Acute Myelogenous Leukemia Stem Research Articles

Immunoglobulin Superfamily Member 8 Is Indispensable for Myeloid Leukemia Via Wnt/β-Catenin Signaling Pathway

Immunoglobulin superfamily member 8 (IGSF8, also known as EWI-2, PGRL, and CD316), is a cell surface protein containing 4 immunoglobulin domains. IGSF8 directly binds to the tetraspanin molecules, CD9 and CD81, and modulates cell adhesion, migration, and growth. Previous studies demonstrated that IGSF8 was associated with prognosis and metastasis in several solid tumors. However, the role of IGSF8 in normal hematopoiesis and myeloid leukemia is still unclear. First, we examined the expression levels of Igsf8 in various hematopoietic fraction of wild-type murine bone marrow cells, and found that Igsf8 is expressed in all hematopoietic lineages. To investigate hematopoietic functions of Igsf8, we generated hematopoietic cells specific Igsf8 deleted mice (Igsf8fl/fl; Vav-Cre) and tamoxifen induced Igsf8 deleted mice (Igsf8fl/fl; Rosa26-CreERT). Igsf8fl/fl, Vav-Cre (denoted as Igsf8-/-) mice represented normal maturation. Deletion of Igsf8 did not significantly affect adult hematopoiesis in peripheral blood and bone marrow. Igsf8-/- long-term hematopoietic stem cells (LT-HSCs: CD34- Flk2- c-Kit+ Sca-1+ Lineage- cells) reduced colony forming ability in vitro, and serial competitive transplantation assay showed comparable donor chimerism by 3 months, but led to decrease Igsf8-/- donor chimerism at 4 months and those after second transplantation in vivo. These results suggest that Igsf8 does not affect the adult hematopoiesis, but it can affect their proliferative and reconstitutive capacity of HSCs. To investigate the effects of Igsf8 on myeloid leukemia, we generated MLL-AF9 and NRASG12V-driven acute myelogenous leukemia (AML), or BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) mice models. Igsf8-/- led to a dramatic reduction in the number of leukemic colonies formed in vitro (Figure 1A). Igsf8-/- leukemia mice showed significantly longer survival in vivo (Figure 1B). This effect was also observed by eliminating Igsf8 expression after leukemia establishment using conditionally deletion. Igsf8-/- AML cells showed decreased S phase fraction. Igsf8-/- leukemia stem cells (LSCs: c-Kit+ Lineage- cells) triggered an increment of the apoptosis, which contribute to significantly lower proportion of LSCs in spleen of Igsf8-/- leukemic mice. Given that Igsf8-/- did not affect homing ability of leukemia cells, these results indicate that Igsf8 is required for propagation of myeloid leukemia and maintenance of LSC. To understand the Igsf8-mediated regulation of myeloid leukemia, we conducted RNA sequencing analysis of LT-HSCs, and LSCs of AML and CML-BC. Gene set enrichment analysis exhibited increase apoptosis related genes and decrease Wnt/β-catenin related genes in Igsf8-/- leukemic cells, but not in LT-HSCs (Figure 1C). Increment of pro-apoptosis genes, and decrement of anti-apoptosis genes and Wnt/β-catenin target genes in Igsf8-/- AML stem cells were validated in quantitative polymerase chain reaction analysis. Further, expression levels of β-catenin protein in Igsf8-/- leukemic cells were significantly lower compared to Igsf8+/+ leukemic cells, but not in normal hematopoietic stem and progenitor cells (Figure 1D). These results suggest that Igsf8 might be critical for myeloid leukemia maintenance via Wnt/β-catenin signaling pathway. Then, we investigated the effects of IGSF8 on human myeloid leukemia. We confirmed IGSF8 expression in several human myeloid leukemia cell line and primary patient-derived leukemia cells. Knockdown of IGSF8 by small hairpin RNA in myeloid leukemia cell lines (THP-1, MV4-11, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro. Knockdown of IGSF8 also caused decrease expression of β-CATENIN in AML cell lines. These results indicate that IGSF8 is also required for propagation of human myeloid leukemia cells. Taken together, our present study reveals that Igsf8 is indispensable for myeloid leukemia, but not adult hematopoiesis, suggesting that IGSF8 inhibition should be considered for targeting myeloid leukemia. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Konuma:SGH Foundation: Research Funding; The Japanese Society of Hematology: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Ito:Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

Ring1A and Ring1B inhibit expression of Glis2 to maintain murine MOZ-TIF2 AML stem cells

AbstractEradication of chemotherapy-resistant leukemia stem cells is expected to improve treatment outcomes in patients with acute myelogenous leukemia (AML). In a mouse model of AML expressing the MOZ-TIF2 fusion, we found that Ring1A and Ring1B, components of Polycomb repressive complex 1, play crucial roles in maintaining AML stem cells. Deletion of Ring1A and Ring1B (Ring1A/B) from MOZ-TIF2 AML cells diminished self-renewal capacity and induced the expression of numerous genes, including Glis2. Overexpression of Glis2 caused MOZ-TIF2 AML cells to differentiate into mature cells, whereas Glis2 knockdown in Ring1A/B-deficient MOZ-TIF2 cells inhibited differentiation. Thus, Ring1A/B regulate and maintain AML stem cells in part by repressing Glis2 expression, which promotes their differentiation. These findings provide new insights into the mechanism of AML stem cell homeostasis and reveal novel targets for cancer stem cell therapy.

