Phenotype Of Leukemic Stem Cells Research Articles

Leukemic Stem Cells: From Leukemic Niche Biology to Treatment Opportunities.

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults. While complete remission can be obtained with intensive chemotherapy in young and fit patients, relapse is frequent and prognosis remains poor. Leukemic cells are thought to arise from a pool of leukemic stem cells (LSCs) which sit at the top of the hierarchy. Since their discovery, more than 30 years ago, LSCs have been a topic of intense research and their identification paved the way for cancer stem cell research. LSCs are defined by their ability to self-renew, to engraft into recipient mice and to give rise to leukemia. Compared to healthy hematopoietic stem cells (HSCs), LSCs display specific mutations, epigenetic modifications, and a specific metabolic profile. LSCs are usually considered resistant to chemotherapy and are therefore the drivers of relapse. Similar to their HSC counterpart, LSCs reside in a highly specialized microenvironment referred to as the “niche”. Bidirectional interactions between leukemic cells and the microenvironment favor leukemic progression at the expense of healthy hematopoiesis. Within the niche, LSCs are thought to be protected from genotoxic insults. Improvement in our understanding of LSC gene expression profile and phenotype has led to the development of prognosis signatures and the identification of potential therapeutic targets. In this review, we will discuss LSC biology in the context of their specific microenvironment and how a better understanding of LSC niche biology could pave the way for new therapies that target AML.

CD96 as a Leukemic Stem Cell Marker in Acute Myeloid Leukemia Patients: Relation to Remission Induction Chemotherapy

Abstract Background: Leukemic stem cells (LSCs) play a crucial role in chemotherapy resistance in acute myeloid leukemia (AML), the classical LSCs phenotype is CD34+/CD38-. Successful eradication of LSCs requires combination of different strategies including targeting LSC specific surface molecules. Aim of Study: To investigate the role of CD96, as a stem cell marker, in AML and its relation to induction therapy outcome. Material and Methods: The current study was conducted on 50 patients with de-novo AML, in addition to 20 controls. Using multicolor flow cytometry, we analyzed the expression of CD96, a potential marker for LSCs among CD34+/CD38- cell population in AML patients at initial diagnosis and correlate it to the response of induction therapy. Results: Percentage of CD96 among CD34+/CD38- was significantly higher in AML patients compared to control group (p < 0.001). However, no statistical significant difference was observed between complete remission (CR) and non-remission (NCR) groups. Also the correlation studies of LSC markers with laboratory findings revealed no significant correlation. Conclusions: CD96 expression among CD34+/CD38- cells may be used as a useful marker for detection of LSCs in AML patients. However its expression is not correlated to post induction therapy outcome.

OMIP 072: A 15-color panel for immunophenotypic identification, quantification, and characterization of leukemic stem cells in children with acute myeloid leukemia.

This panel was designed to identify, quantify and phenotypically characterize putative leukemic stem cells (LSCs) in bone marrow (BM) samples from individual pediatric patients diagnosed with acute myeloid leukemia (AML). Based on an aberrant expression on immunophenotypically defined hematopoietic stem cells (HSCs), several antigens have been proposed as LSC markers in AML research, using healthy adult BM samples as reference material. Generally, these antigens have been evaluated individually in smaller panels (e.g. 8-color panels). This necessitates several tubes to characterize the LSC phenotype and compromises the ability to evaluate LSC heterogeneity. The present 15-color OMIP incorporates nine suggested LSC markers to comprehensively capture LSC immunophenotypes and to explore heterogenic marker-patterns within LSC populations in a single tube. Importantly, this single tube approach requires less input material, which is essential when sampling BM aspirates from pediatric patients where sample volumes often are sparse. As knowledge on normal expression levels of the included LSC markers in HSCs from hematologically healthy children are a prerequisite for labelling a phenotype as abnormal, we have evaluated the applicability of the panel on cryopreserved mononuclear cells (MNCs) isolated from BM samples from pediatric patients without hematological disorders as well as pediatric AML patients. The panel is optimized for cryopreserved BM MNCs, but could in principle, be utilized for LSC detection in any biological material containing human hematopoietic cells.

