Acute Myeloid Leukemia Leukemic Stem Cells Research Articles

Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leukemic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo.

Activating mutations of receptor tyrosine kinases (RTKs) are involved in the pathophysiology of various types of cancers. Among them, FLT3 internal tandem duplications (ITD) and a KIT point mutation at D816V are frequently found in acute myeloid leukemia (AML) and associated with poor prognosis. Although a number of clinical trials were conducted, an effective RTK inhibitor with acceptable toxicity hasn’t been developed.Recent studies have demonstrated these oncogenic RTKs are mislocalized in the cytoplasm, where they transmit aberrant leukemogenic signals to downstream molecules in a compartment-specific manner. So, we speculated that manipulation of their intracellular trafficking between intracellular compartments might be promising as a new therapeutic approach for AML with mutant RTKs. In this study, we evaluated the in vitro and in vivo anti-leukemic effects of chlorpromazine (CPZ) as an inhibitor of membrane trafficking, which is a well-established antipsychotic drug. All of the protocols were approved by the Institutional Review Board of Kinki University Faculty of Medicine, and we obtained AML samples after the written informed consent was given from all the patients.First, to examine the effect of CPZ on the intracellular trafficking of RTK mutants, we introduced murine FLT3-ITD and KIT-D814V (corresponding to human KIT D816V) into murine embryonic fibroblasts (MEFs) isolated from E14.5 fetuses, respectively. Both FLT3-ITD and KIT-D814V were distributed in cytoplasm with a punctate pattern, both of which were mainly located close to endoplasmic reticulum (ER) and Golgi. In contrast, when treated with CPZ, both RTK mutants were predominantly localized in the compartment apart from ER and Golgi in a diffused pattern, indicating that the intracellular trafficking system that retains mutated RTKs at ER or Golgi is disrupted by CPZ treatment.Next, we evaluated the effect of CPZ on cell growth and survival using several human leukemia cell lines and primary AML cells with or without mutant RTK. As a result, CPZ severely inhibited the growth and survival of leukemia cell lines and primary AML cells with mutant RTK in a dose-dependent manner (up to 95% inhibition; IC50: between 1.5 μM and 10.7 μM). In addition, CPZ inhibited the growth of the cells without mutant RTK, whereas the IC50 levels were 3- to 4-fold higher than those seen in the cells with mutant RTK. We also examined the effects of CPZ on the growth and survival of CD34+38- leukemic stem cells (LSCs) derived from AML patients (n=4), and found that CPZ effectively inhibited the growth and/or survival of LSCs, especially in LSCs with mutant RTK (IC50=7.5 μM). As for this mechanism, we found that CPZ significantly suppressed the activities of mutant RTKs and their downstream molecules such as Stat5 in all tested FLT3-ITD(+) LSCs. These results indicate that CPZ inhibits cell growth and/or survival of AML cells and LSCs harboring mutant RTKs. Next, we evaluated the in vivo anti-leukemic effect of CPZ with a xenograft model. Three days after the transplantation of human AML cells with mutant RTKs (n=8), NOD/Scid/IL2Rg-KO (NOG) mice were treated with normal saline or 10 mg/kg CPZ intraperitoneally on everyday schedule. At first, we confirmed that plasma concentration levels of CPZ on this schedule were within the non-toxic range as previously reported (0.03 to 0.71 μM, n=3) and that CPZ doesn’t affect normal hematopoiesis (n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ AML cells and CD34+38- LSCs were significantly reduced by CPZ treatment in the bone marrow (BM) of the recipient mice compared with normal saline-treated control (ctrl) mice (CD45+ 58.2% vs 8.47%, p<.001; LSCs 0.51% vs 0.07%, p<.005), indicating that CPZ can inhibit the growth and survival of both AML cells and LSCs in vivo. We further tested the anti-leukemic activities of CPZ on established human AML in a zenograft model. After confirming the engraftment of human AML cells by BM sampling six weeks after transplantation of FLT3-ITD(+) AML cells (n=3), these mice were treated with CPZ or normal saline. In all cases tested, CPZ treatment for 4 weeks significantly reduced human AML fraction in the BM. (18.4% vs 1.99%, p<.005)Together, these results suggest that CPZ, an inhibitor of the intracellular trafficking of leukemogenic RTKs, would be a promising therapeutic drug to eradicate AML cells with an established safety profile. DisclosuresRai:Shionogi & Co., Ltd. : Research Funding. Tanaka:Shionogi & Co., Ltd. : Research Funding. Tanimura:Shionogi & Co., Ltd. : Research Funding. Matsui:Shionogi & Co., Ltd. : Research Funding. Kanakura:Shionogi & Co., Ltd. : Research Funding. Matsumura:Shionogi & Co., Ltd. : Research Funding.

TIM-3 and Its Ligand, Galectin-9 Constitute a Pan-Myeloid Autocrine Loop to Develop and Maintain Malignant Stem Cells in Most Human Myeloid Leukemia

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on the surface of LSCs in most types of AML, and that TIM-3-targeted therapy could eradicate AML LSCs, based on in vivo xenograft experiments using patients’ AML samples (Kikushige et al, Cell Stem Cell, 2010). Since only the TIM-3+ but not the TIM-3- fraction of human AML cells can reconstitute human AML in immunodeficient mice, we hypothesized that the TIM-3 plays indispensable function to maintain AML LSCs. Galectin-9 is a ligand for TIM-3, and ligation of TIM-3 by galectin-9 has been shown to phosphorylate tyrosine residues of TIM-3 and activate Src family kinases through its SH2 domain in T cells and monocytes.We found that serum galectin-9 concentration was significantly elevated in AML patients (431.1+58.6pg/ml, n=13) but not in the healthy individuals (25.2+6.8pg/ml, n=7) or patients with B cell malignancies (36.1+15.5pg/ml, n=10). Primitive CD34+ AML cells had abundant galectin-9 protein in their cytoplasms. We then transplanted human CD34+ primitive AML cells into irradiated immunodeficient mice. Strikingly, only in mice reconstituted with human AML, but not in those with normal cord blood or human ALL cells had elevated serum levels of human galectin-9: Serum galectin-9 levels were 234.7+69.0pg/ml (n=8) in mice reconstituted with primary human AML cells, whereas 4.64+4.64pg/ml (n=12) in mice with normal human hematopoiesis. These results collectively suggested that AML cells secreted galectin-9 in vivo in an autocrine manner. We then performed transcriptome analyses of primary CD34+TIM-3+ AML cells after galectin-9 ligation. Results were further evaluated by a pathway enrichment analysis, which demonstrated that both NF-κB and β-catenin pathways were activated. In fact, galectin-9 ligation of TIM-3+ human AML cells induced activation of the NF-κB pathway via ERK and AKT phosphorylation. Activation of ERK and AKT pathways are known to inhibit the GSK3β activity, and to promote the nucleus translocation of β-catenin in several cancers. To demonstrate significant nucleus translocation of β-catenin of primary AML cells, we established a quantitation system by utilizing the Array Scan VTI system. By this system, we could formally prove that TIM-3 ligation by galectin-9 significantly promoted the nucleus translocation of β-catenin in primary AML cells.We also have extensively analyzed TIM-3 and galectin-9 interaction in cells from patients with myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and chronic myelogeneous leukemia. Strikingly, in all cases, frequencies of CD34+CD38-TIM-3+ cells dramatically increased along with disease progression from early/chronic phase to overt leukemias. Furthermore, serum levels of galectin-9 were also dramatically elevated after leukemic transformation. Significant nucleus translocation of β-catenin by galectin-9 ligation was also found in these diseases after leukemic transformation. A recent study has shown that NF-κB and β-catenin pathways could play a cooperative role in conferring the cancer-stem-cell properties to non stem cells in intestinal cancer model. These data collectively suggest that TIM-3 and galectin-9 constitutes a pan-myeloid autocrine loop to develop malignant stem cells in the vast majority of human myeloid malignancies. Thus, signaling molecules downstream of TIM-3 and galectin-9 ligation, as well as surface TIM-3 itself might be good candidates for cancer stem cell-target therapy common to most myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Expression of CD123 (IL-3R-alpha), a Therapeutic Target of SL-401, on Myeloproliferative Neoplasms

Background: CD123 (alpha chain of the interleukin-3 receptor [IL-3RA]) heterodimerizes with CD131 (βc, common beta chain) to constitute the high-affinity receptor for IL-3. CD123 is not expressed on hematopoietic stem/progenitor cells from normal bone marrow (BM). In contrast, CD123 is highly expressed in CD34+/CD38- cells from acute myeloid leukemia (AML) patients, which recapitulate the leukemic phenotype in NOD/SCID mice; CD123, therefore, is a marker for leukemic stem cells in AML (Jordan CT et al., Leukemia 2000). CD123 is also highly expressed on CD4+/56+ leukemic cells in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematodermic neoplasm. The validity of CD123 as a rational therapeutic target was illustrated by clinical efficacy data for SL-401, a biologic target therapy directed to IL-3R, in patients with BPDCN and AML, respectively (Frankel AE et al., Blood 2014 andFrankel AE et al., J Clin Oncol 31, 2013 (suppl; abstr 7029)).Objectives: To study CD123 expression as a potential therapeutic target in myeloproliferative neoplasms (MPN).Methods: The study was approved by our institutional review board and patients provided written consent for sample collection. MPN diagnosis was based on WHO 2008 criteria. Surface antigen expression on hematopoietic cells of various lineages was interrogated using the 4-color multiparametric flow cytometer, FACSCaliber TM (BD Biosciences, San Jose, CA). Data analysis was performed using CellQuest Pro Software (BD Biosciences).Results: We studied a total of 21 MPN patients and 3 normal controls. Of the former, 14 patients had systemic mastocytosis (SM) (indolent SM=9, SM with associated myeloid neoplasm=4 and aggressive SM=1), 6 had primary myelofibrosis (PMF) and 1 had eosinophilic leukemia transformed to AML.Normal controls: Three normal BM samples were studied; the number of CD123 positive cells in the total population was <1%. Rare CD34+/CD38- cells were uniformly CD123-. Of the CD123+ cells, only a minor subset (<10%) coexpressed myeloid/granulocyte lineage (CD13+, CD15+ or CD16+) or monocyte/macrophage lineage (CD14+ or CD11b+) markers.Systemic mastocytosis: The data were informative for 6 patients (3 with peripheral blood [PB], 1 with BM, and 2 with paired PB and BM samples) for whom a sufficient number of mast cells (MC) were identified for immunophenotyping. Neoplastic MC were defined as CD117 hi/SSC hi/CD45 hi/CD34-/CD25+/FcεRI+. CD123 expression was seen in the majority of MC for 4 patients (#1-4, CD123 percentage positive, PB/BM): 91%/n.a. (#1), 75%/81% (#2), 84%/n.a. (#3), and 88%/n.a. (#4). In contrast, one patient (#5) had a minority of CD123+ MC (33%/28%).Clonal eosinophilia: PB was studied; the WBC differential count showed 41% leukemic blasts and 57% eosinophils (both confirmed to be clonally related based on FISH studies). Both cell populations were predominantly CD123+ (≥90%).Primary myelofibrosis: We studied PB samples from 6 PMF patients. Approximately 1-2% of circulating cells marked as CD123+; of these, expression was notable on CD34-/CD38+ cells (median 54%; range 41-74% positive). We found a larger proportion (30-50%) of CD123+ cells that coexpressed CD13+, CD16+ or CD11b+ representing monocytes, immature myeloid cells and granulocytes, as compared to normal BM controls. Studies on BM samples from PMF patients are ongoing.Conclusions: CD123 (IL-3RA) is expressed on relevant primary cells of interest in select MPNs, namely neoplastic mast cells in SM patients and eosinophils in clonal eosinophilia patients. In PMF, a minor population of circulating cells was CD123+ with a biased distribution on myeloid lineage cells as compared to normal BM samples. We are currently studying CD123 expression in additional MPN patients, and also analyzing CD123 expression on BM trephine biopsies by immunohistochemistry. In addition, the cytotoxicity assessment of SL-401 against MPN cell lines and primary cells is ongoing. Overall, the aforementioned data support the clinical development of SL-401 in patients with MPNs and clinical trials are currently being planned. DisclosuresBrooks:Stemline Therapeutics: Employment, Equity Ownership. Pardanani:Stemline Therapeutics Inc.: Research Funding.

In-Depth Quantitative Multiplex Proteomics Reveal Subtype-Specific Differences Among Functionally Validated AML Stem Cell Populations

Acute Myeloid Leukemia (AML) is a heterogeneous group of myeloid malignancies. The classification of AML subtypes is based on recurrent genetic abnormalities and typical histopathological features, which impact on the patient’s prognosis. AML is also the model disease for the cancer stem cell model with leukemia stem cells (LSCs) residing at the top of a hierarchical organization. LSCs have self-renewal activity and generate leukemic progeny, which make up the majority of leukemic cells. Because LSCs can be quiescent and reside in specific niches in the bone marrow, rendering them resistant to conventional chemotherapy approaches, they are considered the source of relapse. Hence, further strategies to eradicate LSCs are pivotal to improve patient outcomes of this dismal disease.LSCs present within cell populations can be detected by their capacity to re-initiate the leukemia after xenotransplantation into immuno-compromised mice. However, using current methods, it is neither possible to prospectively isolate pure functional LSCs nor to distinguish them reliably from normal hematopoietic stem cells (HSCs). To define functional LSCs we FACS-sorted primary patient samples of different AML subtypes according to surface-expression of CD34 and CD38 and transplanted each of the resulting four cell populations into conditioned NSG recipients. Several AML samples showed human leukemic engraftment in at least one of the subsets, dissecting LSC-containing and LSC-free subpopulations within the same patient. AML engraftment was mainly observed within the CD34+CD38- fraction, but several cases showed LSC activity also in the CD34+CD38+ fraction or even in the CD34- subsets. As age-matched healthy controls, we collected bone marrow hematopoietic stem and progenitor cells (HSPCs, Lineage -CD34+CD38-) from individuals older than 60 years, undergoing hip replacement surgery.In-depth quantitative multiplex proteomic analysis was performed by employing tandem mass tag (TMT) labeling and high-resolution mass spectrometry. Using this approach, more than 7,000 proteins were quantified from 10 LSC-containing and 8 LSC-free fractions from six individual AML samples of different subtypes. Importantly, our data include many low abundance proteins or others that tend to be difficult to detect by mass spectrometry, such as transcription factors and membrane proteins. We identified up to 1500 differentially expressed proteins between LSC-containing and LSC-free fractions. Most interestingly, differentially expressed proteins also clustered according to AML subtype, indicating subtype-specific proteome differences at the level of AML LSCs. Along the same lines, Gene Ontology and Gene Set Enrichment Analyses showed distinct (in some instances even opposing) differences between AML subtypes. For example, interferon- and integrin-signaling were enriched in LSC-fractions of FLT3-ITD, NPM1-mutated AMLs while reduced in LSC-fractions of FLT3-wt, NPM1-wt AML samples. In summary, our data indicate that also at the level of functionally validated LSC populations, subtype-specific differences in protein expression are remarkably evident. This heterogeneity should be taken into account with respect to the development of targeting strategies for LSCs aiming to improve the clinical outcome of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Signature of Leukemia Stem Cells Defines Subgroups Associated with Clinical Outcome and Cell of Origin in AML

Acute myeloid leukemia (AML) is a hematologic malignancy initiated by leukemia-initiating or leukemia stem cells (LSC) which can differentiate into clonally related leukemic blast cells. This leukemia stem cell model proposes that functional properties of LSC and their blast progeny must be derived by epigenetic differences. Here, we examined genome wide DNA methylation of LSC-enriched populations and blast cells from 15 AML patients, along with 6 well-defined hematopoietic stem and progenitor cell (HSPC) populations from 5 normal controls using Illumina Infinium Human Methylation 450 BeadChip array. Strikingly, LSC-enriched populations exhibited global hypomethylation compared to non-engrafting blast cells, demonstrating that epigenetic change could drive the functional difference of LSC and their blast progeny. We defined an LSC epigenetic signature by integrating DNA methylation and gene expression analysis. The signature independently predicted overall survival of patients in both DNA methylation and gene expression data sets. Finally, we identified that LSC-enriched populations formed two major clusters when compared to normal HSPC: a granulocyte-macrophage progenitor (GMP)-like and a lymphoid-primed multipotential progenitor (L-MPP)-like subgroup that may reflect the cell of origin for these cases. These subgroups showed strong association with cytogenetic abnormalities and molecular mutations associated with the cell of origin. These results provide the first evidence for epigenetic variation between LSC and their blast progeny that are prognostic, and for epigenetically defined cell of origin of AML LSC. DisclosuresNo relevant conflicts of interest to declare.

JMJD1c, a Histone Demethylase, Is Required for the Maintenance of MLL-AF9 AML Leukemic Stem Cells

While most leukemic cells are initially sensitive to chemo- and radiotherapy, leukemic stem cells (LSC) persist and are therefore considered to be the basis for disease relapse in acute myeloid leukemia (AML) (Nat Biotechnol, 25: 1315-1321, 2007). The discovery of epigenetic mutations being the cause of a number of cancers has increased interest in their therapeutic potential (Nat Rev Genet, 7:21-33, 2006; Cell, 128: 683-692, 2007). Although the clinical importance of epigenetic dysregulation has been recognised in various cancers, including MLL-rearranged AML, this topic is currently a novel area of study.Our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified JMJD1c, a histone demethylase, as a potential LSC-specific target. An analysis of a comprehensive patient outcome database showed that high levels of JMJD1c were associated with significantly poorer survival in patients with AML (P = 0.0029), making it clinically relevant and emphasising the therapeutic potential of JMJD1c. Furthermore, the JMJD1c mRNA level was significantly higher in MLL-rearranged than in non-MLL AML patient xenograft samples.Western blot and quantitative PCR confirmed the microarray results, where JMJD1c was significantly upregulated in MLL-AF9 LSC compared to Hoxa9/Meis1a derived LSC and HSC. Overexpression of JMJD1c in Hoxa9/Meis1a pre-LSC significantly increased the clonogenic activity and proliferation compared to control cells. Subsequent microarray experiment identified Hox genes as downstream targets of this epigenetic regulator (fold change > 2, P < 0.05), suggesting the regulatory role of JMJD1c in the self-renewal pathway. Consistent with in vitro observations, there was a significant increase in disease progression as shown by leukemia onset in mice with Hoxa9/Meisa1 pre-LSC transduced with JMJD1c cDNA compared to control (P < 0.0001; at least 12 mice per cohort). Stable knockdown of JMJD1c in MLL-AF9 cells using shRNA significantly impaired the proliferation, clonogenic activity and induced cell differentiation in vitro. In vivo survival studies in mice showed that JMJD1c knockdown severely impaired the maintenance of MLL-AF9 induced leukemia (P < 0.0001; 12 mice per cohort).In conclusion, our studies have identified JMJD1c as an important driver in leukemia maintenance and strongly suggest that JMJD1c possesses an oncogenic role in MLL-AF9 leukemogenesis. Targeting this oncogene may enable us to directly target LSC as a strategy for overcoming drug resistance in AML patients. DisclosuresNo relevant conflicts of interest to declare.

Telomerase Inhibition Effectively Targets Mouse and Human AML Stem Cells and Delays Relapse following Chemotherapy

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell-cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc(-/-) LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs.

GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis

Abstractβ-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell–derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML.

SIRT1 Activation by a c-MYC Oncogenic Network Promotes the Maintenance and Drug Resistance of Human FLT3-ITD Acute Myeloid Leukemia Stem Cells

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes.

Pharmacological Inhibition Of The SIRT1 Deacetylase With The Small Molecule Inhibitor Tenovin-6 Enhances Ablation Of FLT3-ITD+ LSC In Combination With TKI Treatment

Acute myeloid leukemia (AML) is propagated by small populations of leukemia stem cells (LSC). Internal tandem duplication (ITD) in the FMS-like tyrosine kinase-3 (FLT3) is the most frequent mutation seen in AML patients, and is associated with poor prognosis. However, FLT3 tyrosine kinase inhibitors (TKI) demonstrate only moderate clinical activity in FLT3-ITD AML patients. Persistent FLT3-ITD+ AML LSC may represent a source of relapse after treatment. Additional therapeutic strategies are required to target LSC and improve outcomes for FLT3-ITD+ AML patients. We have shown that the stress-related deacetylase SIRT1 is expressed at high levels in FLT3-ITD+ AML stem/progenitor cells and in cord blood CD34+ cells ectopically expressing FLT3-ITD. Inhibition of SIRT1 using RNAi or a potent small molecule inhibitor Tenovin-6 (TV-6) induces apoptosis in FLT3-ITD+ AML progenitors via p53-dependent mechanisms while sparing normal CD34+ cells suggesting a potential role for SIRT1 in FLT3-ITD+ AML LSC maintenance (Blood 2012,120: A1368). We now show that treatment of FLT3-ITD+ AML CD34+ cells with the TKI AC220 (20 nM) did not significantly reduce SIRT1 levels, increase p53 acetylation, or increase p53 target gene expression (p21, Bax, Puma, DR5, Noxa), suggesting that kinase independent mechanisms maintain SIRT1 expression and activity in TKI-treated cells. The combination of TV-6 (1μM) with AC220 (20nM) significantly reduced survival of primary human FLT3-ITD+ AML CD34+ cells compared with AC220 or TV-6 alone (combination 47±8% inhibition, AC220 alone 21±7%, TV-6 alone 25±5%, p=0.03, n=7), and significantly increased inhibition of AML CD34+ cell growth compared to AC220 and TV-6 alone (TV-6 44±7% inhibition; AC220 30±6%, TV+AC220 59±8%, p=0.04, n=7). TV-6 treatment increased p53 expression and activity in AC220-treated FLT3-ITD+ AML CD34+ cells, but did not affect FLT3 phosphorylation or STAT5 activation. Interestingly, TV-6 treatment also reduced survival of FLT3-ITD+ AML CD34+ cells expressing the D835Y mutation associated with TKI resistance (AC220 5±5% inhibition, TV 33±8%, p=0.04, n=3], suggesting SIRT1 inhibition can target TKI-resistant FLT3-ITD+ AML cells. We next tested the effect of TV-6, AC220 and the combination on primary cells from one untreated cytogenetically normal and FLT3-ITD+ patient engrafted in NSG mice (2×106 cells/mouse).12 weeks after transplantation, mice were treated with vehicle, AC220 (10mg/kg, gavage), TV-6 (100mg/kg, intraperitoneal), or the combination (n=7 per group) for 4 weeks. Treatment with TV reduced human cell engrafted in NSG mice (TV-6 2.9*107±1.2*107 human CD45+ cells/2 femurs vs. Control 5.3*107±2.6*107, after 4 weeks treatment, p=0.04). Importantly, the combination of TV and AC220 significantly reduced AML cell engraftment compared with AC220 alone (AC220 2.4*107±0.9*107 human CD45+ cells. Combination 1.2*107±0.6*107, p=0.01). Residual functional LSC were assessed by transplantation into secondary recipients. BM cells from the combination treatment group demonstrated significantly reduced engraftment of leukemia cells after secondary transplantation compared to AC220 treatment alone (16 weeks after secondary transplantation, AC220 1.8*106±0.6*106 human CD45+ cells/2 femurs, Combination 0.4*106±0.1*106, p=0.02, n=6). To determine the potential of this combination for FLT3-ITD+ AML patients refractory to standard chemotherapy, AML xenografts established using cells from a chemotherapy-resistant patient (n = 36) were treated with TV-6, AC220, or the combination vs. Standard of care (SOC, Ara-C + DNR), and vehicle control. Molecular imaging of primary patient xenografts with fluorescently labeled monoclonal antibodies after 4 weeks of therapy revealed significant differences between combination and single arm groups (vs. TV-6, p=0.0005, n=8; vs. AC220, p=0.003, n=7). Furthermore, TV-6 alone increased survival over controls (TV-6 vs. Control, p=0.02, n=6), and the combination demonstrated significantly improved survival over AC220 (p=0.02) and SOC (p=0.006. n=6). In conclusion, our studies indicate that elevated SIRT1 expression in FLT3-ITD AML cells is maintained after FLT3 TKI treatment. Inhibition of SIRT1 with TV-6 can enhance ablation of FLT3-ITD+ AML LSC in combination with FLT3 TKI treatment. Our results support further evaluation of inhibition of SIRT1 as a therapeutic strategy in FLT3-ITD+ AML. Disclosures:Popa:KinN Therapeutics AS: Employment.

TIM-3, a Leukemia Stem Cell Marker, Plays a Role In Leukemic Transformation Through Autocrine Stimulatory Signaling By Its Ligand, Galectin-9

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs) (Kikushige et al, Cell Stem Cell, 2010). We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38- LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although CD34+CD38- normal bone marrow stem cells do not express TIM-3, TIM-3 is expressed in the CD34+CD38- population in MDS, and is further up-regulated with progression into leukemia. The average percentages of TIM-3+ cells in the CD34+CD38- population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is a type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. To understand the function of TIM-3, we investigated the interaction between TIM-3 and its ligand galectin-9 in AML LSCs. We found that AML patients showed significantly higher serum galectin-9 concentration than healthy individuals (healthy controls: 18.3+4.3 pg/ml, AML patients: 139.1+33.4 pg/ml, P<0.05). Unexpectedly, we found that leukemic cells expressed a high level of galectin-9 protein, as compared to other hematopoietic cells including T cells, B cells and monocytes. Using KASUMI-3 (TIM-3+ AML cell line) and primary AML samples, we confirmed that AML cells could secrete galectin-9 after TLR stimulation in vitro. Furthermore, microarray analysis demonstrated that TIM-3 stimulation by the physiological concentration of galectin-9 induced significant gene expression changes toward pro-survival axis including up-regulation of MCL-1, the important survival factor for HSCs and LSCs. These results collectively suggest that AML cells can produce and secrete galectin-9, and galectin-9 can bind and stimulate TIM-3-expressing AML cells including LSCs in an autocrine manner to support their survival or leukemia progression. Disclosures:Miyamoto:Kyushu University Hospital: Employment.

Leukemia Stem Cell Marker CD123 (IL-3R alpha) Predicts Minimal Residual Disease and Relapse, Providing a Valid Target For SL-101 In Acute Myeloid Leukemia With FLT3-ITD Mutations

Leukemia stem cells (LSC) play critical role in initiation and progression of acute myeloid leukemia (AML). CD123 (IL-3R alpha) is one of the established LSC markers, and the frequency of CD123+ LSCs at diagnosis is associated with response to treatment (Vergez F et al, Haematologica 2011). However, it remains unclear whether CD123+ LSCs contribute to minimal residual disease (MRD) and disease recurrence.In this study, we first analyzed expression of CD123 on CD34+ blast cells in a cohort of 95 newly diagnosed AML patients (pts) treated with induction regimens between 02/2011 and 10/2012. In these pts, standard MRD assessment was routinely performed at the time of complete remission (CR) as described (Ravandi et al, Leukemia 2012). CD123 was highly expressed in diagnostic BM samples (67%, range 26.8%-91.7%). Higher expression of CD123+ cells on blasts and within CD34+ stem/progenitor cells correlated with poor prognosis cytogenetics (p=0.016 and 0.034) and unmutated NPM1 (p<0.001 and p=0.003, respectively). Altogether, 62.1% (59/95) pts achieved negative MRD, which in a multivariate analysis strongly correlated with longer time to relapse (HR: 0.71; 95%CI: 0.58-0.87; p<0.001). A higher proportion of CD123+CD34+ cells in remission marrows (ratio of %CD34+CD123+/%CD34+ greater than 0.83) was associated with higher risk to remain MRD-positive in both univariate (OR, 0.11-0.96, p=0.043) and multivariate Logistic Regression models (p=0.027). Although higher frequencies of CD34+CD123+ blasts significantly correlated with shorter time to relapse in the univariate Cox proportional hazard model (p=0.037), only white blood count, FLT3-ITD mutation and time to achieve MRD remained significant in a multivariate analysis.We next analyzed expression of CD123 in enriched CD34+CD38-CD99+ LSC fraction (Jan et al, Sci Transl Med. 2012) in 10 newly diagnosed AML BM samples. As controls, we used 3 normal bone marrow (NBM) samples. Cell surface characteristics and signaling pathways activation were defined by the time-of-flight mass cytometry (CyTOF) (Bendall et al, Science. 2011) using 11 cell surface markers (CD34, CD38, CD123, CD99, CD45, CD33, CD117, CD7, CD4, CD90 and CD133) and 8 intracellular markers (p-4EBP1, p-NF¦ÊB, p-STAT3, p-AKT, p-mTOR, p-ERK, p-S6 and p-STAT5). The frequency of CD123+ cells within CD34+CD38-CD99+ LSCs was significantly higher in AML samples at diagnosis (median 78.1%, range 38.4%-96.3%) compared to NBM (median 25.1%, range 22.8%-39.6%; p<0.001). FLT3-mutated AML expressed higher levels of CD123 within LSCs (median 90.9%, range 72.9%-96.3%) compared to FLT3-wt (median 69.5%, range 38.4%-73.5%; p=0.013). Multiple intracellular signaling pathways were highly activated in CD123+ LSCs in AML with FLT3-ITD compared to NBM stem cells, including p-4EBP1, p-NF¦ÊB, p-AKT, p-mTOR and p-ERK (Figure 1). This data indicate that targeting CD123-expressing LSC may have anti-leukemia effects in AML, especially those with FLT3 mutations. [Display omitted] To this end, we tested anti-leukemia activity of SL-101, a novel targeted therapy directed to CD123, in AML cell lines. SL-101 is an anti-CD123 scFv antibody conjugate. CD123 is highly expressed in AML cell lines (n=12) except for U937 and HL-60 cells (median 75.2%, range 0.9%-99.8%). SL101-induced killing strongly correlated with CD123 expression (r=-0.8220, p=0.001), suggesting that SL101 induces target-specific cell killing at low nanomolar range (median IC50 10.9 nM, range 0.1 nM-249.9 nM). We next analyzed the combined effects of SL101 and FLT3 inhibitor sorafenib in three FLT3-ITD cell lines. Strikingly, exogenous IL-3 significantly protected FLT3 mutant cells from sorafenib-induced cell killing in MV4;11 (p<0.01) and MOLM13 cells (p<0.001) but not in MOLM14 cells. In turn, combination with SL-101 revoked IL-3 mediated cytoprotection in MV4;11 (p<0.001) and MOLM13 cells (p<0.01).In conclusion, expression of CD123 on CD34+ blast cells is associated with persistent MRD in newly diagnosed AML. Multiple signaling pathways are activated in these CD123+ LSCs, supporting the feasibility of targeting CD123 to eliminate AML LSCs. SL-101 induces apoptotic cell death in CD123+ AML cell lines at low nanomolar concentrations and abrogates IL-3 mediated cytoprotection in FLT3-mutant cells. Ongoing studies are focusing on the anti-leukemia efficacy of SL-101 in primitive LSCs in primary AML samples, especially those with FLT3-ITD. Disclosures:Brooks:Stemline Therapeutics: Employment, Equity Ownership. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Selective Anti Leukemic Activity Of Low Dose Decitabine In Combination With Ruxolitinib Against Stem/Progenitor Cells From Elderly AML Patients

Acute myeloid leukemia (AML) is a clonal hematopoietic disorder characterized by an accumulation of immature leukemic cells. The incidence of AML sharply increases with age. However outcomes for AML in the elderly are dismal with low response rates and high rates of relapse. Primitive AML leukemia stem cells (LSC) are resistant to conventional therapy, and potentially contribute to propensity to relapse. Therefore, new strategies to enhance targeting of AML LSCs and are required to improve outcomes. AML cells demonstrate several epigenetic alterations including enhanced promoter hypermethylation that may contribute to altered growth and self-renewal of LSC. The DNA methyltransferase (DNMT) inhibitor decitabine (DAC) has shown activity against AML LSC in preclinical studies. In clinical studies DAC is well tolerated as monotherapy and induces responses in approximately 20% of AML patients who are not candidates for conventional chemotherapy. Therefore there is considerable interest in developing combinations of DAC with other therapies for elderly patients with AML. We have previously shown that LSC from AML patients with poor risk cytogenetics demonstrate increased JAK2 tyrosine kinase activity and enhanced sensitivity to JAK2 inhibition. Since cross regulation between JAK/STAT and DNMT pathways has been reported, we evaluated the activity of the combination of DAC with the JAK2 tyrosine kinase inhibitor (TKI) Ruxolitinib (RUX) on CD34+ cells from elderly AML patients (>60 years). Exposure to low dose DAC (10 to 200nM) for 72 hours significantly inhibited the growth of primary AML CD34+ cells in a CellTitre Glo assay in a dose dependent manner (n=12, DAC 10nM 88±9% of control, 50nM 77±9%, 100nM 64±11% and 200nM 56±15%). DAC did not induce significant apoptosis in AML CD34+ cells (n=10, control, 22±5% of Annexin V positive cells vs 24±7% for the samples treated with 200nM of DAC) but dramatically and significantly reduced AML CD34+ cell growth in CFC assays (33±3%, 57±14%, 75±3% and 86±7% inhibition with 10, 50, 100 and 200nM of DAC respectively). The combination of RUX (100nM) and DAC (10 and 50nM) significantly enhanced inhibition of AML CD34+ cell growth compared to RUX or DAC alone (DAC 10nM 87±10% of control for, RUX 62±20%, DAC10+RUX 46±20%, DAC50nM 75±10%, and DAC50+RUX 43±20%, n=7, p<0.009). Combination of RUX (100nM) with 10 and 50nM DAC did not significantly enhance apoptosis in AML (n=10) or normal stem/progenitor cells (n=8, Ctrl 20.6±5%, DAC10nM 21.6±5%, RUX100nM 31.5±5%, DAC10-RUX 32.1±5%, DAC50nM 22.6±5% and DAC50-RUX 32.4±8%), but resulted in significantly enhanced inhibition of AML CD34+ CFC compared to RUX or DAC alone (n=4, CFC inhibition with DAC10 34±4%, RUX 46±15%, DAC10+RUX 68±17%, DAC50 56±15%, and DAC50+RUX 77±7%, p<0.004), but did not enhance inhibition of CB CFU-GM (n=4, CFC inhibition with DAC10 0±12%, RUX 24.3±16%, DAC10+RUX 4±9%, DAC50 9±16%, and DAC50+RUX 25±11%). These results indicate that combined targeting of both JAK/STAT and DNMT pathways can selectively inhibit the growth capacity of AML compared with normal stem/progenitor cells. We performed Western blotting on AML CD34+ cells (n=5) treated for 3 days with RUX 100nM, DAC 50nM or the combination of RUX and DAC to evaluate expression of DNMT1, DNMT3a, DNMT3b, phosphorylated STAT3 (Y705) and STAT5 (Y694) and SOCS3. Compared to the individual drugs, the combination resulted in greater reduction of p-STAT3 in 4 out 5 AML patient samples that was associated with increase in SOCS3 in 2 samples, suggesting a crosstalk between the JAK/STAT and DNMT pathways. Interestingly we did not observe enhanced inhibition of p-STAT5 with the combination. Two of five AML samples displayed decrease of DNMT1 expression and 3 of 5 samples decreased DNMT3a and DNMT3b expression upon treatment with the combination. In conclusion, our studies suggest that there may be a crosstalk between the JAK/STAT/SOCS3 signalling and DNA methylation mechanisms in CD34+ cells from elderly AML patients, and indicate that JAK2 TKI in combination with low doses of DAC selectively and significantly enhance inhibition of stem/progenitor cells from elderly AML patients compared to DNMT inhibitors alone. These results support further evaluation of this novel combination for the treatment of AML in the elderly. Disclosures:No relevant conflicts of interest to declare.

Leukemia Stem/Progenitor Cells From AML Patients Treated With The Multi-Kinase Inhibitor TG02 Demonstrate Increased Proliferation and Are Sensitized To Chemotherapeutic Agents

