Differentiation In Acute Myeloid Leukemia Research Articles

Arrested Differentiation in Acute Myeloid Leukemia (AML) with Silenced Ankyrin Repeat and SOCS Box Protein 3 (ASB3) Expression

Abstract 1232Acute myeloid leukemia (AML) is a hematological malignancy of the myeloid lineage characterized by an accumulation of abnormal immature white blood cells as a result of arrested myeloid differentiation. In the clinical setting, promyelocytic leukemic cells can be induced to differentiate into mature granulocytes by all-trans retinoic acid (ATRA), which acts by targeting oncogenic transcription factors. Expression profiling has previously identified ASB2, a member of the ankyrin repeat and SOCS box protein family as an ATRA target gene. ASBs negatively regulate cytokine signaling by mediating the ubiquitination of a broad range of target proteins. Furthermore, ASB proteins may regulate hematopoietic differentiation as suggested by ASB2 mediated degradation of the mixed lineage leukemia (MLL) protein and HOX gene down-regulation. MLL fusion proteins, present in approximately 10% of AML cases, have been found to be resistant to ASB2 mediated degradation which may contribute to leukemogenesis. Given that ASB2 plays a role in modulating differentiation and is ATRA induced it is feasible that other family members behave similarly.To understand the role of ASB proteins in hematopoietic differentiation we examined the expression profiles of ASB genes during normal myeloid differentiation using cells isolated at each stage of differentiation towards the granulocyte lineage. Alongside this, both ATRA-induced and DMSO-induced differentiation in AML cell lines (HL-60 and NB4) was assessed. In addition, ASB expression was analysed in the MILE study, which encompasses expression profiling of 16 leukemic subtypes, an independent cohort of clinically phenotyped patient samples, and normal bone marrow. Candidate ASBs who exhibited significantly different expression were identified by bioinformatic analysis of the MILE study and patient samples. One candidate (ASB3) was selected for silencing by shRNA and the resultant phenotype was assessed by proliferation ability, apoptosis potential and differentiation capacity. Furthermore, ASB3 silencing was analysed by Affymetrix microarrays to identify potential targets of ASB3.ASBs were found to be differentially expressed throughout normal hematopoietic differentiation with the highest expression in immature cells. In particular, ASB3 exhibited a two-fold decrease (p<0.001) in expression as myeloid cells matured. Several ASBs showed differential expression between AML patient samples and normal bone marrow with ASB3 exhibiting aberrant over-expression in AML (p≤0.005). Furthermore, Kaplan Meier plots using the MILE dataset indicated AML patients with FAB AML M0-M2 possess lower ASB3 expression which was associated with reduced overall survival and a poorer prognosis (p<0.05). ATRA or DMSO induced differentiation of HL-60 and NB4 cells altered ASB3 expression ten-fold (p<0.001) suggesting that the association of ASB3 with differentiation is not ATRA specific. Silencing of ASB3 expression (70% ±10%) in HL-60 and NB4 cells resulted in decreased proliferation and a 17% ±6% increase in apoptosis (p<0.01). In addition, reduced ASB3 expression markedly inhibited ATRA-induced differentiation of HL-60 and NB4 cells and ASB3 expression levels remained at a low level. Microarray analysis identified 78 differentially expressed genes between control and ASB3 silencing (p<0.05) including key genes specifically involved in apoptosis and hematopoietic differentiation e.g. PBX1, RUNX2 and CD44 were up-regulated during ASB3 silencing while ZNRF1, HOXA7 and CSRP2 were down-regulated when ASB3 was silenced.As ASB3 is highly expressed in myeloid progenitors and loss of expression blocks ATRA-induced differentiation it is possible ASB3 may play a role in myeloid differentiation. Little is known about the function of ASB3 although it has been suggested that it is a negative regulator of tumor necrosis factor receptor II (TNF-R2). Our study, and others, would suggest that it may have a role in the regulation of transcription factors important for differentiation e.g. HOX genes, as with ASB2. Furthermore, poor prognosis AML exhibited lower ASB3 expression predominantly in the most immature AML subtypes which suggests that without ASB3 the AML cells are unable to differentiate. Additional studies are required to develop our understanding of the function of ASB3 in myeloid differentiation and its contribution to leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

LSD1 Inhibitor Activates Retinoic Acid Pathway in MLL Fusion Positive Acute Myeloid Leukemia