Delineation of target expression profiles in CD34+/CD38− and CD34+/CD38+ stem and progenitor cells in AML and CML

AbstractIn an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38− and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38− and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38− LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably expressed “aberrant” membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication–mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38− LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.

Leukemic stem cell phenotype is associated with mutational profile in acute myeloid leukemia.

Background/AimsUnderstanding leukemic stem cell (LSC) is important for acute myeloid leukemia (AML) treatment. However, association of LSC with patient prognosis and genetic information in AML patients is unclear.MethodsHere we investigated the associations between genetic information and the various LSC phenotypes, namely multipotent progenitor (MPP)-like, lymphoid primed multipotent progenitor (LMPP)-like and granulocyte-macrophage progenitors (GMP)-like LSC in 52 AML patients.ResultsIn secondary AML patients, MPP-like LSC was significantly higher than de novo AML (p = 0.0037). The proportion of MPP-like LSC was especially high in post-myeloproliferative neoplasm AML (p = 0.0485). There was no correlation between age and LSC phenotype. Mutations of KRAS and NRAS were observed in MPP-like LSC dominant patients, TP53 and ASXL1 mutations in LMPP-like LSC dominant patients, and CEBPA, DNMT3A and IDH1 mutations in GMP-like LSC dominant patients. Furthermore, KRAS mutation was significantly associated with MPP-like LSC expression (p = 0.0540), and TP53 mutation with LMPP-like LSC expression (p = 0.0276). When the patients were separated according to the combined risk including next generation sequencing data, the poorer the prognosis, the higher the LMPP-like LSC expression (p = 0.0052). This suggests that the dominant phenotype of LSC is one of the important factors in predicting the prognosis and treatment of AML.ConclusionsLSC phenotype in AML is closely associated with the recurrent mutations which has prognostic implication. Further research to confirm the meaning of LSC phenotype in the context of genetic aberration is warranted.

Impact of Leukemia Stem Cells Phenotype Expression on Response to Induction Therapy in Acute Myeloid Leukemia Patients.

Laboratory data suggest that acute myeloid leukemia AML originates from a rare population of cells, termed Leukemic Stem Cells (LSCs) or leukemia-initiating cells, which are capable of self-renewal, proliferation and differentiation into malignant blasts. There is a universal agreement that LSCs lie within the CD34+ compartment of hemopoietic cells and most of leukemic stem cells express the interleukin-3 alpha chain receptor, CD123 and lack CD38. This study aimed to estimate the expression of LSC phenotype in AML patients and to correlate it with response to induction therapy. A cohort of 41 patients older than 15 years with newly diagnosed de novo AML were enrolled in this study. They were obtained from the National center of hematology in Baghdad and Baghdad teaching hospital between February and July 2013. The expression of CD34, CD38 and CD123 was assessed by multi-color flow cytometry. LSC positive (LSC+) samples must express CD34 and CD123 and lack the expression of CD38 in >1% of cells. French American British (FAB) classification system was used in this study. After four weeks of induction therapy; three groups were found: those who reached the Complete morphological Remission (CR), those who failed to reach CR and those who died before the assessment of morphological remission. The last two groups were merged for statistical purposes. After the course of induction therapy, 41.46% of patients had complete morphological remission while 58.54% of the studied patients failed to reach complete remission. The Complete Remission (CR) rate was higher (53.33%) in patients who were negative for LSC phenotype than patients who were positive for LSC phenotype (34.61%). LSCs were expressed in 63.41% of AML cases and were in approximate distribution in FAB M3 and non-M3 patients. The expression of LSC phenotype was associated with poor response to induction therapy in AML patients.