Extracellular Vesicle microRNAs from Acute Myeloid Leukemia Are Involved in the Regulation of Adherence Junction in Bone Marrow Microenvironment.

Background. It has been shown that bone marrow (BM) microenvironment and physiological hematopoiesis were disturbed during development of acute myelogenous leukemia (AML). However, little is known the molecular mechanism involved in this process. We have recently demonstrated that high number of extracellular vesicles (EVs) including exosomes exist in BM interstitial fluid. Myeloid neoplasm derived EVs carry miRNAs and transmitted into mesenchymal stem cells (MSCs) (Highlights of ASH 2015, GSE64029). A subset of extracellular vesicular miRNAs including miR-7977 was increased in BM cavity and regulate the expression level of mRNA stabilizer poly(rC) binding protein 1 (PCBP1) and modulated the function of hematopoietic supporting capacity of MSCs. On the other hand, a relatively large subset of extra vesicular miRNAs was actually decreased in BM cavity of AML as compared with that of healthy volunteer. However, the clinical significance of these decreased miRNAs is unresolved. In the present study, miRNA pathway analysis was conducted to elucidate the role of decreased miRNAs in BM environment of AML.Methods. To harvest EVs from 2 x 105primary AML CD34+ cells, normal BM CD34+ cells and leukemic cell lines including TF-1 and Kasumi-1, cells were cultured in serum-free StemPro®-34 medium with a cytokine cocktail on plates coated with fibronectin fragments. EV miRNA from the supernatant of CD34+ hematopoietic and leukemic cells were prepared. Microarray analysis of the miRNA profiles was done with the human miRNA Oligo chip (Human_miRNA_V20) and the 3D-Gene® miRNA labeling kit. For analysis of transcriptome in CD34+/CD38- normal and AML cells, data sets (GSE24395) was downloaded as a matrix by GEOquery package (Bioconductor, R commander version 3.2.2). The data sets (GSE24395) and our data sets (GSE64029) were normalized by rma method using limma package before analysis. Clustering analysis were performed with amap package to find a subset of miRNA decreased. For miRNA pathway analysis, DIANA-mirPath version 3 was utilized.Results. Thirty EV miRNAs derived from AML were decreased as compared with those from normal CD34+ cells. Especially, miR-92a-3p, miR-125a-3p, miR-4448, miR-4484 and miR-4270 were remarkably reduced. The miRNA pathway analysis indicated that a KEGG pathway (hsa04520), adherens junction strongly correlated with these decreased miRNAs (P=0.00000025). The miRNAs including miR-4448, miR-4484 and miR-4270 could be associated with downstream molecules of adherens junction including beta-catenin, IGF-1R, WAVE, Vinculin, TGF-beta and Smad4. Importantly, we found that CD34+CD38- AML stem cells highly expressed JAM family molecules (JAM2 and JAM3) associated with adherens junction (GSE24395). These results suggested that these miRNAs regulate the pathway of adherens junction in BM microenvironment. In addition, adherens junction could be enhanced concomitant with reduction of miRNAs released from AML.Conclusion. EV miRNAs derived from AML are involved in the regulation of the pathway of adherens junction in BM microenvironment. Adherens junction is known to contribute to quiescence, apoptosis and drug resistance via HIPPO signaling pathway, EV miRNAs may have an important role on the development and drug resistance of AML. DisclosuresNo relevant conflicts of interest to declare.

Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells from Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo.

Current understanding suggests that malignant stem and progenitor cells must be reduced or eliminated for prolonged remissions in myeloid neoplasms such as acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). Multicolor flow cytometry has been widely used to distinguish stem and myeloid progenitor cells from other populations in normal and malignant bone marrow. In this study, we present a method for assessing drug sensitivity in MDS and AML patient hematopoietic stem and myeloid progenitor cell populations ex vivo using the investigational Nedd8‐activating enzyme inhibitor MLN4924 and standard‐of‐care agent cytarabine as examples. Utilizing a multicolor flow cytometry antibody panel for identification of hematopoietic stem cells, multipotent progenitors, common myeloid progenitors, granulocyte‐monocyte progenitors, and megakaryocyte‐erythroid progenitors present in mononuclear cell fractions isolated from bone marrow aspirates, we compare stem and progenitor cell counts after treatment for 24 hours with drug versus diluent. We demonstrate that MLN4924 exerts a cytotoxic effect on MDS and AML stem and progenitor cell populations, whereas cytarabine has more limited effects. Further application of this method for evaluating drug effects on these populations ex vivo and in vivo may inform rational design and selection of therapies in the clinical setting. Stem Cells Translational Medicine 2017;6:840–850

Rational Design of a Parthenolide-based Drug Regimen That Selectively Eradicates Acute Myelogenous Leukemia Stem Cells