Abstract 1345 Background:Among the subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) responds dramatically to differentiation therapy with all-trans retinoic acid (ATRA). However, ATRA is not sufficient to induce differentiation in non-APL AML. Although the molecular basis for the poor response of non-APL AML to ATRA was poorly understood, Lysine-specific demethylase 1 (LSD1), the histone demetylase, was found to inhibit the retinoic acid pathway by chromatin modification through H3K4 demethylation, resulting in silencing of gene expression targeted by retinoic acid. Herein, we first evaluated whether MLL fusion partners, such as MLL-AF9 and MLL-AF4/AF5q31, affect the sensitivity of ATRA in human and murine MLL fusion positive AML cells, which is one of the most aggressive pediatric AML. In addition, we also assess whether the LSD1 inhibitor affects the ATRA sensitivity in MLL fusion positive AML cells. Methods:Three human AML cell lines with MLL fusion (THP-1 and MOLM-13 expressing MLL-AF9, and KOCL48 expressing MLL-AF4) and two murine leukemic cell lines derived from murine Lin- hematopoietic progenitors transduced by retroviral vector expressing MLL fusion genes, such as MLL-AF9 and MLL-AF5q31 were used in this study. To test the sensitivity of ATRA, all cell lines were treated with 1 μM ATRA for three days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis, NBT reduction test and flow cytometric analysis (FCM) of CD11b expression. FCM analysis was also carried out to evaluate cell cycle and annexin V assay. Quantitative RT-PCR (qRT-PCR) analysis and western blotting was carried out to measure the RARα, C/EBPα, C/EBPε, and PU.1 expressions. To determine whether Tranylcypromine (TCP), which is a nonreversible LSD1 inhibitor, could decrease the IC50 of ATRA in MLL-AF4/AF5q31 positive cells, KOCL48 and murine MLL-AF5q31 expressing cells were treated with 0μM or 10μM TCP and titrating doses of ATRA (ranging from 0μM to 10μM). After three days, cell count was analyzed by counting nuclei using a Coulter counter to evaluate IC50 of ATRA in each cell lines. Results:We first determined that morphological changes characteristic of monocytic differentiation, CD11b expression and NBT reduction are more readily induced by ATRA in human and murine MLL-AF9 positive cells than human and murine MLL-AF4/AF5q31 positive cells The NBT reduction percentage was 17.6±1.69 in THP-1, but 2.7±1.2 in KOCL48 cells (p<0.01). The ATRA treatment also induced growth inhibition accompanied with G0/G1 arrest and apoptosis more efficiency in MLL-AF9 positive cells than MLL-AF4/AF5q31 cells. The IC50 of ATRA for THP-1 cells was 0.21±0.04 μM, but 5.31±1.50 μM for KOCL48 cells (p<0.01) The percentage of cells arrested in G0/G1 phase and Annexin/PI positive cells were 84% and 17.8% in THP-1 but 40% and 4.8% in KOCL48, respectively. Furthermore, qRT-PCR analysis and western blot analysis revealed that ATRA increased expression level of RARα, C/EBPα, C/EBPε, and PU.1, which is involved in monocytic differentiation through retinoic acid pathway, in MLL-AF9 positive cells, but not in MLL-AF4/AF5q31 positive cells. Collectively, retinoic acid pathway is more impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells. Next, we also determined that ATRA and TCP combination treatment suppressed cell growth and decreased the IC50 of ATRA in KOCL48 and murine MLL-AF5q31 expressing cells (IC50 of ATRA: 0.20±0.10 μM and 0.20±0.09 μM with TCP, vs 5.5±3.2 μM and over 10 μM without TCP, p<0.05), accompanied with morphological changes and CD11b expression, suggesting that inhibition of LSD1 restores ATRA sensitivity in both cell lines. Conclusions:Our data demonstrate that retinoic acid pathway was more profoundly impaired in MLL-AF4/AF5q31 positive cell than MLL-AF9 positive cells, suggesting MLL-AF4/AF5q31 contributes inactivation of retinoic acid pathway. Our data also demonstrate TCP restore the sensitivity of ATRA in ATRA-resistant MLL-AF4/AF5q31 positive cell lines, suggesting LSD1 plays a major role in inactivation of retinoic acid pathway in MLL-AF4/AF5q31 positive cells. Therefore, LSD1 inhibitor might be important novel therapeutic option for differentiation therapy of MLL-fusion positive AML, especially for ATRA resistant MLL-AF4/AF5q31 positive cells. Disclosures:No relevant conflicts of interest to declare.

MiRNA let-7c promotes granulocytic differentiation in acute myeloid leukemia

MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression post-transcriptionally, are involved in many complex cellular processes. Several miRNAs are differentially expressed in hematopoietic tissues and play important roles in normal differentiation, but, when aberrantly regulated, contribute to the abnormal proliferation and differentiation of leukemic cells. Recently, we reported that a small subset of miRNAs is differentially expressed in acute promyelocytic leukemia (APL) blasts and is modulated by treatment with all-trans-retinoic acid (ATRA). In particular, PML/RARα-positive blasts from APL patients display lower levels of miRNA let-7c, a member of the let-7 family, than normal promyelocytes and its expression increases after ATRA treatment. In this study, we investigated the effects of let-7c in acute myeloid leukemia (AML) cells. We found that ectopic expression of let-7c promotes granulocytic differentiation of AML cell lines and primary blasts. Moreover, we identified PBX2, a well-known homeodomain protein whose aberrant expression enhances HoxA9-dependent leukemogenesis, as a novel let-7c target that may contribute to the AML phenotype. Together, these studies raise the possibility that perturbation of the let-7c-PBX2 pathway may have a therapeutic value in AML.

ATP Depletion Triggers Acute Myeloid Leukemia Differentiation through an ATR/Chk1 Protein-dependent and p53 Protein-independent Pathway

Despite advances in oncology drug development, most commonly used cancer therapeutics exhibit serious adverse effects. Often the toxicities of chemotherapeutics are due to the induction of significant DNA damage that is necessary for their ability to kill cancer cells. In some clinical situations, the direct induction of significant cytotoxicity is not a requirement to meet clinical goals. For example, differentiation, growth arrest, and/or senescence is a valuable outcome in some cases. In fact, in the case of acute myeloid leukemia (AML), the use of the differentiation agent all-trans-retinoic acid (ATRA) has revolutionized the therapy for a subset of leukemia patients and led to a dramatic survival improvement. Remarkably, this therapeutic approach is possible even in many elderly patients, who would not be able to tolerate therapy with traditional cytotoxic chemotherapy. Because of the success of ATRA, there is widespread interest in identifying differentiation strategies that may be effective for the 90-95% of AML patients who do not clinically respond to ATRA. Utilizing an AML differentiation agent that is in development, we found that AML differentiation can be induced through ATP depletion and the subsequent activation of DNA damage signaling through an ATR/Chk1-dependent and p53-independent pathway. This study not only reveals mechanisms of AML differentiation but also suggests that further investigation is warranted to investigate the potential clinical use of low dose chemotherapeutics to induce differentiation instead of cytotoxicity. This therapeutic approach may be of particular benefit to patients, such as elderly AML patients, who often cannot tolerate traditional AML chemotherapy.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Cell-Type-Specific Effects of Silibinin on Vitamin D-Induced Differentiation of Acute Myeloid Leukemia Cells Are Associated with Differential Modulation of RXRα Levels