Leukemic Stem Cell Phenotype Is Associated with Mutational Profile in Acute Myeloid Leukemia

Background: Understanding leukemic stem cell (LSC) is important for acute myeloid leukemia (AML) treatment. As such, understanding the relationship between LSC and genetically defined sub-clones can, in turn, help to understand the heterogeneity of AML. However, to date, there are only a few reports specifically focusing on this topic. To this end, we conducted this study to (1) examine the phenotypic diversity of AML-LSC, (2) explore the association between AML-LSC phenotypes and gene mutations, and (3) investigate the prognostic implications of AML-LSCs. Methods: Mononuclear cells (MNCs) were isolated from the patient's bone marrow aspirates by ficoll gradient centrifugation and cryopreserved in serum-free medium. Stored cells were thawed to Iscove's Modified Dulbecco's Medium (IMDM) and washed with fluorescence-activated cell sorting (FACS) buffer [1% FBS, Dulbecco's Phosphate-Buffered Saline (DPBS)]. Cells were stained with following anti-human monoclonal antibodies: CD45-APC/cy7, CD34-APC, CD38-BV421, CD90-PE, CD123-PE/Cy7, CD45RA-PerCP/Cy5.5. Analyses were performed on a FACSCanto II (HTS) (BD Bioscience) and FlowJo V 10.0 (BD Bioscience) program. For sequencing, the DNA capture probes for 76 target genes were designed using the Agilent SureDesign web-based application. The target regions included protein coding exons with 10 bp intron flanking regions and hot spot regions of the 20 genes involved in recurrent translocations. DNA was extracted on a Chemagic 360 instrument (Perkin Elmer, Baesweiler, Germany). The genomic DNA was sheared using Covaris S220 focused‐ultrasonicator (Covaris, Woburn, MA). We used 50ng of total input genomic DNA. A library preparation was performed according to Agilent's SureSelectQXT Target Enrichment protocol. Paired-end 150-bp sequencing was using NextSeq 550 Dx platform (Illumina, San Diego, CA). Targeted sequencing raw data was obtained in FASTQ format. Results: In secondary AML patients, MPP-like LSC was significantly higher than de-novo AML (p=0.0037), and was higher in MPN-AML than in MDS-AML (p=0.0485). There was no correlation between age and LSC phenotype, though CD34+CD38- subpopulation was enriched in younger patients (&lt;65 yrs). Mutations of KRAS and NRAS were frequently observed in MPP-like LSC dominant patients (3/14 and 4/14), TP53 and ASXL1 mutations in LMPP-like LSC dominant patients (4/12 and 4/12) , and CEBPA, DNMT3A and IDH1 (6/12, 4/12, and 3/12) mutations in GMP-like LSC dominant patients. Furthermore, as shown in Figure, KRAS mutation was significantly associated with the percentage of MPP-like LSC phenotype (p=0.0540), and TP53 mutation with the percentage of LMPP-like LSC phenotype (p=0.0276). When the patients were separated according to the combined risk including next generation sequencing data, the poorer the prognosis, the higher the LMPP-like LSC expression (p=0.0052). The importance of our study lies in that we showed for a given AML patients there is a dominant LSC phenotype and LSCs are associated with clinical outcomes, supporting the significance of cancer stem cell model for human AML. First of all, based on detailed characterization of the surface immunophenotype of AML LSCs we found that AML show evidence of a hierarchical cellular organization. We also recognized that the composition of LSC phenotypes is associated with AML phenotypes. For example, secondary AML patients showed higher fraction of MPP-like LSCs compared to de novo AML patients. In this regard, the higher expression of MPP-like LSCs could explain the poor response to standard treatments traditionally associated with secondary AML. Furthermore, the higher expression of MPP-like LSCs in post-MPN AML compared to post-MDS AML could explain the dismal prognosis associated with post-MPN AML, despite the relative indolent clinical course in their chronic phase and the presence of druggable target. Conclusion: In conclusion, our findings provide better insights into the characteristics and clinical implications of LSC. Although in a small scale, we provide evidence that specific LSC phenotypes are associated with certain mutations thus should be in the AML risk stratification process. Figure Disclosures Yoon: Janssen: Consultancy; Kyowa Hako Kirin: Research Funding; Genentech, Inc.: Research Funding; Yuhan Pharma: Research Funding; MSD: Consultancy; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Ludwig Boltzmann Cluster Oncology (LBC ONC): first 10years and future perspectives.