Although multidrug approaches to cancer therapy are common, few strategies are based on rigorous scientific principles. Rather, drug combinations are largely dictated by empirical or clinical parameters. In the present study we developed a strategy for rational design of a regimen that selectively targets human acute myelogenous leukemia (AML) stem cells. As a starting point, we used parthenolide, an agent shown to target critical mechanisms of redox balance in primary AML cells. Next, using proteomic, genomic, and metabolomic methods, we determined that treatment with parthenolide leads to induction of compensatory mechanisms that include up-regulated NADPH production via the pentose phosphate pathway as well as activation of the Nrf2-mediated oxidative stress response pathway. Using this knowledge we identified 2-deoxyglucose and temsirolimus as agents that can be added to a parthenolide regimen as a means to inhibit such compensatory events and thereby further enhance eradication of AML cells. We demonstrate that the parthenolide, 2-deoxyglucose, temsirolimus (termed PDT) regimen is a potent means of targeting AML stem cells but has little to no effect on normal stem cells. Taken together our findings illustrate a comprehensive approach to designing combination anticancer drug regimens.

15-Methylene-Eburnamonine Kills Leukemic Stem Cells and Reduces Engraftment in a Humanized Bone Marrow Xenograft Mouse Model of Leukemia.

Recent studies suggest that leukemia stem cells (LSCs) play a critical role in the initiation, propagation, and relapse of leukemia. Herein we show that (-)-15-methylene-eburnamonine, a derivative of the alkaloid (-)-eburnamonine, is cytotoxic against acute and chronic lymphocytic leukemias (ALL and CLL) and acute myelogenous leukemia (AML). The agent also decreases primary LSC frequency in vitro. The cytotoxic effects appear to be mediated via the oxidative stress pathways. Furthermore, we show that the compound kills AML, ALL, and CLL stem cells. By the use of a novel humanized bone marrow murine model of leukemia (huBM/NSG), it was found to decrease progenitor cell engraftment.

Characterization and Targeting of Myelodysplastic Syndrome Stem Cells

Abstract Myelodysplastic syndrome (MDS) is a malignant disorder of myeloid hematopoietic cells, resulting in ineffective hematopoiesis leading to bone marrow dysfunction and failure, and frequently evolves to acute myelogenous leukemia (AML). While chemotherapy can on occasion induce transient remissions, the disease is typically progressive, with the vast majority dying of MDS complications or AML. While MDS is thought to arise from a malignant stem cell population, the properties of such cells are not well understood. Thus, we performed studies to better characterize candidate MDS stem cells and allow insights into the development of novel stem cell directed targeted therapies. One line of investigation examined the cytokine receptor CD123, which we have previously shown to be a marker of AML stem cells. Analysis of primary human specimens demonstrates that CD123 is not expressed in the normal stem cell population (phenotypically identified as CD34+/CD38-) or in stem cells(CD34+/CD38-) from low risk (<5% blasts and/or ≤ intermediate risk IPSS-R) MDS patients, but is readily detected in high-risk patients (>5% blasts and/or ≥ high risk IPSS-R) as shown in figure 1. These data suggest that up-regulation of CD123 may be a hallmark of pathogenesis as MDS progresses toward AML. To better understand the properties of CD123+ vs. CD123- stem cells, we performed global expression studies (RNA-seq) on sorted primary cell populations derived from high-risk MDS patients. The data indicate strong up-regulation of protein synthesis machinery, which indicates a major change in underlying cellular physiology and metabolism. Increased protein synthesis in CD123+ MDS stem cells was verified using the fluorescent protein substrate OP-puro, which identifies newly synthesized polypeptides. We found a ~13 fold difference in OP-puro staining of the CD123+ MDS stem cells versus CD123- cells. Based on these findings, we pursued two related lines of investigation to develop targeted therapies against MDS stem cells. First, we tested the protein synthesis inhibitors anisomycin and homoharringtonine for their ability to target the candidate MDS stem cell population; both agents demonstrated highly selective eradication of CD123+ stem cells, with a significant differential toxicity observed in multiple samples (up to an 80-fold difference in viability after treatment) as shown in figure 2. Second, because protein synthesis inhibition is known to rapidly down-regulate Bcl-2, we tested a selective Bcl-2 inhibitor. This agent also selectively eradicated CD123+ MDS stem cells in 4 of 10 patient samples tested. Finally, analysis of protein synthesis inhibitors or a Bcl-2 inhibitor in combination with the hypomethylating agent 5-azacytidine demonstrated potent efficacy in targeting the CD123+ stem cell population with greater then additive toxicity when compared to single agent treatment. We found significant differential sensitivity in the CD123+ population to the Bcl-2 inhibitor + 5-azacytidine in 9 out of 10 in samples tested and found significant toxicity to the combination of anisomycin or homoharringtonine with 5-azacytidine in 4 out of 4 samples. Taken together, these data show that we have identified two novel pharmacological approaches that may effectively target the MDS stem cell population. Further, these agents may function well in conjunction with commonly used agents used in the treatment of MDS. Disclosures Pollyea: GlycoMimetics: Other: Member of data safety monitoring board; Pfizer: Consultancy; Ariad: Consultancy; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34(+) cells, especially the LSC-enriched CD34(+)CD38(-) subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34(+) cells but not those from normal CD34(+) cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide-down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.

HSP70 Inhibitor, YK5, Synergizes with Chemotherapeutic Agents and Prevents Chemoresistance in Acute Myelogenous Leukemia (AML).

Abstract 2476Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance and poor prognosis. YK5, a novel inhibitor of tumor-HSP70, has been shown to induce potent cell death in AML blast, progenitor, and stem cell populations with minimal effects in normal hematopoietic cells. Due to the role of HSP70 in drug resistance, we examined the effect of combining YK5 with other chemotherapeutic agents, including arsenic trioxide, cytarabine, suberoylanilide hydroxamic acid (SAHA) and PU-H71, a novel tumor-specific HSP90 inhibitor.We tested the ability of YK5 to synergize with either AsO3, AraC, SAHA, or PU-H71 in primary AML samples. Using multiparameter flow cytometry to measure viability after 48 hours of treatment, we found that combining 1μM YK5 with either 500nM AsO3 or PU-H71 resulted in a significant increase in cell death when compared to either agent alone (n=9, mean viability: 51.8, 67.2, and 13.4% for AsO3, YK5, and AsO3/YK5, respectively, P = 0.0018; mean viability: 57.1 and 20.8% for PU-H71 and PU-H71/YK5, respectively, P = 0.0029). A synergistic relationship between YK5 and both AsO3 and PU-H71 was found in all nine primary samples (combination indexes 0.29 – 0.76 with YK5/AsO3, 0.33 – 0.83 with YK5/PU-H71). In contrast, the combination of YK5 with either AsO3 or PU-H71 in CD34+ cord blood mononuclear cells did not result in a significant increase in cell death when compared to either agent alone (mean viability: 42.4, 72.4, and 37.2% for AsO3, YK5, and AsO3/YK5, respectively; mean viability: 61.1 and 51.1% for PU-H71 and PU-H71/YK5, respectively). YK5 in combination with either AraC or SAHA, however, did not result in a significant increase in cell death when compared to either drug alone, with an additive effect being demonstrated with a 1:1 YK5 to AraC/SAHA drug ratio (Mean CI = 0.9918).To determine the mechanism of the observed synergistic activity, intracellular HSP70 and active caspase-3, a client of HSP70, were measured using flow cytometry. Both AsO3 and PU-H71 significantly increased intracellular HSP70 and caspase-3 (Mean fold change = 18.3, 21.0 of HSP70 and 9.9, 8.3 of Caspase-3 for AsO3 and PU-H71 treatment, respectively), while treatment with AraC or SAHA resulted in no change in HSP70 levels. Furthermore, quantitative PCR revealed that treatment with either AsO3 or PU-H71 strongly upregulated HSPA1A and HSPA6, the main stress-inducible isoforms of HSP70 (Mean fold change = 15.9, 14.1 of HSPA1A, and 20.8, 23.4 of HSPA6 for AsO3 and PU-H71 treatment, respectively). AraC and SAHA had no significant upregulation of these genes. We have previously shown that increased levels of HSPA1A correlate with sensitivity to HSP70 inhibition via YK5.To further explore the mechanism of this observed synergy, flow cytometry was used to measure the levels of reactive oxygen species (ROS). Treatment with AsO3, PU-H71, AraC, or SAHA resulted in a significant increase in ROS (Mean fold change = 2.75, 1.92, 2.89, 1.67, respectively). Quantitative PCR also confirmed the activation of the oxidative stress response by the upregulation of heme oxygenase 1 (HMOX1) by treatment with these drugs (Mean fold change = 10.9, 8.7, 11.2, 7.7, respectively). YK5, however, did not induce ROS or upregulate HMOX1. Interestingly, pretreatment with NAC in primary AML samples (n=4) resulted in no protection from YK5 synergistic effect when combined with either AsO3 or PU-H71. These results suggest that YK5 synergizes with AsO3 and PU-H71 due to the increase in intracellular HSP70 caused by these drugs. This synergy is most likely due to the activation of the heat shock response and independent of the production of ROS due to drug treatment.In summary, we have found that the novel tumor-HSP70 inhibitor YK5 can synergize with AsO3 and PU-H71 in primary human AML, and that the basis of this synergism is due to the increase in intracellular HSP70 caused by these chemotherapeutic agents. HSP70 inhibition represents a novel approach in AML treatment and can be particularly significant to drug-resistant patients when combined with other chemotherapy. Disclosures:Roboz:Astex Pharmaceuticals: Research Funding.

The First Demonstration of Sequential Acquisition of Class I and Class II Gene Mutations in Formation of Human Acute Myeloid Leukemia Stem Cells

Abstract 859Hematopoietic stem cells (HSCs) should be the main target for accumulation of mutational events, which eventually leads to formation of leukemic stem cells. These leukemogenic mutations have been classified at least into class I (providing the proliferative and survival advantage) and class II (impairing the differentiation activity) gene abnormalities. It has been proposed that acquisition of both class I and class II mutation are essential for the development of leukemia. Although several experimental animal studies suggest this model, there is no direct evidence that class I and class II mutations collaborate to contribute to development of human leukemias. Here we demonstrate that the acquisition of 8;21 translocation, which encodes the AML1-ETO (a class II chimeric fusion gene), and of mutational c-Kit (a class I mutation) is sequentially occurred in human acute myelogenous leukemia (AML). It has been shown that in t(8;21) AML patients treated with chemotherapy, a small amount of AML1-ETO mRNA was never disappeared even in patients maintaining remission for more than 10 years. We have demonstrated that this AML1-ETO mRNA in “cured” patients is derived from t(8;21)+ HSCs that consisted only a few percent of HSCs in remission (Miyamoto et al., PNAS 2000; 97: 7521–7526). The t(8;21)+ HSCs possessed normal differentiation at least into myeloerythroid cells and B cells. These data strongly suggest that acquisition of the AML1-ETO fusion is not sufficient for development of t(8;21) AML, and that t(8;21)+ HSCs are preleukemic HSCs.We hypothesized that acquisition of additional class I mutation might transform the AML1-ETO+ preleukemic HSCs into AML stem cells. We therefore searched for class I mutations in t(8;21) AML samples, and found that in 13 out of 33 t(8;21) AML patients, AML cells have c-Kit mutations (but not other class I such as FLT3-ITD and N-Ras mutations) at diagnosis. We then tested whether the AML1-ETO+ preleukemic HSCs in remission marrow have the c-Kit mutation. Six out of these 13 t(8;21) AML patients with c-Kit mutation maintaining long-term remission were enrolled in this study. To confirm the coexistence of AML1-ETO and c-Kit mutation in single leukemic stem cells, CD34+CD38− AML cells were purified from the bone marrow of patients at diagnosis, and tested for the presence of AML1-ETO and c-Kit mutation by single cell PCR. In all of 910 single CD34+CD38− AML cells, both AML1-ETO and c-Kit mutations were detected. Then, CD34+CD38− HSCs in remission were tested for the presence of AML1-ETO and c-Kit mutation. In 1728 single CD34+CD38− HSCs of remission marrow, 0.9% (16 cells) of these cells expressed AML1-ETO. Surprisingly, none of these AML1-ETO+ preleukemic HSCs possessed c-Kit mutation, indicating that AML1-ETO+ clones in long-term remission are independent from the original t(8;21) AML clones in terms of the presence of c-Kit mutation. We then performed colony-forming assays to evaluate the differentiation potential of these AML1-ETO+ preleukemic HSCs. HSCs of remission marrow-derived colonies were picked up, and tested for the presence of AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, 1.2% (89 colonies) of these colonies were positive for AML1-ETO, and all of these colonies were negative for c-Kit mutation. These data collectively suggest that the acquisition of c-Kit mutation is the second step for formation of t(8;21) AML stem cells: Normal HSCs acquire t(8;21) and express resultant AML1-ETO (Class II) but it is not sufficient for full transformation into AML stem cells. These preleukemic HSCs possess normal differentiation activity, but additional c-Kit mutation (Class I) might be critical in transforming into AML stem cells. This is the first clear-cut evidence that HSCs transform into AML stem cells by stepwise acquisition of Class I and Class II mutations. Disclosures:No relevant conflicts of interest to declare.

Guaianolide Sesquiterpene Lactones, a Source To Discover Agents That Selectively Inhibit Acute Myelogenous Leukemia Stem and Progenitor Cells

Small molecules that can selectively target cancer stem cells (CSCs) remain rare currently and exhibit no common structural features. Here we report a series of guaianolide sesquiterpene lactones (GSLs) and their derivatives that can selectively eradicate acute myelogenous leukemia (AML) stem or progenitor cells. Natural GSL compounds arglabin, an anticancer clinical drug, and micheliolide (MCL), are able to reduce the proportion of AML stem cells (CD34⁺CD38⁻) in primary AML cells. Targeting of AML stem cells is further confirmed by a sharp reduction of colony-forming units of primary AML cells upon MCL treatment. Moreover, DMAMCL, the dimethylamino Michael adduct of MCL, slowly releases MCL in plasma and in vivo and demonstrates remarkable therapeutic efficacy in the nonobese diabetic/severe combined immunodeficiency AML models. These findings indicate that GSL is an ample source for chemical agents against AML stem or progenitor cells and that GSL is potentially highly useful to explore anti-CSC approaches.

FLT3-ITD+ AML Blast, Progenitor and Stem Cell Populations Demonstrate Higher Sensitivity to the Hsp90 Inhibitor PU-H71,

Abstract 3500Hsp90, one of the best-characterized molecular chaperones, plays indispensable roles in folding and assembly, intracellular transport, stabilization, and degradation of proteins, and therefore, facilitating cell signaling. Hsp90 is also involved in tumorigenesis by stabilizing oncogenic client proteins. Thus, Hsp90 inhibition has been considered a promising therapeutic strategy for different types of cancer including leukemia. PU-H71 is a novel HSP90 inhibitor with high specificity for oncogenic Hsp90. We investigated the effect of PU-H71 in acute myelogenous leukemia (AML), in particularly, AML stem cells (AML-SCs) that are known to give rise to AML blasts, are refractory to conventional therapies, and thus likely to account for AML relapses.The effect of PU-H71 was evaluated using a panel of 12 leukemia cell lines. Among the 12 leukemia cell lines tested, MOLM-13 and MV4-11 cells were the most sensitive (LD50s 253 nM and 120 nM respectively). Both MV4-11 and MOLM-13 carry FLT3-ITD mutation (occurring in ∼40% AML cases) and MLL translocations (occurring in ∼20% AML cases). Both MLL and FLT3 have been reported as client proteins of Hsp90. However, other leukemia cell lines with MLL rearrangements such as THP-1 and a MLL-ENL cell line derived from MLL-ENL transformed human CD34+ cord blood cells exhibited resistance to PU-H71 treatment (LD50 > 2 μM). The data suggested that FLT3-ITD+ AML samples may display higher sensitivity to Hsp90 inhibition.To confirm the higher sensitivity of FLT3-ITD+ AML cells to Hsp90 targeted therapy, 15 primary AML patient samples (8 FLT3-ITD mutants and 7 wild type FLT3) were treated with increasing concentrations of PU-H71. Cell viability on different cell populations was evaluated using multiparameter flow cytometry at 48 hours after treatment with PU-H71. The average LD50 of PU-H71 in FLT3-ITD+ AML cells was 492 nM (95% CI, 127.636 – 856.364). In contrast, the average LD50 in AML samples with wild type FLT3 was 2.795 μM (95% CI, 1.058 – 4.532). The near 6-fold difference between LD50s for PU-H71 was significant (p=0.0068). Importantly PU-H71 also killed FLT3-ITD+ AML stem and progenitor cells more effectively. Furthermore, PU-H71 treatment decreased the ability to form colonies in FLT3-ITD+ AML specimens more effectively than FLT3 WT AMLs (97.6% and 79.3% decrease relative to control respectively; N=3; P=0.0236). Importantly, PU-H71 had minor toxicity to normal blood mononuclear cells and normal cord blood hematopoietic stem cells.FLT3-ITD+ cell lines and primary AML cells treated with 0.5 μM PU-H71 showed a substantial decrease of phosphorylated forms of Erk1/2, JNK, AKT, p70RSK, NF-κB(p65) and Stat5, which was observed within 4 hours post PU-H71 treatment, whereas the phosphorylation levels of MAPK p38 remained unaffected. Immunoblotting and phosphoflow assays corroborated the inhibition of AKT and Stat5 signaling by PU-H71 in stem and progenitor populations.In summary, FLT3-ITD+ AML cells display a stronger response to PU-H71, suggesting that the FLT3-ITD mutation results in a higher dependency on Hsp90 to stabilize the aberrant signaling elicited by constitutive activation of FLT3. Our data suggests that PU-H71 represents a novel therapy for FLT3-ITD+ AML patients with the potential to ablate AML-SCs. Disclosures:Roboz:EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,

Abstract 3584The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo. Disclosures:No relevant conflicts of interest to declare.

Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice

Human leukemic stem cells, like other cancer stem cells, are hypothesized to be rare, capable of incomplete differentiation, and restricted to a phenotype associated with early hematopoietic progenitors or stem cells. However, recent work in other types of tumors has challenged the cancer stem cell model. Using a robust model of xenotransplantation based on NOD/SCID/IL2Rγc-deficient mice, we confirmed that human leukemic stem cells, functionally defined by us as SCID leukemia-initiating cells (SL-ICs), are rare in acute myelogenous leukemia (AML). In contrast to previous results, SL-ICs were found among cells expressing lineage markers (i.e., among Lin+ cells), CD38, or CD45RA, all markers associated with normal committed progenitors. Remarkably, each engrafting fraction consistently recapitulated the original phenotypic diversity of the primary AML specimen and contained self-renewing leukemic stem cells, as demonstrated by secondary transplants. While SL-ICs were enriched in the Lin-CD38- fraction compared with the other fractions analyzed, SL-ICs in this fraction represented only one-third of all SL-ICs present in the unfractionated specimen. These results indicate that human AML stem cells are rare and enriched but not restricted to the phenotype associated with normal primitive hematopoietic cells. These results suggest a plasticity of the cancer stem cell phenotype that we believe has not been previously described.

Abstract 4334: Tracking of leukemic stem cells from frozen and fresh samples in a mouse model of human AML using bioluminescent imaging