TG02 is a multi-kinase inhibitor that targets cyclin-dependent kinases (CDKs), ERK5, JAK2, and Flt3. In vitro studies of TG02 have shown robust induction of apoptosis in both acute myeloid leukemia (AML) cell lines and primary cells (Goh et al, 2011). Leukemia stem cells (LSCs) comprise a largely quiescent, highly chemotherapy-resistant cell population and are believed to initiate and maintain AML, as well as contribute to its poor prognosis. Thus, we sought to investigate the impact of TG02 on LSCs collected from patients with relapsed/refractory AML enrolled in a phase I dose escalation trial. Patients ≥ 18 years with advanced hematological malignancies or newly diagnosed AML pts ≥ 65 years unfit for intensive therapy were enrolled onto daily (A) and intermittent (B, 5 days on 2 days off X 2 weeks) schedules in 28-day cycles. Pts had acceptable organ function and ECOG PS 0-2. Dose levels were 10 - 70 mg on arm A and 30 - 150 mg on arm B.We evaluated immunophenotypically defined leukemia stem and progenitor cells (LSPCs) by flow cytometry, cell cycle status and colony forming assays. A total of 16 patients were evaluated with treatment doses ranging from 10-150 mg of TG02.Clinically, treatment with TG02 did not have an effect in AML tumor burden, and most patients at our center only received one cycle of treatment (Roboz et al ASCO 2012 Annual Meeting Abstract #6557, J Clin. Oncol. 30, 2012). However, we found that 8 patient samples showed increased LSPCs in both the bone marrow and peripheral blood. Interestingly, we observed an increase in LSPC cell proliferation, as determined by Ki-67 positive staining. AML colony forming assays also showed increased colony formation (n=5) after one cycle of treatment, which suggests an increase in the frequency of LSPCs. The increase in colony formation in peripheral blood samples suggests mobilization of LSPCs from the marrow into the circulation. Thus, we hypothesized that exposure to TG02 in vivo may result in sensitization to other chemotherapeutic agents, such as Ara-C.We evaluated the effects of Ara-C and other chemotherapeutics, such as vincristine, in primary AML cells obtained from patients before and after treatment with TG02. We found that in vivo exposure to TG02 resulted in significantly increased sensitivity to Ara-C in vitro in 3 out of 4 samples testedTogether, our data suggest that TG02 induces an effect in LSCs resulting in increased proliferation and, thus, sensitization to other chemotherapeutic drugs, such as Ara-C. Importantly, although no patients at our center receiving single agent TG02 met the criteria for an objective response, by performing correlative studies in association with the clinical trial, we found the TG02 has a marked effect in AML LSCs that could potentially be exploited by combining it with other agents. Disclosures:Burrows:Tragara Pharmaceuticals: Employment, Equity Ownership. Feldman:Tragara Pharmaceuticals: Consultancy.

Evaluation Of CD33 Expression and Functional Analysis Of The CD33/CD3 Bispecific BiTE® Antibody AMG 330 In Primary AML Samples

Antibody-based immunotherapy represents a promising strategy to specifically target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). We evaluated a single-chain bispecific CD33/CD3 BiTE® antibody (AMG 330) for its suitability as immunotherapy in AML. A prerequisite for successful immunotherapeutic approaches using this molecule is the expression of CD33 on AML blasts including leukemic stem cells (LSCs). Therefore, we quantified CD33 expression on AML blasts and LSCs by flow cytometry (mean fluorescence intensity, MFI) and correlated expression intensity with cytogenetic and molecular disease characteristics in order to identify patient cohorts possibly most suited for CD33-targeted therapies. CD33 expression was detected in >99% of patient samples (n=621, MFI ratio ≥ 1.5) although highly variable. A strong correlation between high CD33 expression levels and NPM1 mutations (p<0.001) was found. In contrast, low CD33 expression levels were significantly associated with complex karyotypes and t(8,21) translocations (p<0.001). Furthermore, LSCs within the CD34+/CD38- compartment displayed CD33 at higher levels than healthy donor stem cells (p=0.047). Importantly, colony formation of CD34+/Lin-cells from healthy donors was not affected after pre-incubation with AMG 330 and T-cells.A major hurdle for measuring cytotoxic effects on AML blasts has long been that primary AML patient samples show progressive cell death within a few days ex-vivo. To simulate the natural setting of target and T-cells in AML patients, we developed a long-term culture system for AML blasts that allowed us to observe these co-cultures for up to 5 weeks. Thus, we were able to show effective elimination of AML blasts within primary samples by AMG 330-activated and expanded residual CD3+/CD45RA-/CCR7+ memory T-lymphocytes. While the functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT vs. CD33DIM AML cell lines in an in-vitro cytotoxicity assay potent anti-leukemic activity on primary AML blasts was observed irrespective of CD33 expression level. At low effector to target ratios (up to 1:79), the recruited T-cells lysed autologous blasts completely in the majority of samples. Further T-cell analysis showed that naive T-cells (CD45RA+/CCR7+) were not expanded by AMG 330; neither were terminally differentiated T-cells (CD45RA+/CCR7-), probably due to their poor proliferative capacity. We did not observe an increase in percentage of CD3+/CD4+/CD25+/FoxP3+regulatory T-cells in the presence of AMG 330, suggesting that these cells may not have impacted AMG 330-mediated T-cell activity in our experiments. Compared to control cultures, T-cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3 upon response to AMG 330, which was partially reversible after complete target cell elimination. However, PD-1 up-regulation did not correlate with an up-regulation of PD-L1 on AML blasts despite substantial INFγ secretion by activated T-cells.This study provides the first correlation of CD33 expression levels to a comprehensive genotype analysis in adult AML patients. While CD33 expression may vary by AML biologic subgroup, AMG 330 exposure led to lysis of AML blasts even in samples with low levels of expression. Targeting CD33 using AMG 330 in primary AML samples led to efficient T-cell activation and expansion as expected from the mechanism of action of BiTE® antibodies. The remarkable ex-vivo activity of AMG 330 supports further development of AMG 330 as an immunotherapy for patients with AML. Disclosures:Kufer:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Baeuerle:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership.

Normal Hematopoietic Stem Cells within the AML Bone Marrow Have a Distinct and Higher ALDH Activity Level than Co-Existing Leukemic Stem Cells

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDHbright). The ALDHbright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDHlow. In CD34-positive AML, LSC are also ALDHlow but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.

A stem cell-like gene expression signature associates with inferior outcomes and a distinct microRNA expression profile in adults with primary cytogenetically normal acute myeloid leukemia

Acute myeloid leukemia (AML) is hypothesized to be sustained by self-renewing leukemia stem cells (LSCs). Recently, gene expression signatures (GES) from functionally defined AML LSC populations were reported, and expression of a 'core enriched' (CE) GES, representing 44 genes activated in LCSs, conferred shorter survival in cytogenetically normal (CN) AML. The prognostic impact of the CE GES in the context of other molecular markers, including gene mutations and microRNA (miR) expression alterations, is unknown and its clinical utility is unclear. We studied associations of the CE GES with known molecular prognosticators, miR expression profiles, and outcomes in 364 well-characterized CN-AML patients. A high CE score (CE(high)) associated with FLT3-internal tandem duplication, WT1 and RUNX1 mutations, wild-type CEBPA and TET2, and high ERG, BAALC and miR-155 expression. CE(high) patients had a lower complete remission (CR) rate (P=0.003) and shorter disease-free (DFS, P<0.001) and overall survival (OS, P<0.001) than CE(low) patients. These associations persisted in multivariable analyses adjusting for other prognosticators (CR, P=0.02; DFS, P<0.001; and OS, P<0.001). CE(high) status was accompanied by a characteristic miR expression signature. Fifteen miRs were upregulated in both younger and older CE(high) patients, including miRs relevant for stem cell function. Our results support the clinical relevance of LSCs and improve risk stratification in AML.