SummaryIn 2008 the Ludwig Boltzmann Cluster Oncology (LBC ONC) was established on the basis of two previous Ludwig Boltzmann Institutes working in the field of hematology and cancer research. The general aim of the LBC ONC is to improve treatment of hematopoietic neoplasms by eradicating cancer-initiating and disease-propagating cells, also known as leukemic stem cells (LSC) in the context of leukemia. In a first phase, the LBC ONC characterized the phenotype and molecular aberration profiles of LSC in various malignancies. The LSC phenotypes were established in acute and chronic myeloid leukemia, in acute lymphoblastic leukemia and in chronic lymphocytic leukemia. In addition, the concept of preleukemic (premalignant) neoplastic stem cells (pre-L-NSC) was coined by the LBC ONC and was tested in myelodysplastic syndromes and myeloproliferative neoplasms. Phenotypic characterization of LSC provided a solid basis for their purification and for the characterization of specific target expression profiles. In a second phase, molecular markers and targets were validated. This second phase is ongoing and should result in the development of new diagnostics parameters and novel, more effective, LSC-eradicating, treatment strategies; however, many issues still remain to be solved, such as sub-clonal evolution, LSC niche interactions, immunologic control of LSC, and LSC resistance. In the forthcoming years, the LBC ONC will concentrate on developing LSC-eradicating strategies, with special focus on LSC resistance, precision medicine and translation of LSC-eradicating concepts into clinical application.

Evaluation of Cell Surface Markers and Targets in Leukemic Stem Cells (LSC) Reveals Distinct Expression Profiles, Unique Drug Effects, and Specific Checkpoint Regulation in AML LSC and CML LSC

In an attempt to identify novel cell surface markers and targets in leukemic stem cells (LSC) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples of patients with AML (n=257), CML (n=134), and controls (n=256: other BM neoplasms, n=116; normal/reactive BM, n=29; idiopathic cytopenia, n=31; lymphoma patients without BM involvement, n=80) for expression of cell surface markers and targets on CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells (PC) by multi-color flow cytometry. In addition, we examined mRNA expression profiles in highly purified CD34+/CD38− stem cells and CD34+/CD38+ PC by gene array- and qPCR analyses. Aberrant LSC expression profiles were identified in all patients examined. In patients with CML, CD34+/CD38− LSC expressed an almost invariable aberration profile defined as CD25+/CD26+/CD56+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably displayed aberrant surface antigens, including CD25 (55%), CD96 (35%), CD371 (CLL-1) (75%), and IL-1RAP (60%). With the exception of a subset of FLT3 ITD+ patients (45% of all FLT3-mutated cases), AML LSC did not exhibit CD26. All other markers and targets identified on AML and/or CML LSC, including CD9, CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, neural proliferation and differentiation control-1 antigen (NPDC1), and roundabout-4 (ROBO4), were also detectable on normal hematopoietic stem cells (HSC). However, several surface-targets, including CD33 and CD52, were expressed at higher levels on CD34+/CD38− LSC compared to normal HSC, and antibody-mediated targeting resulted in their selective depletion and in a significantly reduced engraftment of LSC in NSG mice. Since certain surface antigens, like CD47 (IAP), CD243 (MDR1), and CD274 (PD-L1) may contribute to intrinsic drug resistance of LSC, we also examined the expression of these antigens in AML and CML LSC. Regardless of the type of leukemia, LSC were found to express CD47 in all patients examined. MDR-1 was found to be expressed on LSC in 11/39 patients with AML (28%) and 2/22 patients with CML (9%). PD-L1 was not detectable on LSC in untreated BM samples. Exposure of LSC to interferon-gamma (IFN-G, 100 U/ml, 48 hours) resulted in expression of PD-L1 on LSC in all patients with Ph+ CML (control: 100%; IFN-G: 183±40%, p<0.05). Unexpectedly, however, IFN-G was found to induce PD-L1 expression in only 1 out of 11 AML patients tested. IFN-G showed no effects on expression of CD47 or MDR-1 in LSC. We next examined the potential mechanisms of IFN-G-mediated expression of PD-L1 on LSC and screened for drugs capable of counteracting the expression of this resistance-inducing checkpoint molecule. Of all targeted drugs examined (n=10) only the BRD4/MYC inhibitor JQ1 was identified as a regulator of IFN-G-induced PD-L1 expression on CML LSC. In particular, JQ1 (2.5 µM, 48 hours) was found to suppress IFN-G-induced (100 U/ml, 48 hours) upregulation of PD-L1 in primary CML LSC in all donors tested (IFN-G: 183±40% versus IFN-G+JQ1: 136±27% of control) suggesting that checkpoint expression in LSC is regulated by epigenetic mechanisms and MYC activity. Together, we have established cell surface marker- and target expression profiles in CD34+/CD38− AML LSC and CML LSC which should facilitate their enrichment and may support the development of LSC-eradicating treatment concepts. In addition, the unique expression profiles of LSC should provide a powerful basis for diagnostic LSC phenotyping in patients with CML and AML. DisclosuresHoermann:Novartis: Honoraria; Gilead: Research Funding; Ariad: Honoraria; Amgen: Honoraria. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Valent:Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria.

