant activities, and only 1.1–2.7 fold activity reductions were observed when compared with the activities against HL-60, while doxorubicin showed 20-fold activity reduction. Our study demonstrated that the C4 hydroxyl group of MCL might not only be a suitable position for structural modifications, but also be a starting point for the design of appropriate molecular probes to explore the specific targets in the progenitor cell line KG-1a.

RNA Trafficking by Acute Myelogenous Leukemia Exosomes

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML.

T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE antibody construct

Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML.