Acute Myeloid Cell Lines Research Articles

Targeting EYA1 Activity in MLL- Rearranged Leukemia

The Mixed Lineage Leukemia gene (also known as MLL or KMT2A) is involved in chromosomal translocations in a subtype of acute myeloid (AML) and lymphoblastic leukemias (ALL). MLL translocations have been identified with more than 80 partner genes, and the most common AML translocation partner is MLLT3 (AF9). MLL-rearranged (MLL-r) leukemia is associated with a sudden onset, aggressive progression, and poor prognosis in comparison to non-MLL-r (MLL-nr) leukemias, and there is a need for more effective therapies. Previous work determined that AF9 directly interacts with multiple different regulatory proteins, including the BCL6 corepressor (BCOR). An AF9 point mutation (E531R) was identified that selectively disrupts BCOR binding to AF9 and MLL-AF9. The loss of BCOR binding to MLL-AF9 abrogated its leukemogenic ability in a mouse transplant model. RNA-seq analysis determined that of the MLL-AF9 direct target genes, EYA1 was the most down-regulated in the MLL-AF9 (E531R) point mutant-transformed cells. Addback of EYA1 into MLL-AF9 (E531R) cells partially rescued its leukemogenic ability, suggesting that EYA1 has an important role in MLL-AF9 leukemogenesis. EYA1 is a transcriptional coactivator and protein tyrosine phosphatase that is expressed during embryonic development and its re-expression has been implicated in several cancers, including MLL-r leukemia. The objective of this study is to investigate the in vitroand in vivo effects of EYA1 inhibition in MLL-AF9 transformed murine bone marrow stem/progenitor cells and in human MLL-rearranged (MLL-r) and non-rearranged (MLL-nr) leukemia cell lines. Cells were treated with small molecule inhibitors at varied concentrations and time points. Effects of inhibition were assessed with viability, cell morphology, cell cycle, surface and intracellular marker expression analyses, and in vivo transplant studies. EYA1 inhibition reduced cell viability by 50% - 90% in MLL-AF9 transformed murine bone marrow (mBM) cells and multiple human AML cell lines. EYA1 inhibition did not affect some human B-ALL cell lines, which also express undetectable levels of EYA1, indicating a correlation between the response to EYA1 inhibition and EYA1 expression. In MLL-r and MLL-nr cells, cell cycle analysis indicated a reduction in cells entering S-phase, stalling in G0/G1, with EYA1 inhibition as well as an induction in cellular senescence. Inhibiting EYA1 activity induced cell morphology changes toward a more differentiated state in MLL-AF9 mBM and several MLL-r and -nr cell lines. To confirm these qualitative results, cells were stained for myeloid differentiation markers, and cell surface expression of CD11b, Gr1, and CD14 increased in a dose- and time-dependent manner in all cell types, while HSC markers CD117 (c-KIT) and CD34 were reduced in MLL-AF9 mBM and human AML cells, respectively. In assessing the mechanism of EYA1 in MLL-r leukemia, MLL-AF9 cells treated with EYA1 inhibitors showed increased RNA pol II CTD Tyr1 phosphorylation levels (known EYA1 substrate) compared to vehicle-treated cells. Thus, unregulated EYA1 phosphatase activity targeting RNA Pol II CTD may contribute to leukemia development. Mice that received MLL-AF9 mBM cells treated with an EYA1 inhibitor had a significantly decreased rate of disease progression and increased survival compared to control. Bone marrow cells isolated from these mice showed increased expression of myeloid differentiation markers CD11b and Gr1 compared to vehicle-treated mice, confirming that EYA1 activity contributes significantly to leukemogenesis. The inhibition of EYA1 induces leukemia cell differentiation, cell cycle arrest and senescence, and cell death. Ongoing experiments include additional in vivo murine leukemia, further mechanistic analyses, and combinatorial experiments to evaluate the efficacy of a multi-target approach with EYA1 inhibition and other targeted-therapies. This work may provide a promising, novel therapeutic approach to MLL-r and MLL-nr leukemias that will help improve patient outcomes.

Arsenic Trioxide and Thalidomide Combination Induces Autophagy Along with Apoptosis in Acute Myeloid Cell Lines.