Plant polyphenols have been shown to enhance the differentiation of acute myeloid leukemia (AML) cells induced by the hormonal form of vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D). However, how these agents modulate 1,25D effects in different subtypes of AML cells remains poorly understood. Here, we show that both carnosic acid (CA) and silibinin (SIL) synergistically enhancd 1,25D-induced differentiation of myeloblastic HL60 cells. However, in promonocytic U937 cells, only CA caused potentiation while SIL attenuated 1,25D effect. The enhanced effect of 1,25D+CA was accompanied by increases in both the vitamin D receptor (VDR) and retinoid X receptor alpha (RXRα) protein levels and vitamin D response element (VDRE) transactivation in both cell lines. Similar increases were observed in HL60 cells treated with 1,25D + SIL. In U937 cells, however, SIL inhibited 1,25D-induced VDRE transactivation concomitant with downregulation of RXRα at both transcriptional and posttranscriptional levels. These inhibitory effects correlated with the inability of SIL, with or without 1,25D, to activate the Nrf2/antioxidant response element signaling pathway in U937 cells. These results suggest that opposite effects of SIL on 1,25D-induced differentiation of HL60 and U937 cells may be determined by cell-type-specific signaling and transcriptional responses to this polyphenol resulting in differential modulation of RXRα expression.

Abstract 5392: Angiocidin induces differentiation of acute myeloid leukemia (AML) cells

Abstract Acute myeloid leukemia (AML) is a malignant proliferative disorder where blast stage cells fail to terminally differentiate and accumulate in the blood and bone marrow. AML represents a very significant health risk in the general population with rising incidence in aging adults. From 2004-2008 the median age at diagnosis was 67.8 years and it is estimated 12,950 adults will be diagnosed with 9,050 deaths from AML leukemia in 2011 (http://seer.cancer.gov/statfacts/html/amyl.html, accessed November 2011). Standard AML therapy requires intensive combination chemotherapy with a low rate of durable remissions often requiring stem cell transplantation and associated with significant treatment-related toxicity, especially in elderly patients. Due to this toxicity, the majority of elderly patients are not eligible for therapy. Therefore, new therapeutic options for the treatment of AML are urgently needed. In this study, we present evidence that the human protein angiocidin, discovered in our laboratory, has the potential to be used as a novel, non-toxic therapy for AML. We found that angiocidin can induce both AML cell lines and primary patient cells to increase expression of surface markers characteristic of a more normal differentiated phenotype. We evaluated expression of CD14, a major receptor for the innate immune system in macrophages, CD54 or ICAM-1, the major adhesion receptor used by macrophages for endothelial transmigration, and CD15, a neutrophil phagocytosis receptor. Expression of these receptors was measured by flow cytometry after cells were cultured for 24 hours in media alone or media containing 10 μg/ml angiocidin. Angiocidin up-regulated these receptors by more than 10 fold in 50% of the AML cells tested. The AML cells included the cell line THP-1 and 11 primary AML patient cells. Angiocidin up-regulated CD14 and CD54 in THP-1 cells and in 3 out of 5 M5 phenotype AML patient cells tested and 1 M4 AML phenotype out of 6 M4 samples tested. Angiocidin also up-regulated CD15 in 1 M4 AML patient phenotype out of 6 M4 patient cells tested. These results suggest that angiocidin can differentiate AML cells and therefore has the potential to be developed as differentiation therapy for AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5392. doi:1538-7445.AM2012-5392

High expression of lactotriaosylceramide, a differentiation-associated glycosphingolipid, in the bone marrow of acute myeloid leukemia patients

Glycosphingolipids (GSLs) are information-bearing biomolecules that play critical roles in embryonic development, signal transduction and carcinogenesis. Previous studies indicate that certain GSLs are associated with differentiation in acute myeloid leukemia (AML) cells. In this study, we collected bone marrow samples from healthy donors and AML patients and analyzed the GSL expression profiles comprehensively using electrospray ionization linear ion-trap mass spectrometry. The results showed that AML patients had higher expression of the GSL lactotriaosylceramide (Lc3), GM3 and neolactotetraosylceramide (nLc4) in their bone marrow than did the healthy donors (P < 0.05), especially the M1 subtype of AML. To further explore the molecular mechanisms of Lc3, we examined the expression of the Lc3 synthase β1,3-N-acetylglucosaminyltransferase5 (β3Gn-T5) and found that the bone marrow samples of AML patients had 16-fold higher expression of β3Gn-T5 than those of healthy donors (P < 0.05). Our results suggest that AML-associated GSLs Lc3, GM3 and nLc4 are possibly involved in initiation and differentiation of AML.

The intersection of genetic and chemical genomic screens identifies GSK-3α as a target in human acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML.

GSK3 is a regulator of RAR-mediated differentiation

Acute myeloid leukemia (AML) is the most common form of leukemia in adults. Unfortunately, the standard therapeutic agents used for this disease have high toxicities and poor efficacy. The one exception to these poor outcomes is the use of the retinoid, all-trans retinoic acid (ATRA), for a rare subtype of AML (APL). The use of the differentiation agent, ATRA, in combination with low-dose chemotherapy leads to the long-term survival and presumed cure of 75-85% of patients. Unfortunately ATRA has not been clinically useful for other subtypes of AML. Though many non-APL leukemic cells respond to ATRA, they require significantly higher concentrations of ATRA for effective differentiation. Here we show that the combination of ATRA with glycogen synthase kinase 3 (GSK3) inhibition significantly enhances ATRA-mediated AML differentiation and growth inhibition. These studies have revealed that ATRA's receptor, the retinoic acid receptor (RAR), is a novel target of GSK3 phosphorylation and that GSK3 can impact the expression and transcriptional activity of the RAR. Overall, our studies suggest the clinical potential of ATRA and GSK3 inhibition for AML and provide a mechanistic framework to explain the promising activity of this combination regimen.

A Targeted shRNA Knockdown Screen of Phosphoinositide Modulator Genes Reveals INPP5B As Required for Cell Cycle Progression of MLL-AF9 Acute Myeloid Leukemia Cells

Abstract 2444Phosphoinositides are pivotal regulatory and scaffolding lipids that coordinate key aspects of cellular physiology. For example, phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates endocytosis, exocytosis, membrane ruffling and the function of actin, and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), downstream of phosphoinositide-3-kinase (PI3K), is a critical mediator of signal transduction. In acute myeloid leukemia (AML) mutations of FLT3 or KIT induce constitutive activation of PI3K/AKT signaling; and PTEN and SHIP1, two PI3K antagonists, have been linked to hematopoietic stem cell maintenance, with experimental deletion of these genes leading to leukemia. Little is known about the role of other phosphoinositide modulators in normal or leukemic hematopoiesis. We hypothesized that a genetic knockdown screen, utilising lentivirally delivered short hairpin RNAs (345 pLKO.1 puro vectors) targeting 92 genes with known or supposed roles in the regulation of phosphoinositide metabolism, would reveal novel regulators of proliferation and differentiation in AML, representing candidate therapeutic targets.In an initial screen, hairpins were tested for their ability to impact on proliferation or survival of human THP-1 AML cells using fold change in fluorescence of the cell viability indicator alamarBlue relative to control cells (infected with a non-targeting control hairpin) over four days as a surrogate. THP-1 cells harbor a t(9;11) chromosomal translocation which is the cytogenetic hallmark of MLL-AF9. Lentiviral supernatants, prepared individually in 96 well plates with one vector per well, were used to infect target cells, again in 96 well plate format with one lentiviral supernatant per well. 24 hours following infection puromycin was added. The alamarBlue dye assay was performed on replicate samples 48 hours and six days later. All non-infected cells were killed by puromycin exposure within 48 hours. 38 shRNAs, including two targeting PTEN, increased proliferation or enhanced survival, whereas 158, including multiple constructs targeting AKT1, AKT2 and PI3KR2, reduced proliferation or induced apoptosis (versus control cells fold change± 2SD). These results confirmed that the screening strategy was effective because it correctly identified known pro- and anti-proliferative regulators of phosphoinositide metabolism.Next, we expanded the screen to include six other AML cell lines (MonoMac1, HL60, K562, NB4, Kasumi-1 and U937). 14 genes met our criteria for a “hit”: at least two knockdown constructs per gene needed to reduce proliferation on average by at least 55% compared to non-targeting shRNA control across all cell lines screened. We focused our further investigations on type II inositol-1,4,5-trisphosphate 5-phosphatase (INPP5B), because Inpp5b−/- mice are viable into adulthood with no known hematopoietic phenotype, suggesting INPP5B might selectively regulate leukemia cells. INPP5B catalyses the conversion of inositol-1,4,5-trisphosphate (IP3) to inositol-1,4-bisphphate (IP2) and hence contributes to cellular calcium signalling mediated through phospholipase C. Knockdown of Inpp5b also reduced proliferation of murine MLL-AF9 AML cells in liquid culture using three separate shRNA constructs that reduced transcript levels by 70–80%, and largely abolished colony formation in methylcellulose; residual colonies were much smaller than control colonies. While wild-type KIT+ bone marrow stem and progenitor cells were robustly immortalized by infection with an MLL-AF9 retroviral vector, Inpp5b−/− KIT+ cells either failed to immortalize or exhibited weak immortalisation, with fewer, smaller colonies, reminiscent of the knockdown phenotype. In cell cycle analyses of cells harvested from the third round of serial replating in methylcellulose, 90% of Inpp5b−/− MLL-AF9 cells were in G1 versus 41% of WT cells. No increase in apoptosis was observed.These data implicate INPP5B as a novel and selective regulator of leukemic versus normal hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Pharmacological Inhibition or Genetic Knockdown of Kdm1a (Lsd1 or Aof2) Induces Differentiation of MLL Acute Myeloid Leukaemia Stem Cells

Abstract 58Histone demethylases are candidate therapeutic targets in cancer. We analysed a previously published microarray expression dataset of murine acute myeloid leukemias (AML) experimentally initiated with human MLL fusion oncogenes to identify histone demethylases whose expression correlated with the frequency of leukemia stem cells (LSCs). Only expression of Kdm1a correlated positively and significantly (p<0.001) with LSC frequency, a finding confirmed by quantitative PCR of selected leukemias. To investigate whether Kdm1a has a critical role in MLL LSCs, we targeted Kdm1a transcripts for knockdown in murine MLL-AF9 AML cells using lentivirally expressed shRNAs. Knockdown AML cells formed significantly fewer colonies in semi-solid culture compared with AML cells expressing a non-targeting control shRNA (12±7% of control; n=4; p=0.01), and the extent of Kdm1a knockdown correlated significantly (six shRNAs tested) with loss of AML cell colony forming (CFC) potential (p<0.005). Residual Kdm1a knockdown colonies predominantly exhibited “Type 2” morphology (i.e. containing macrophages), a finding confirmed by examination of cytospin preparations. The phenotype was rescued by forced expression of human KDM1A. Kdm1a knockdown MLL-AF9 AML cells were unable to initiate leukemia in secondary transplant assays, in contrast to control cells which readily induced short latency AML. These data demonstrate that Kdm1a is required to prevent differentiation of murine MLL-AF9 AML LSCs.Tranylcypromine (TCP) is a licensed monoamine oxidase inhibitor that inhibits KDM1A, with an IC50 of 5–10uM. Treatment of murine MLL-AF9 AML cells, or human THP1 MLL-AF9 cells, with TCP phenocopied Kdm1a knockdown at a dose close to the KDM1A IC50; both murine and human MLL-AF9 cells exhibited reduced proliferation, fewer colonies and increased macrophage differentiation. Next, we tested the effect of KDM1A knockdown or TCP treatment (25uM) on primary human MLL leukemia cells. KDM1A knockdown reduced proliferation, induced morphological differentiation and induced apoptosis in the two samples of MLL-AF6 leukemia tested (5–20 fold fewer cells remained after a ten day culture versus control cells). Likewise, TCP treatment reduced proliferation 4-fold over five days (MLL-AF6 n=2; MLL-AF10 n=1), blocked CFC formation, reduced the proportion of blasts and increased the proportion of cells with features of monocyte/macrophage differentiation. Of note, addition of 25uM TCP to normal murine KIT+ BM stem and progenitor cells cultured in methylcellulose did not significantly reduce CFC frequency or size or induce macrophage differentiation, suggesting that MLL LSCs are more sensitive than normal BM cells to a given level of KDM1A inhibition.To investigate the mechanism by which KDM1A inhibits a differentiation programme in MLL-AF9 LSCs, we performed exon array analysis of Kdm1a knockdown versus control LSCs 48 hours following lentiviral infection, prior to appearance of morphologically differentiated cells. Down regulated transcriptional regulators included Id2, Hmgb3 and Egr1, but not Hoxa, Meis1 or Myb. Up regulated transcriptional regulators included Irf8. Knockdown of Id2 in murine MLL-AF9 AML cells reduced CFC frequency. In contrast, forced expression of Id2 enhanced CFC frequency both of control MLL-AF9 AML cells and of Kdm1a knockdown LSCs, the latter representing a partial phenotypic rescue of Kdm1a knockdown.These data demonstrate that KDM1A prevents differentiation of MLL LSCs by regulating the relative expression of pro- versus anti-differentiation transcriptional regulators and highlight it as a novel therapeutic target in MLL leukemias. Disclosures:No relevant conflicts of interest to declare.

Src Kinase Inhibition by Dasatinib Enhances Targeting of Human AML Stem/Progenitor Cells by Chemotherapeutic Agents

Abstract 4270Tyrosine kinases play a critical role in regulating cellular proliferation, differentiation and survival. We have previously shown that Src family tyrosine kinases (SFKs) including Lyn, Hck and Fgr are abnormally activated in Acute Myeloid Leukemia (AML) cells compared to normal CD34+ hematopoietic progenitors (Dos Santos et al., Blood 2008, 111:2269). Dasatinib is a potent small molecule inhibitor of SFKs and the ABL kinase that is approved for clinical use in Chronic Myeloid Leukemia. However the ability of Dasatinib to target AML is poorly documented. Here we evaluated the potential activity of Dasatanib on AML stem/progenitor cell maintenance and drug sensitivity. We assessed SFK phosphorylation in stem/progenitor populations from AML patients by flow cytometry (n=42). We observed significant increase of SFK phosphorylation in AML compared to normal Lin− CD34+CD38− cells (3.5±1.8 versus 1.7±0.48 fold increase compared to isotype control, p=0.008), Lin− CD34+ CD38+ (3.6±1.6 versus 1.6±0.37, p=0.001) and Lin− CD34− cells (2.9±1.4 versus 1.1±0.4, p=0.001). Treatment with Dasatinib significantly reduced AML stem cell growth in LTCIC assays (56±23% inhibition with 200nM Dasatinib, p=0.003, n=5), and reduced growth of committed progenitors in CFC assays (73.6±13% inhibition with 500nM Dasatinib, and 41.7±10.8% inhibition with 10nM Dasatinib, n=9, p<0,0001), but did not reduce normal LTC-IC (n=4) or CFU-GM (23±9.1% inhibition with 500nM Dasatinib, and 1.3±11.3% with 10nM Dasatinib, n=6, p=0,007). Inhibition of growth was associated with increased apoptosis (41.5% ±10.7 with 200nM Dasatinib versus 26%±8.2 for the control, p=0,004, n=5), reduced proliferation (as evaluated with CFSE labeling, proliferation index 3.02±0.3 for the control and 2.27±0.6 with 200 nM Dasatinib, p=0.01 (n=5), and enhanced differentiation of AML stem/progenitor cells with significant increase in CD11b+ and CD15+ cells and reduction in CD34+, CD33+ and CD71+ cells, (Dasatinib 200 nM n=7). Exposure of AML cells (n=4) to Dasatinib (200nM) resulted in reduced phosphorylation of STAT3 (Y705), STAT5 (Y694) and Akt (Thr308) but did not affect MAPK (Thr202/Y204) phosphorylation. Interestingly, Dasatinib treatment strongly enhanced expression of p53 protein and its downstream targets, including p21 and Bax, in AML specimen (n=7), suggesting that Dasatinib treatment resulted in activation of p53 signaling in AML stem/progenitor cells. As compared to treatment with individual agents, the combination of Dasatinib with either daunorubicin (DNR) or Ara-C resulted in significantly increased apoptosis (14.9%±11 with Dasatinib, 15.8%±8.6 with 0.05μ M DNR and 25% with DNR+ Dasatinib=7, p=0.005), decreased proliferation (proliferation index 3.02±0.3 for control, 2.27±0.6 with 200 nM Dasatinib, 2.32±0.8 with 0.05μ M DNR and 1.29±0.4 for the combination n=5, p=0.01) and reduced CFC growth (n=6, 56.2±6.4% inhibition with 200nM Dasatinib, 35%±5.6 with 0.01μ M DNR, p=0.001, 82%±9.5 with DNR + Dasatinib, p=0.001, 51.8%±8.5 with 0.01μ M Ara-C and 83.2%±4.7 with Ara-C + Dasatinib, p=0.0002). The combination of Dasatinib with chemotherapeutic agents significantly enhanced expression of p53 and downstream targets compared with these agents alone. In conclusion, SFK activity is increased in primary human AML stem and progenitor cells. SFK blockade with Dasatinib reduces maintenance of AML stem and progenitor cells through inhibition of proliferation, induction of apoptosis and enhancement of differentiation. Dasatinib treatment enhances sensitivity of AML stem/progenitor cells to chemotherapeutic agents, potentially through inhibition of Akt and STAT signaling and activation of p53. These results support further preclinical and clinical evaluation of SFK blockade with Dasatinib to enhance AML stem and progenitor cells targeting by chemotherapeutic agents. Disclosures:No relevant conflicts of interest to declare.

CpG methylation patterns and decitabine treatment response in acute myeloid leukemia cells and normal hematopoietic precursors

The DNA hypomethylating drug decitabine maintains normal hematopoietic stem cell (HSC) self-renewal but induces terminal differentiation in acute myeloid leukemia (AML) cells. The basis for these contrasting cell-fates, and for selective CpG hypomethylation by decitabine, is poorly understood. Promoter CpGs, with methylation measured by microarray, were classified by the direction of methylation change with normal myeloid maturation. In AML cells, the methylation pattern at maturation-responsive CpG suggested at least partial maturation. Consistent with partial maturation, in gene expression analyses, AML cells expressed high levels of the key lineage-specifying factor CEBPA, but relatively low levels of the key late-differentiation driver CEBPE. In methylation analysis by mass-spectrometry, CEBPE promoter CpG that are usually hypomethylated during granulocyte maturation were significantly hypermethylated in AML cells. Decitabine treatment induced cellular differentiation of AML cells, and the largest methylation decreases were at CpG that are hypomethylated with myeloid maturation, including CEBPE promoter CpG. In contrast, decitabine-treated normal HSC retained immature morphology, and methylation significantly decreased at CpG that are less methylated in immature cells. High expression of lineage-specifying factor and aberrant epigenetic repression of some key late-differentiation genes distinguishes AML cells from normal HSC and could explain the contrasting differentiation and methylation responses to decitabine.

Intersecting Chemical Genomic and Genetic Screens Identifies Glycogen Synthase Kinase-3α (GSK-3α) as a Modulator of Differentiation In Acute Myeloid Leukemia

AbstractAbstract 1000The treatment of acute myeloid leukemia (AML) poses a vexing challenge despite an improved understanding of its molecular pathogenesis. A two-hit theory has been proposed for the pathogenesis of AML where the first hit imparts a proliferation defect and the second a block in differentiation. Drug discovery efforts for AML, however, have largely focused on the proliferation defect. Using the intersection of chemical biology and high-throughput genetic screening we sought to identify new AML differentiation targets by measuring the induction of a complex gene expression signature of myeloid maturation. We performed two independent small molecule library screens and a high-throughput shRNA screen for perturbations that induce differentiation in AML cells. We measured this differentiation signature using the previously described gene expression-based high-throughput screening (GE-HTS) approach in which gene expression signatures serve as surrogates for different biological states. Glycogen Synthase Kinase-3 (GSK-3) emerged as a target at the intersection of these three screens. GSK-3 is a multifunctional serine threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation and proliferation, with an emerging role in human leukemia. We demonstrate that the GSK-3 inhibitors scoring in the primary screens indeed induce the differentiation signature with a dose-response in AML cell lines. In order to further validate GSK-3 as a target, we extended testing to lithium chloride and SB216763, two commonly used GSK-3 inhibitors not in the original screens. Both of these molecules induced AML differentiation as measured by gene expression and morphological changes in multiple AML cell lines and in primary patient blasts in vitro. GSK-3 is expressed as two highly homologous but non-redundant isoforms, GSK-3α and GSK-3β, with small molecule inhibitors non-selectively reported to target both. In order to further validate the findings of the small molecule library screens, we performed a high-throughput shRNA screen targeting the human kinome for shRNAs that induce differentiation. Multiple hairpins against GSK-3α scored. In secondary testing of four AML cell lines, we found that genetic loss of GSK-3α induced differentiation as measured by induction of the complex gene expression signature, alterations in genome-wide expression, and morphological changes associated with maturation. Moreover, colony formation in methylcellulose was impeded. These effects could be rescued with a GSK-3α cDNA immune to the effects of the shRNA, further supporting the on-target activity of the hairpin. In contrast, GSK-3β-directed hairpins induced minimal differentiation. We next extended testing to an in vivo U937 orthotopic model of AML. While pan-GSK-3 inhibition with lithium chloride treatment did not demonstrate efficacy in this model, inhibition of GSK-3α with shRNA attenuated development of disease compared to an shRNA control. We hypothesize that the rise in β-catenin with pan-inhibition of GSK-3 may attenuate the response to lithium in vivo. In contrast, isolated knockdown of GSK-3α does not induce β-catenin. While much of the prior cancer literature has focused on the role of GSK-3β in human malignancy, these studies suggest a role for GSK-3α in AML differentiation and support a role for GSK-3α-directed targeted therapy. Disclosures:No relevant conflicts of interest to declare.

RAD001, An Inhibitor of mTOR, Enhances Monocytic Differentiation Induced by ATRA through Phosphorylation of C/EBPα In AML Cell Lines

AbstractAbstract 2152 Background:CCAAT/ enhancer binding protein alpha (C/EBPα) is a critical transcription factor that controls monocytic and granulocytic differentiation. Several recent studies have reported that C/EBPα expression is down-regulated in acute myeloid leukemia (AML), leading to suppression of monocytic differentiation. All-trans-retinoic acid (ATRA) induces numerous transcriptional factors, including C/EBPα; however, ATRA alone is not sufficient to induce monocytic differentiation in AML. The purpose of this study was to identify agents that increase the efficacy of ATRA. RAD001 (Everolimus; provided by Novartis), a rapamycin analog, is a relatively new drug that inhibits the Akt/ PI3K/ mTOR pathway. To assess the utility of differentiation therapy as a treatment for types of AML other than acute promyelocytic leukemia, we evaluated the effects of RAD001 and ATRA combination treatment in several AML cell lines. Methods:Three AML cell lines (U-937, THP-1, and KOCL48) and two primary AML samples were treated with 2.5–5.0 nM RAD001 and 1 μM ATRA for five days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis and flow cytometric analysis (FCM) of CD11b expression. An Annexin V assay was carried out to measure apoptosis. Microarray analysis using an Agilent expression array was employed to determine changes in gene expression associated with ATRA and RAD001 combination treatment. Quantitative RT-PCR (qRT-PCR) analysis was performed to validate the microarray results. Western blotting was carried out to measure the phosphorylation of C/EBPα at Ser 21. Results:We determined that ATRA and RAD001 treatment induced morphological changes characteristic of monocytic differentiation. Microarray analysis of THP-1 revealed that ATRA and RAD001 induced expression of a set of genes associated with monocytic differentiation, including MPEG1, CD11b, CD115 and CD14. FCM analysis confirmed that ATRA and RAD001 intensified CD11b expression in the three cell lines tested, especially in the two ATRA-resistant cell lines (KOCL48 and U937). qRT-PCR analysis also revealed that ATRA and RAD001 treatment increased expression of C/EBPα and C/EBPε, which is involved in the terminal stages of monocytic differentiation, in all three cell lines and two primary samples compared to treatment with ATRA only. Expression of PU.1 was also increased by combination treatment in all cells tested except the U937 cell line. Western blot analysis revealed that ATRA and RAD001 decreased phosphorylation of C/EBPα at serine 21. ATRA and RAD001 combination treatment also suppressed cell growth in two ATRA-resistant cell lines (growth inhibition rate: 70–80%). The Annexin V assay demonstrated that ATRA and RAD001 combination treatment strongly induced apoptosis in the three cell lines tested. Microarray analysis revealed that FasL,FADD, and caspase 8, which are associated with apoptotic pathways, showed the greatest degree of up-regulation in THP-1 cells treated with ATRA and RAD001. qRT-PCR analysis confirmed up-regulation of these genes in all three cell lines and in both primary AML samples, indicating that ATRA and RAD001 induce apoptosis in AML cells through the extrinsic cell death signaling pathway. Conclusions:RAD001 induced monocytic differentiation through induction of a set of genes associated with monocytic differentiation and phosphorylation of C/EBPα at Ser 21 when combined with ATRA. This combination therapy also induced apoptosis in AML cells through activation of the extrinsic cell death signaling pathway. Disclosures:No relevant conflicts of interest to declare.

The Autophagy Gene ULK1 Plays a Role In AML Differentiation and Is Negatively Regulated by the Oncogenic MicroRNA 106a

AbstractAbstract 503Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents allowing for example survival upon a variety of stress stimuli. Although often referred to as type II programmed cell death, autophagy is primarily a pro-survival mechanism rather than an important cell death effector. During autophagy, part of the cytoplasm or entire organelles are sequestered into double-membraned vesicles, called autophagosomes, that ultimately fuse with lysosomes and degrade their contents. Regarding hematopoiesis there is accumulating evidence that autophagic mechanisms are operative in the development of different hematopoietic lineages. To identify ATG genes with a function in neutrophil differentiation, we employed an shRNA library targeting autophagy regulators in acute myeloid leukemia (AML) cells as a model for neutrophil differentiation. Among others, we found that inhibiting the key autophagy kinase ULK1 (also known as ATG1) using RNAi significantly attenuated all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 and HL60 AML cells. In agreement, we observed significantly elevated ULK1 mRNA and protein levels during neutrophil differentiation of these cells. Moreover, ULK1 expression was significantly reduced in primary AML patient samples as compared to mature myeloid cells from healthy donors. Given the aberrant microRNAs (miRNAs) expression in myeloid malignancies, we wondered if altered miRNA expression in AML might contribute to the low ULK1 levels seen. MiRNAs are 19–25 nucleotide, non-protein coding RNAs that regulate gene expression by directly binding to partially complementary sequences in the 3′-untranslated regions (3′-UTR) of their target mRNAs. Several target prediction software's identified a putative miR-106a binding site in the ULK1 3′-UTR. Interestingly, miR-106a is part of a highly oncogenic miRNA family. We then confirmed direct inhibition of ULK1 by miR-106a using wild-type and miR-106a mutated 3′-UTR ULK1 reporters. Moreover, a decrease of miR-106a expression was seen during ATRA-induced differentiation of AML cells paralleled by increased ULK1 mRNA levels. Lastly, stable overexpression of miR-106a in NB4 cells resulted in decreased ULK1 levels and markedly impaired neutrophil differentiation upon ATRA treatment. In summary, we show for the first time a significant involvement of the autophagy gene ULK1 in neutrophil differentiation and an association of low ULK1 levels with primary AML. Our studies indicate that ULK1 is a putative tumor suppressor in AML that is targeted by the oncogenic miR-106a. Disclosures:No relevant conflicts of interest to declare.

Activation of Myeloid Differentiation-Associated Autophagy In Combination with ATRA-Therapy Enhances Neutrophil Differentiation of AML Cells.

AbstractAbstract 1046Autophagy is a self-digesting recycling mechanism for cellular organelles and macromolecules with cytoprotective functions. On the other hand, autophagy might also lead to caspase-independent, type II cell-death. Of interest, there is accumulating evidence for non-metabolic, cell specific functions of autophagy in mature hematopoietic cells but also during hematopoietic differentiation. We therefore aimed at investigating the involvement of autophagy in the molecular pathogenesis of acute myeloid leukemia (AML) and, in particular, in acute promyelocytic leukemia (APL). We found significantly increased autophagic activity during ATRA-induced neutrophil differentiation of NB4 and HL60 AML cells as evidenced by increased autophagosome formation (appearance of LC3-GFP dots), LC3-I to -II conversion and degradation of p62 (SQSTM1), which is degraded in the autophagosome. Most importantly, ATRA treatment induced a novel type of autophagy that was independent of the key autophagy gene Beclin-1 as opposed to autophagy induced by starvation or chemotherapeutics. Moreover, autophagy seen during neutrophil differentiation of AML cells was clearly less intense than, for example, starvation-induced autophagy as measured by the number of LC3-GFP dots per cell. We have previously shown that the autophagy gene ATG5 as well as death-associated protein kinase 2 (DAPK2) are crucial in all-trans retinoic (ATRA)-induced neutrophil differentiation. Since it was found earlier that overexpression of DAPK2 can induce autophagy in epithelial cells, we were asking whether there might be a direct link from DAPK2 to ATG5. Indeed, we found direct DAPK2-ATG5 binding when overexpressed in 293T HEK cells. Moreover, we were able to pull down endogenous DAPK2 together with ATG5 in ATRA-treated NB4 APL cells. Using DAPK2 overexpressing NB4 cells, we found increased DAPK2-ATG5 interaction with progressive neutrophil differentiation. Further, knocking down DAPK2 caused decreased ATG5-ATG12 complex formation during AML differentiation. Lastly, we found that pharmacological activation of autophagy in combination with ATRA significantly boosted AML differentiation, representing a potential new strategy for APL therapy. In summary, we show that autophagy is needed for neutrophil differentiation of AML cells, possibly via the newly identified DAPK2-ATG5 pathway, and would like to refer to this new type of Beclin-1-independent, non-canonical autophagy as myeloid differentiation-associated autophagy (MDAA). Disclosures:No relevant conflicts of interest to declare.

Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia

Differentiating agents have been proposed to overcome the impaired cellular differentiation in acute myeloid leukemia (AML). However, only the combinations of all-trans retinoic acid or arsenic trioxide with chemotherapy have been successful, and only in treating acute promyelocytic leukemia (also called AML3). We show that iron homeostasis is an effective target in the treatment of AML. Iron chelating therapy induces the differentiation of leukemia blasts and normal bone marrow precursors into monocytes/macrophages in a manner involving modulation of reactive oxygen species expression and the activation of mitogen-activated protein kinases (MAPKs). 30% of the genes most strongly induced by iron deprivation are also targeted by vitamin D3 (VD), a well known differentiating agent. Iron chelating agents induce expression and phosphorylation of the VD receptor (VDR), and iron deprivation and VD act synergistically. VD magnifies activation of MAPK JNK and the induction of VDR target genes. When used to treat one AML patient refractory to chemotherapy, the combination of iron-chelating agents and VD resulted in reversal of pancytopenia and in blast differentiation. We propose that iron availability modulates myeloid cell commitment and that targeting this cellular differentiation pathway together with conventional differentiating agents provides new therapeutic modalities for AML.

Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia

Meisoindigo, a derivative of Indigo naturalis, has been used in China for chronic myeloid leukemia. In vitro cell line studies have shown that this agent might induce apoptosis and myeloid differentiation of acute myeloid leukemia (AML). In this study, we explored its mechanisms and potential in AML. NB4, HL-60, and U937 cells and primary AML cells were used to examine its effects and the NOD/SCID animal model was used to evaluate its in vivo activity. Meisoindigo inhibited the growth of leukemic cells by inducing marked apoptosis and moderate cell-cycle arrest at the G0/G1 phase. It down-regulated anti-apoptotic Bcl-2, and up-regulated pro-apoptotic Bak and Bax and cell-cycle related proteins, p21and p27. Furthermore, it induced myeloid differentiation, as demonstrated by morphologic changes, up-regulation of CD11b, and increased nitroblue tetrazolium reduction activity in all cell lines tested. In addition, meisoindigo down-regulated the expression of human telomerase reverse transcriptase and enhanced the cytotoxicity of conventional chemotherapeutic agents, cytarabine and idarubicin. As with the results from cell lines, meisoindigo also induced apoptosis, up-regulated p21 and p27, and down-regulated Bcl-2 in primary AML cells. The in vivo anti-leukemic activity of meisoindigo was also demonstrated by decreased spleen size in a dose-dependent manner. Taking these results together, meisoindigo is a potential agent for AML.