Abstract The aim of this study was to develop and use in vivo bioluminescent imaging to track luciferase-labeled acute myelogenous leukemia (AML) stem cells in immunedeficient mice using AML blood samples collected from patients and subsequently frozen. The ability to utilize frozen pools as viable reservoirs for AML stem cells will allow the freezing back of samples from the clinic for later use as well as the use of previously frozen samples for engraftment and therapeutic studies. Using thawed, isolated cells, we assessed AML stem cell engraftment and quantitatively compared it to fresh AML stem cell engraftment in a xenogeneic transplant model using in vivo bioluminescent imaging. Frozen AML stem cell engraftment was also compared to both fresh and frozen normal human hematopoietic stem cell (HSC) engraftment. Human hematopoietic stem cells (CD34+CD38-) were isolated from both normal frozen cord blood and from peripheral blood of AML patients using modified Miltenyi Biotec (Auburn, CA) magnetic bead isolation kits. Isolated cells were transduced with a lentiviral vector (HIV-luc) pseudotyped with the VSV envelope able to express the firefly luciferase (fluc) gene in human HSC. Using a Caliper Life Sciences/Xenogen IVIS 200 optical imaging system, cells were assayed for fluc expression 48 hours after transduction. Transduced HSC and AML cells (6 × 104 each) were transplanted intravenously into sublethally irradiated adult NOD/SCID/IL2Rγnull mice and mice were imaged serially from day 1 up to day 160 post transplantation. Observed bioluminescent signal and patterns from thawed AML stem cell engraftment are similar to our results obtained using fresh AML stem cells in mice. Luminescent signal from peripheral blood and spleen engraftment of frozen AML stem cells was detectable within one day post-transplantation. Both fresh and frozen AML stem cell populations exhibited diffuse whole body signal, indicating luciferase-labeled stem cells in the peripheral blood, similar to a hallmark of the disease in humans. The presence of blast cells consistent with AML disease was observed by blood smears taken from mice engrafted with frozen AML stem cells. To our knowledge, we have demonstrated for the first time that frozen AML stem cells can be efficiently labeled with a luciferase enzyme for dynamic tracking in vivo. This work has resulted in a useful animal model that will enable us to elucidate the engraftment patterns of AML cells in living animals, learn the dynamics of the interplay of different stem cell populations, and provide information on the efficacy of new treatments in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4334.

CD47 Is An Independent Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells

A growing body of evidence has added to our fundamental knowledge by demonstrating that human acute myelogenous leukemia (AML) is organized as a cellular hierarchy initiated and maintained by rare self-renewing leukemia stem cells (LSC). One implication of this cancer stem cell model is that in order to eradicate the leukemia and cure the patient, therapies must target and eliminate the leukemia stem cells. For the development of such LSC-targeted therapies, it is necessary to identify molecules that are preferentially expressed in LSC compared to their normal counterparts and that are critical for their function. We report here the identification of increased expression of CD47 on human AML LSC compared to normal hematopoietic stem cells (HSC). Furthermore, we demonstrate that differential CD47 expression on Lin-CD34+CD38− cells can be utilized to prospectively separate normal (CD47lo) from leukemic (CD47hi) progenitors from the same patient sample. CD47 serves as the ligand for signal regulatory protein alpha (SIRP-alpha) on phagocytic cells, which in turn delivers an inhibitory signal for phagocytosis. We hypothesize that increased CD47 expression on human AML contributes to pathogenesis by inhibiting phagocytosis of leukemia cells through the interaction of CD47 with SIRP-alpha. One prediction from this hypothesis is that increased expression of CD47 will be associated with a worse clinical outcome. We analyzed microarray gene expression data and associated clinical outcomes for a cohort of 123 AML patients with normal cytogenetics. Patients were stratified into low CD47 and high CD47 expression groups, based on expression relative to the median. Increased CD47 expression was found to be associated with worse event-free and overall survival. Patients in the low CD47 expression group had a median event-free survival of 18.2 months compared to 9.9 months in the high CD47 expression group, corresponding to a hazard ratio of 1.61 (p=0.04). For overall survival, patients in the low CD47 expression group had a median of 24.3 months compared to 14.1 months in the high CD47 expression group, corresponding to a hazard ratio of 1.70 (p=0.02). In multivariable analysis considering age, FLT3-ITD status, and CD47 expression as a continuous variable, increased CD47 expression remained associated with worse event-free survival with a hazard ratio of 1.33 (p=0.03) and overall survival with a hazard ratio of 1.31 (p=0.05). A second prediction from our hypothesis is that disruption of the CD47-SIRP-alpha interaction with a monoclonal antibody directed against CD47 will enable the phagocytosis of AML LSC. We obtained mouse anti-human CD47 monoclonal antibodies that are able to disrupt the CD47-SIRP-alpha interaction and tested their ability to enable phagocytosis of AML LSC both in vitro and in vivo. Unlike isotype control or an anti-CD45 antibody, anti-CD47 antibodies enabled phagocytosis of AML LSC by both mouse and human macrophages in vitro. Furthermore, unlike isotype control or anti-CD45 antibody, coating of AML LSC with anti-CD47 antibody completely eliminated in vivo engraftment upon xenotransplantation into NOG mice. Finally, treatment of NOG mice already engrafted with human AML LSC with daily intraperitoneal injections of anti-CD47 antibody for two weeks, resulted in complete elimination of circulating leukemia and a statistically significant reduction in bone marrow leukemia compared to control IgG. In summary, increased CD47 expression is an independent poor prognostic factor that can be targeted on AML stem cells with a monoclonal antibody capable of stimulating phagocytosis and in vivo elimination of LSC.