Objective Autophagy and apoptosis play key roles in cancer survival and pathogenesis and are governed by specific genes which have a dual role in both cell death and survival. Arsenic trioxide (ATO) and thalidomide (THAL) are used for treatment of many types of hematologic malignancies. ATO prevents the proliferation of cells and induces apoptosis in some cancer cells. Moreover, THAL has immunomodulatory and antiangiogenic effects in malignant cells. The aim of present study was to examine the effects of ATO and THAL on U937 and KG-1 cells, and evaluation of mRNA expression level of VEGFs genes, PI3K genes and some of autophagy genes. Materials and Methods In this in vitro experimental study, U937 and KG-1 cells were treated by ATO (0.4-5 µM) and THAL (5-100 µM) for 24, 48 and 72 hours. Cell viability was measured by MTT assay. The apoptosis rate and cell cycle arrest were evaluated by flow cytometry (Annexin/PI) and cell cycle flow cytometry analysis, respectively. The effect of ATO/THAL on mRNAs expression was evaluated by real-time polymerase chain reaction (PCR). Results ATO/THAL combination enhanced cell apoptosis in a dose-dependent manner. Also, ATO/THAL induced SubG1/ G1 phase arrest. mRNA expression levels of VEGFC (contrary to other VEGFs isoform), PI3K, AKT, mTOR, MEK1, PTEN, IL6, LC3 and P62 genes were upregulated in acute myeloid leukemia (AML) cells following treatment with ATO/THAL. ConclusionCombined treatment with ATO and THAL can inhibit proliferation and invasion of AML cells by down-regulating ULK1 and BECLIN1 and up-regulating PTEN and IL6, and this effect was more marked than the effects of ATO and THAL alone.

The impact of inflammation and cytokine expression of PM2.5 in AML.

Environmental and health issues have become a major focus of research worldwide in recent years. Particulate matter with diameter ≤2.5 µm (PM2.5) is a common air pollutant that has been demonstrated to be associated with various diseases, including acute myeloid leukemia (AML). In the present study, the effects of PM2.5 on the proliferation and inflammation were assessed using three human acute myeloid cell lines (U937, HL-60 and KG-1a) in vitro. Additionally, the levels of several cytokines [interleukin (IL)-2, IL-10, IL-17A and tumor necrosis factor (TNF)α] in AML cells and Sprague Dawley rats were evaluated to investigate the effects of PM2.5 on cytokine expression in AML. The results revealed that PM2.5 was capable of enhancing inflammatory responses in AML cells, and increasing IL-2, IL-10, IL-17A and TNFα mRNA expression in AML cells to different degrees. Furthermore, PM2.5 increased IL-2 and IL-10 contents in rats following 12 weeks of exposure. These results suggested that PM2.5 may serve a role in promoting the occurrence and progression of leukemia by affecting cytokine expression, and that there may be various mechanisms active in different AML subtypes.

The Combination of Flavokawain B and Daunorubicin Induces Apoptosis in Human Myeloid Leukemic Cells by Modifying NF-κB.

Flavokawain B (FKB), is a natural chalcone isolated from kava root that induces apoptosis in cancer cells. Herein we investigated the effects of combination of FKB and daunorubicin (DNR) on human leukemic cells. Cell viability and death were assessed by the MTS assay and flow cytometry. NK-κB was detected by western blotting. FKB alone and in combination with DNR reduced the viable cell numbers of four leukemic cell lines. FKB itself induced apoptosis of an acute myeloid cell line, HL-60. Because the additive effect of DNR and FKB was most obvious in HL-60 cells, subsequent experiments were performed with HL-60 cells. Combined treatment of the two compounds increased NF-κB activation at 12 h. A combination treatment of DNR and FKB may improve the anticancer effects of DNR in DNR-resistant acute myeloid leukemia.

Potential Role of OCT4 in Leukemogenesis.

Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.

The Hexane Fraction of Bursera microphylla A. Gray Induces p21-Mediated Anti-Proliferative and Pro-Apoptotic Effects in Human Cancer-Derived Cell Lines.

Bursera microphylla (BM), one of the common elephant trees, is widely distributed in the Sonoran Desert in Mexico. The Seri ethnic group in the Sonoran Desert uses BM as an anti-inflammatory and painkiller drug for the treatment of sore throat, herpes labialis, abscessed tooth, and wound healing. Dried stems and leaves of BM are used in a tea to relieve painful urination and to stimulate bronchial secretion. Furthermore, BM is used for fighting venereal diseases. To investigate the effects of the hexane fraction of resin methanol extract (BM-H) on cell growth, the acute myeloid cell line (OCI-AML3) was treated with 250, 25, or 2.5 µg/mL of BM-H. The first 2 concentrations were able to significantly decrease OCI-AML3 cell number. This reduced cell number was associated with decreased S-phase, blockade of the G2/M phase of the cell cycle, and increased cell death. Similar results were obtained on all tested tumor cell lines of different origins. We found that blockade of the cell cycle was due to upregulation of p21 protein in a p53-independent way. Increase of p21 was possibly due to upstream upregulation of p-ERK (which stabilizes p21 protein) and downregulation of p-38 (which promotes its degradation). Regarding cell death, activation of caspase-3, but not of caspase-8 or -9, was detectable after BM-H treatment. In conclusion, these data suggest that the BM’s hexane fraction inhibited proliferation of cell lines mainly by a p21-dependent, p53-independent mechanism and promoted apoptosis through activation of caspase-3, but not caspase-8 or -9.

The Hexane Fraction of Bursera microphylla A Gray Induces p21-Mediated Antiproliferative and Proapoptotic Effects in Human Cancer–Derived Cell Lines

Bursera microphylla (BM), one of the common elephant trees, is widely distributed in the Sonoran desert in Mexico. The Seri ethnic group in the Sonoran desert uses BM as an anti-inflammatory and painkiller drug for the treatment of sore throat, herpes labialis, abscessed tooth, and wound healing. Dried stems and leaves of BM are used in a tea to relieve painful urination and to stimulate bronchial secretion. Furthermore, BM is used for fighting venereal diseases. To investigate the effects of the hexane fraction of resin methanol extract (BM-H) on cell growth, the acute myeloid cell line (OCI-AML3) was treated with 250, 25, or 2.5 µg/mL of BM-H. The first 2 concentrations were able to significantly decrease OCI-AML3 cell number. This reduced cell number was associated with decreased S-phase, blockade of G2/M phase of the cell cycle, and increased cell death. Similar results were obtained on all tested tumor cell lines of different origins. We found that blockade of the cell cycle was a result of upregulation of p21 protein in a p53-independent way. Increase of p21 was possibly a result of upstream upregulation of p-ERK (which stabilizes p21 protein) and downregulation of p-38 (which promotes its degradation). Regarding cell death, activation of caspase-3, but not of caspase-8 or -9, was detectable after BM-H treatment. In conclusion, these data suggest that BM-H inhibited proliferation of cell lines mainly by a p21-dependent, p53-independent mechanism and promoted apoptosis through activation of caspase-3 but not caspase-8 or -9.

SOX12: a novel potential target for acute myeloid leukaemia.

The role of SRY-related high-mobility-group box (SOX) 12 in leukaemia progression and haematopoiesis remains elusive. This study aimed to examine the expression and function of SOX12 in acute myeloid leukaemia (AML) using human myeloid leukaemia samples and the acute myeloid cell line THP1. Mononuclear cells were isolated from the bone marrow of AML patients and healthy donors. SOX12 expression in haematopoietic cells was evaluated by reverse transcription polymerase chain reaction (RT-PCR). SOX12 short hairpin RNAs (shRNAs) were transduced into THP1 cells, and gene knockdown was confirmed by quantitative RT-PCR and Western blot analysis. SOX12 was preferentially expressed in CD34+ cells in AML patients. The THP1 cells transduced with SOX12 shRNAs exhibited significantly reduced SOX12 expression and cell proliferation. SOX12 knockdown had no effect on apoptosis, but it induced cell cycle arrest at G1 phase and reduced the number of colonies. The transduced THP1 and primary AML cells were reconstituted in non-obese diabetic-severe combined immunodeficient (NOD/SCID) mice, and their numbers were significantly reduced 6-12weeks after transplantation. The mRNA and protein levels of β-catenin were significantly diminished following SOX12 knockdown, accompanied by a decrease in TCF/Wnt activity. SOX12 may be involved in leukaemia progression by regulating the expression of β-catenin and then interfering with TCF/Wnt pathway, which may be a target for AML.