Abstract 2292: Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells.

Abstract We recently demonstrated stem cell gene signatures predict clinical outcome in acute myeloid leukemia (AML) (Eppert et. al., Nature Medicine, 2011). Concomitant to this work, miRNA signatures for hematopoietic stem cells (HSC) and leukemia stem cells (LSC) were also generated. miRNA are small non-coding RNAs that regulate the translation and mRNA stability of protein coding genes with significant roles in the maintenance of human HSC (Lechman et. al., Cell Stem Cell, in press). To understand the functional role of miRNA in normal human blood development, we undertook an in vivo over-expression screen of 10 miRNA candidates over-represented in HSC and LSC. Lineage depleted human umbilical cord blood cells (Lin- CB) were transduced with lentivirus expressing either a candidate miRNA or control vector and xeno-transplanted into NSG mice. Three miRNA displayed a competitive growth advantage while 4 miRNA induced a growth disadvantage along with skewing of lineage output. A top LSC array candidate, miR-125b, showed the most pronounced phenotype with overt expansion of marked cells, enlarged spleens and increased lymphoid and erythroid output. Detailed analysis of miR-125b grafts revealed a greatly expanded MLP population, in comparison to HSC and MPP. Furthermore, upon enforced in vivo expression of miR-125b in 3 AML patient samples, we observed large increases in the CD34+CD117+ populations for all three AML samples, suggesting increased LSC numbers. Secondary LDA experiments revealed up to a 34 fold increase in LSC activity in comparison to control vector transduced AML cells. These data suggest that miR-125b normally functions in the limited self-renewal of lymphoid committed early progenitors and this function may be usurped during leukemogenesis to enhance LSC self-renewal. Citation Format: Eric R. Lechman, Karin G. Hermans, Stephanie Dobson, Kolja Eppert, Mark Minden, John E. Dick. Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2292. doi:10.1158/1538-7445.AM2013-2292

Abstract 605: Apogossypolone derivative -BI97D6 effectively targets MCL1 overexpressing Acute Myeloid Leukemia cells.

Abstract Aberrant expression of anti-apoptotic proteins such as BCL2, MCL1, and BCL-XL is observed in many tumors including Acute Myeloid Leukemia (AML). An obvious strategy for AML therapy would be to target the anti-apoptotic BCL2 family members. While compounds such as ABT-737 have shown promise for treatment of some malignancies, the compound binds poorly to MCL1 and thus cells that overexpress MCL1 usually display high resistance to the drug. Furthermore, ABT-737 activates ERK signaling resulting in MCL1 overexpression in surviving cells (Konopleva, et al, Leukemia 2011). The apogossypolone derivative -BI97D6 binds MCL1 and BCL2, BCL-XL, BFL1 with high affinities (Wei, et al, J Med Chem 2010). We recently reported that -BI97D6 (a) efficiently kills AML cell lines even in the presence of protective mesenchymal stroma cells, (b) interferes with the association between MCL1/BIM and BCL2/BAX, (c) stimulates BAX conformational change, (d) requires BAX or BAK for cell killing, and (e) does not induce ER stress. These data suggests that -BI97D6, unlike other gossypol or apogossypol compounds, acts mainly as a BH3 mimetic. In the current study, we first examined the role of MCL1 in -BI97D6-mediated killing. OCI-AML3 cells are extremely resistant to ABT-737 due to its high expression of MCL1. MCL1 expression was suppressed by lenti-viral shRNA in OCI-AML3 cells and knockdown of MCL1 protein by 80% was achieved as determined by quantitative western blot. Compared to cells with non-specific control shRNA, cells with MCL1 shRNA were greatly sensitized to ABT-737, and significantly sensitized to AraC, while -BI97D6, which binds MCL1 with high affinity, is almost equipotent in knockdown and control cells. Since results with AML cell lines have been promising, we examined the efficacy of -BI97D6 on primary AML samples. Flow cytometry analysis revealed that -BI97D6 potently induced apoptosis in AML blast cells at low nM concentration (EC50&amp;lt;50nM). -BI97D6 also effectively killed putative CD34+CD38- leukemia stem cells (LSC). Importantly, primary AML with FLT3-ITD and FLT3-D835 mutations showed similar sensitivity as those without FLT3 mutations (EC50&amp;lt;50nM). As FLT3 mutations are associated with poor prognosis in AML, these findings suggests that -BI97D6 can target AML LSC and could benefit even high risk patients. In summary, compound -BI97D6 may be a promising drug candidate for MCL1-targeted AML therapy. Citation Format: Rongqing Pan, Vivian Ruvolo, Maurizio Pellecchia, Jun Wei, Marina Konopleva, John C. Reed, Peter Ruvolo, Michael Andreeff. Apogossypolone derivative -BI97D6 effectively targets MCL1 overexpressing Acute Myeloid Leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2013-605

Leukemic Stem Cells Shows the Way for Novel Target of Acute Myeloid Leukemia Therapy

Leukemic stem cells (LSCs) have been identified in acute myeloid leukemia (AML). Similar to haematopoietic stem cells, these LSCs are able to self-renew, differentiate, and proliferate extensively. Recent studies suggest that LSCs are critical for the initiation and maintenance of leukemia. This review will describe the characteristic features of LSCs in AML and the possible targets expressed on the surface of AML, the intracellular targets and the novel molecular and flow cytometry methodologies being used to particularly ablate the LSC population. Studies have shown the potential importance of ablating LSCs when treating leukemia. The unique characteristics of LSCs that differentiate them from their normal counterparts can be applied to specifically target the leukemic population. Current therapeutic strategies may not effectively ablate the LSCs, leaving the potential for disease progression or recurrence. A better understanding of LSCs and molecular biology will allow the design of more effective therapies.