Protein Expression in Acute Myelogenous Leukemia Stem Cells Compared to Bulk AML Cells Using Reverse Phase Proteins Arrays (RPPA)

The majority of studies of Acute Myelogenous Leukemia (AML) cell biology that are performed on patient derived samples utilize the more readily available bulk mononuclear leukemia cells (BULK) instead of rare stem cells (SC), defined as CD34+CD38− for this analysis. However, resistance of SC to therapy is thought to be more clinically relevant with SC leading to relapse and mortality. If the cellular machinery within the SC compartment is significantly different from that of the Bulk cells then these studies could generate results of uncertain veracity arising from studying the wrong cell. The ability to measure gene and protein expression in SC would answer this question but to date conventional methodology has required too many cells to permit the analysis of protein expression and activation patterns within SC. Using RPPA methodology on blood (PB) or marrow (BM) derived AML cells we have previously demonstrated that recurrent protein expression signatures with prognostic implications exist in AML. Our RPPA methodology requires only 333 cells per slide making the analysis of protein expression in stem cells possible for the first time. We therefore set out to determine whether protein expression in AML SC differed from BULK cells. Leukemia enriched (CD3/CD19 depleted) mononuclear cell fractions from 103 AML samples from 87 patients (including 13 paired PB and BM and 5 paired diagnosis and relapse samples) were separated in to CD34− and CD34+ fractions, followed by separation of the CD34+ fraction into CD34+CD38+ and CD34+CD38− fractions using magnetic antibody selection. Among the 103 samples 8 had only CD34+/− samples, 31 had BULK/CD34+/CD34−, 18 had BULK/CD34+/CD34−/CD34+CD38+, and 45 had all 5 samples. Whole cell lysates and total RNA preparations were made from these fractions. RPPA were printed using 5 serial 1:2 dilutions, printed in replicate. Slides were stained with 127 antibodies (predominantly apoptosis, cell cycle and STP regulating proteins) detecting 87 total, 37 phopho and 3 caspase cleavage sites. Spot intensities were quantified using MicroVigene software. Data was analyzed using R, with loading control and topographical background normalization being utilized. Analysis was performed using a mixed-effects model and β-uniform model. We compared expression between the BULK, and SC fractions, between CD34+ and CD34− fractions and between CD34+CD38+ and C34+CD38−(SC) fractions. In univariate analysis, protein expression in SC was higher for Caspase8, ZNF342, α-Catenin, and TRIM24, and lower for, Bax, pP70S6K and pS6RP across the entire population when compared to protein from BULK leukemic cells. The fold differences were subtle ranging from 15 to 27% increases and 13 to 38% decreases in expression in the SC relative to the BULK cells. There was no statistically significant evidence for global differences in individual protein expression for the CD34+/CD34− or CD34+CD38+/CD34+CD38− comparisons. In multivariate analysis, when patterns of expression of all 127 proteins were compared, the majority of samples from individual patients showed close correlation regardless of whether BULK/SC, CD34+/CD34− or CD34+CD38+/CD34+CD38− were compared. Gene expression profiling of these same samples using mRNA amplification techniques is underway. Comparison of differences in GEP and RPPA will be performed at a later date. In summary, the RPPA technique has enabled the analysis of expression of numerous proteins and their activation state in AML stem cells for the first time. We observed that protein expression in SC generally is similar to that of BULK cells. This increases confidence in the ability to generalize the results obtained in studies of bulk AML cells. A few proteins did show consistent differences in expression between stem and bulk cells. Novel agents targeting these should be investigated for the ability to individually or synergistically target AML SC.

Nuclear Factor-κB Modulation in Patients Undergoing Induction Chemotherapy for Acute Myelogenous Leukemia

Nuclear factor-kappaB (NF-kappaB) is constitutively expressed in many acute myelogenous leukemia (AML) cells and AML stem cells. Ex vivo treatment of AML cells with inhibitors of NF-kappaB results in diminished AML cell survival and enhances the cytotoxic effects of chemotherapeutic agents. The purpose of this study was to determine if standard anti-inflammatory agents modulate AML cell nuclear NF-kappaB when administered in conjunction with induction chemotherapy. Patients with newly diagnosed AML were treated with dexamethasone, choline magnesium trisalicylate, or both for 24 hours prior to and 24 hours following initiation of standard induction chemotherapy. AML cell nuclear NF-kappaB was measured at baseline, 24, and 48 hours. Choline magnesium trisalicylate +/- dexamethasone decreased nuclear NF-kappaB, whereas dexamethasone alone was associated with an increase in nuclear NF-kappaB in AML cells. These results show the feasibility of NF-kappaB modulation in conjunction with induction chemotherapy for patients with AML using inexpensive readily available medications. A follow-up study to determine the effects of NF-kappaB modulation on clinical end points is warranted.

Derepression in the Desert: The Third Workshop on Clinical Translation of Epigenetics in Cancer Therapeutics

In the past decade, the scientific and medical community has witnessed dramatic progress in epigenetics and has begun to understand some of the epigenetic mechanisms that contribute to human disease, particularly cancer. In January 2007, the third biannual workshop on Clinical Translation of

Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2r gamma(null) mice carrying a complete null mutation of the cytokine gamma c upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle-dependent cytotoxic therapy. Global transcriptional profiling identified LS cell-specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.