Abstract 3561: Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells

Abstract Human acute myeloid cell lines undergo genetic re-programming in response to differentiation inducers. The M3 AML cell line, HL-60, has served as a model system for studying phorbol ester-induced myeloid differentiation into macrophage-like cells. Macrophage differentiation is preceded by cell cycle arrest and followed by apoptosis. Therefore, the system elucidates the changes in the leukemia cell gene expression program necessary to override the genetic alterations driving continuous cell division, preventing myeloid differentiation, and blocking programmed cell death. We hypothesize that defining the evolving transcriptome as AML cells reverse their proliferative, undifferentiated, and pro-survival phenotype will reveal genes regulating these processes and possibly identify new targets for AML treatment. Whole genome exon DNA microarray analyses identifies 1260 genes whose RNA expression levels change significantly during the reprogramming. Among these genes are members of the BCL-2 family that encode both pro- and anti-apoptotic proteins (BBC3, PMAIP1, BID and BCL2, MCL1, BCL2A1, respectively). Myeloid cell leukemia 1 (MCL1) is a BCL-2 family protein defined as an anti-apoptotic protein, however MCL1 increases in expression following phorbol ester treatment in HL-60 cells, as well as other AML lines including THP-1, Kasumi-1, and KG-1. The MCL1 gene can be alternatively spliced to generate a transcript encoding a BH3-only variant (MCL1S) known to exhibit pro-apoptotic function. Western blotting demonstrates the accumulation of MCL1S protein prior to the initiation of apoptosis in phorbol ester treated cells. Here we present evidence for splice variant switching and explore the possibility that reduction in the pro-survival form, MCL1L, and accumulation of MCL1S mediates the p53-independent initiation of apoptosis. Currently, we are evaluating the effects on the leukemic phenotype and apoptotic response to phorbol ester in HL-60 cell lines overexpressing MCL1L, MCL1S, and PMAIP1, as well as HL-60 lines containing CRISPR-Cas9 mediated knockouts of MCL1 and PMAIP1. Citation Format: Cassandra Holbert, Jeffrey Forrester, Michael Roberts. Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3561.

Abstract 5459: Preclinical efficacy of the novel PIM2 kinase inhibitor, JP_11646, in human acute leukemia models

Abstract PIM kinases (PIM1,2, and 3) are constitutively active serine/threonine protein kinases which regulate cell cycle, survival, and drug resistance and have been strongly implicated in cancer cell survival and tumorigenesis. Although expression levels of PIM family members (PIM1, 2, and 3) differ by cancer type, PIM1 and PIM2 kinases are preferentially overexpressed in hematological malignancies. Here we studied the biological effects of treatment with a novel pan-PIM inhibitor, JP_11646 exhibiting potent inhibition of PIM2 kinase activity, in human acute myeloid (AML), acute lymphocytic leukemia (ALL), and lymphoma cell lines. Human AML cell lines (THP-1, KG-1, HL60-VCR, ML2, MV(4;11)) were evaluated for basal mRNA and protein expression of PIM kinases using real time PCR and western blot analysis, respectively. A panel of thirty additional human AML, ALL (Nalm-6), and lymphoma (RAJI) cell lines were exposed to JP_11646 at concentrations ranging from 0.001 to 10 uM for 48-72 hours followed by measurements of cell proliferation by MTT assay and cell viability by CellTiter-Glo assay. The Hill model was used to fit each concentration-response curve for each cell line and endpoint. Nonlinear regression analysis was performed using Phoenix 64 for estimation of IC50 and Imax. AML (HEL) cells after 72 hour of JP_11646 treatment were analyzed for induction of apoptosis measured via annexin V and 7AAD flow cytometry. AML cell lines exhibited differential basal mRNA expression of PIM kinases. The cell lines that expressed the highest levels of PIM1, PIM2, and PIM3 mRNA were THP-1 and ML2, respectively. Basal expression of the PIM2 protein revealed the presence of all three PIM2 isoforms in AML (HEL, THP-1) cells. JP_11646 treatment resulted in inhibition of proliferation across the entire panel of AML and ALL cell lines, with the overall potency (IC50) ranging from 0.07 to 0.37 uM and maximal inhibition ranging from 70 to 97.5 percent. As the drug exposure time increased from 48 to 72 hours, the maximal inhibition increased 2 to 3-fold. Inhibition of cell viability across a panel of 30 leukemia and lymphoma cell lines demonstrated a similar IC50 range from 0.05 to 0.25 uM. AML (HEL) cells treated with JP_116464 at doses of 0.1 and 0.5 uM for 72 hours exhibited a concentration-dependent induction of apoptosis of 32% and 89% percent, respectively. Taken together, our results demonstrate broad anti-leukemic activity and potency of the novel PIM kinase inhibitor, JP_11646, across a wide panel of human AML and ALL cell lines with induction of apoptosis and cell death at nM concentrations. Further, investigation of the in vivo effects of JP_11646 in representative human AML xenograft models is ongoing. Citation Format: Krista E. Pundt, Carmen M. Baldino, Justin Caserta, Stephane Dumas, Yvonne Flanders, Eunice S. Wang, Gerald J. Fetterly. Preclinical efficacy of the novel PIM2 kinase inhibitor, JP_11646, in human acute leukemia models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2014-5459

Abstract 346: Differentiation induced apoptosis in AML cells: The role of p73 in p53-independent versus p53-mediated apoptosis

Abstract Human acute myeloid cell lines can be induced to differentiate into macrophage-like cells by phorbol esters. Differentiation is accompanied by cessation of cell proliferation and initiation of apoptosis. Many of these cell lines, like HL-60, are p53 deficient and therefore execute a differentiation-induced, p53-independent program of cell death. Evaluating changes in gene expression during a time course of PMA-induced HL-60 cell differentiation by DNA microarray analyses and RT-qPCR reveals up-regulation of several pro-apoptotic genes (ex. TNF, MCL1, EGR2, BCL3) and down-regulation of several anti-apoptotic genes (ex. BCL2, ANXA3, TSC22D3). Interestingly, RNA levels of p73 are unchanged, suggesting p73 does not substitute for the missing p53 function via activation of p73 gene transcription, however, we report here that p73 protein levels increase dramatically during the differentiation time course. By reintroducing p53 into HL-60 cells, we compare the changing gene expression profile during p53-mediated apoptosis versus that which occurs during phorbol ester-induced p73 accumulation through protein stabilization. Common and unique gene expression changes define the putative p73-dependent apoptosis program compared to p53-dependent apoptosis. Citation Format: Michael Roberts, Rizwan Saffie, Harold Salmons, Mansoor Ghoto, Juliana Schneider, Jeffrey Forrester. Differentiation induced apoptosis in AML cells: The role of p73 in p53-independent versus p53-mediated apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2014-346

C-Myc regulates expression of NKG2D ligands ULBP1/2/3 in AML and modulates their susceptibility to NK-mediated lysis

AbstractCytarabine (cytosine arabinoside) is one of the most effective drugs for the treatment of patients diagnosed with acute myeloid leukemia (AML). Despite its efficiency against AML cells, the emergence of drug resistance due to prolonged chemotherapy in most patients is still a major obstacle. Several studies have shown that drug resistance mechanisms alter the sensitivity of leukemia cells to immune system effector cells. To investigate this phenomenon, parental acute myeloid cell lines, HL-60 and KG-1, were continuously exposed to increasing doses of cytarabine in order to establish equivalent resistant cell lines, HL-60(R) and KG-1(R). Our data indicate that cytarabine-resistant cells are more susceptible to natural killer (NK)-mediated cell lysis as compared with parental cytarabine-sensitive cells. The increased susceptibility correlates with the induction of UL-16 binding proteins (ULBP) 1/2/3 and NK group 2, member D (NKG2D) ligands on target cells by a mechanism involving c-Myc induction. More importantly, chromatin immunoprecipitation assay revealed that ULBP1/3 are direct targets of c-Myc. Using drug-resistant primary AML blasts as target cells, inhibition of c-Myc resulted in decreased expression of NKG2D ligands and the subsequent impairment of NK cell lysis. This study provides for the first time, the c-Myc dependent regulation of NKG2D ligands in AML.

Modulation Of The Glycolytic Metabolism In Acute Myeloid Leukemia Cells

Glycolysis is the central axis of cellular metabolism. The cancer cell bioenergetic status heavily relies on high glycolytic rates, even in aerobic conditions, thus sustaining the expensive processes of cell growth and proliferation. Growing evidences show that signaling aberrations - especially those involving PI3K/Akt/mTOR, HIF1a, Ras/Raf/MEK/ERK - are strictly connected to the establishment of a pro-glycolytic metabolism, through a multi-level crosstalk between proteins and metabolites that contribute to the acquisition of an energetic background granting a proliferative advantage. Here we investigated the glycolytic rate of resting and activated normal peripheral blood lymphocytes (NPBLs) and of acute myeloid leukemia (AML) cell lines. In an attempt to modulate the cellular metabolism for therapeutic intervention, we tested the following compounds that directly interfere with major metabolic or signaling pathways: dichloroacetate (DCA), a glycolysis inhibitor; aminooxyacetate (AOA), a glutaminolysis inhibitor; ST1326 (kindly given by Sigma-Tau), a fatty acid oxidation (FAO) inhibitor; and the MEK inhibitor PD0325901 (Selleck Chemicals). The cytotoxic drug effects were evaluated on two human leukemia cell lines, U937 and OCI-AML3, characterized by PI3K/Akt/mTOR and Ras/Raf/MEK/ERK hyperactivation, respectively. Cell counts, apoptosis (AnnexinV), glucose and lactate levels (GEM4000, Instrumentation Laboratory, UK) were measured. The glucose consumption rate (GCR) and lactate production rate (LPR) were calculated according to Li et al. (Biotechol. Appl. Biochem., 2005, 42, 73-80). Resting NPBLs were characterized by a very low glycolytic rate, according to their quiescent state, while cultured phytohemagglutinin-activated NPBLs displayed a remarkable increase in glycolytic rate: the GCR calculated over 72 hours showed a 25 fold-increase, while LPR had a of 10 fold-increase. Acute myeloid cell lines showed a high glucose catabolism: at 24h the U937 cell line, compared to activated NPBLs, had a 6.7 fold higher GCR, while the OCI-AML3 cell line showed a 4-fold increase. DCA exposure showed at 24h no detectable effect on GCR, LPR and apoptosis at concentrations ranging from 0.01 to 0.5mM on the U937 cell line. Apoptosis effects were detected only at higher concentrations: AnnexinV positive cells increased from 4.3 ± 1.5 (control) to 62.8 ± 16.4 (5mM) and 88.1 ± 16.8 (10mM). Exposure to AOA (24h at 1000µM) slightly increased GCR (1.23-fold) and LPR (1.22-fold) on U937 cells, followed by apoptotic effects at 72h: from 6.24 ± 4.2 (control) to 10.4 ± 0.8 at 100µM to 83.5 ± 0.7 at 1000µM. The FAO inhibitor ST1326 (10µM at 24h) induced a 3-fold increase of GCR in the U937 cell line. Apoptotic effects were seen in the U937 cells at 72h, from 5.0 ± 2.3 (control) to 35.9 ± 5.7 at 5µM to 64.1 ± 20.5 at 10µM. Conversely, GCR, LPR and apoptosis did not change on the OCI-AML3 line following ST1326 exposure. The MEK inhibitor PD0325901 caused a reduction of GCR and LPR on OCI-AML3 cells (6-fold GCR decrease, 2-fold LPR decrease at 100nM); apoptosis at 72h ranged from 6.3 ± 1.1 (control) to 15.5 ± 3.9 at 10nM to 45.3 ± 0.1 at 100nM. The U937 cells proved resistant to this compound, showing no metabolic perturbation and absence of apoptotic effects. In summary, this study indicates that exploiting the metabolism as a target for therapeutic intervention appears to be a promising new strategy. In fact, the inhibition of glycolysis by blocking either the activity of the enzymes that directly participate to the metabolic pathway or key components of cell signaling has proven to be effective in inducing apoptosis in AML cells. Interestingly, the opposing response to the various compounds observed in the two AML models may likely reflect their divergent signaling network, prompting further studies to evaluate the correlation between aberrant signal transduction pathways and peculiar metabolic profiles. Disclosures:Nicolai:Sigma Tau Pharmaceuticals: Employment.

The sorafenib plus nutlin-3 combination promotes synergistic cytotoxicity in acute myeloid leukemic cells irrespectively of FLT3 and p53 status

Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia. Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 μM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA. The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA. Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.

Antiproliferative and Proapoptotic Effects of Proteasome Inhibitors and their Combination with Histone Deacetylase Inhibitors on Leukemia Cells

New chemotherapeutic agents are still required to further optimise treatment of leukemia patients. Proteasome inhibition by bortezomib, PR-171 (carfilzomib) and NPI-0052 (salinosporamide A) has been successfully used for the treatment of multiple myeloma and mantle cell lymphoma and is considered also as novel treatment strategy in leukemia. Combination of proteasome inhibitors bortezomib and NPI-0052 induces synergistic anti-multiple myeloma activity both in vitro using multiple myeloma cells and in vivo in a human plasmacytoma xenograft mouse model. Cell death resulting from proteasome inhibition requires caspase activation and increased levels of reactive oxygen species. While bortezomib induces several caspases, NPI-0052 activates predominantly caspase-8-dependent pathway. We studied the effect of bortezomib (10 nM) on DNA synthesis and apoptosis in human acute myeloid cell lines KASUMI-1, ML-1, ML-2 and CTV-1 cells. Bortezomib was potent inhibitor of DNA synthesis in all four types of leukemia cells and induced apoptosis in KASUMI-1, ML-2 and CTV-1 cells but not in ML-1 cells. Other research groups showed that histone deacetylase inhibitors (valproic acid or benzamide derivative MS-275) in combination with NPI-0052 or PR-171 induced greater levels of acute leukemia cell death than in combination with bortezomib. Proteasome inhibition as monotherapy and its combination with many conventional therapies as novel treatment strategies in leukemia are promising. Malignant cells are more sensitive to this treatment than normal hematopoietic cells.

Cotreatment with Pioglitazone, a Synthetic Ligand for Peroxisome Proliferator-Activated Receptor γ (PPARγ), Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis of Human Leukemia Cells.

Imatinib (STI571, Glivec) is the choice treatment for Bcr/Abl-positive malignancies. Emergence of resistance to Imatinib warrants the exploration of novel well-tolerated anticancer agents. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor family, which mainly associates with the adipocyte differentiation, but also appears to facilitate cell differentiation or apoptosis in certain malignant cells. Previous studies imply that the PPARγ activation pathway may be a possible intervention mode for treatment of leukemia, which is resistant to imatinib (STI571). In this study, we investigated the effects of pioglitazone, a synthetic ligand for PPARg, on the cell growth and TRAIL-induced apoptosis in a novel imatinib (STI571) resistant acute myeloid cell line (SR-1), which we have established from an STI571 resistant blast crisis patient, as well as HL-60 cells. HL-60 and SR-1 cells are relatively resistant to TRAIL-induced apoptosis. Pioglitazone alone inhibited the cell growth of SR-1 and HL-60 cells, but did not induce the apoptosis of these cell lines. However, simultaneous exposure of cells to 100 ng/ml TRAIL with either 25 μM pioglitazone or 50 μM piogliazone resulted in a striking increase in apoptosis. To clarify the mechanism of pioglitazone to sensitize the leukemia cells to TRAIL-induced apoptosis, we investigated the change of the proteins related to cell cycle and apoptosis by western blot. As results, we observed the significant decrease of X-linked inhibitor of apoptosis (XIAP) and the increased expression of p21 by cotreatment of pioglitazone with TRAIL. Taken together, these findings indicate that pioglitazone may have promising activity in augmenting TRAIL-induced apoptosis of human acute leukemia cells including the imatinib (STI571) resistant acute myeloid cell line.