Abstract 4702: Combination of BET inhibitor INCB054329 and LSD1 inhibitor INCB059872 is synergistic for the treatment of AML in vitro and in vivo

Abstract Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Indeed, a screen for essential genes in the murine MLL-AF9 retroviral model of leukemia identified the epigenetic regulators BRD4 and LSD1 as potential therapeutic targets. Furthermore, independent studies have demonstrated that inhibition of BRD4 or LSD1 by small molecules induced myeloid differentiation and suppressed leukemic stem cell phenotype in AML models. Recently it has been demonstrated that combinations of inhibitors that target distinct epigenetic regulators can exhibit synergistic effects on target gene transcription and cancer cell growth. Therefore we investigated the potential combinatorial effects of a novel FAD-directed LSD1 inhibitor, INCB059872, together with the BET inhibitor, INCB054329. The effects of single agent LSD1 and BET inhibitors or their combination were assessed using human AML models in vitro and in vivo. INCB054329 as monotherapy inhibited cell proliferation and induced apoptosis in human AML cell lines, while INCB059872 induced cellular differentiation in these cell lines as determined by the induction of myeloid differentiation markers, CD86 and CD11b. The combination of INCB054329 and INCB059872 enhanced myeloid differentiation and apoptosis in human AML cell lines compared with the single agents. Interestingly, c-myc expression was down-regulated to greater extent with the combination of both compounds compared to either agent alone. Enhanced anti-tumor efficacy with favorable tolerability was observed in human AML xenograft models when both agents were administered simultaneously or sequentially, with INCB059872 dosing regimens preceding INCB054329 administration demonstrating the greatest efficacy. These ongoing studies demonstrate that concurrent inhibition of two distinct families of epigenetic regulators, BET and LSD1, is active in preclinical AML models, and provide a rationale for the clinical evaluation of this novel, epigenetic doublet therapy in AML. Citation Format: Xuesong Liu, Matthew Stubbs, Min Ye, Roberts Collins, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Yvonne Lo, Chunhong He, Liangxing Wu, Andrew Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Phillip Liu, Sang Hyun Lee. Combination of BET inhibitor INCB054329 and LSD1 inhibitor INCB059872 is synergistic for the treatment of AML in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4702.

Quantitative assessment of the CD26+ leukemic stem cell compartment in chronic myeloid leukemia: patient-subgroups, prognostic impact, and technical aspects

Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26− HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors.

The Novel Leukemia Stem Cell Marker GPR56 Discriminates Leukemic Subclones with Divergent Stem Cell Properties in Human Acute Myeloid Leukemia

Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal.We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection.To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a >50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker.Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones.In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment. DisclosuresNo relevant conflicts of interest to declare.

Identification of Existing Bioactive Compounds That Target Acute Myeloid Leukemia Stem Cells

Current therapies for acute myeloid leukemia (AML) are able to achieve complete remission in 80-85% of patients; however large portions of these cases relapse. The 5-year survival rate is estimated at 30%. Thus, the development of more efficient approaches to treat AML is absolutely needed. Similarly to normal hematopoiesis, leukemias are organized as cellular hierarchies supported by leukemic stem cells (LSCs). LSCs are relatively resistant to chemotherapy and may act as a cancer cell reservoir that leads to relapse. The discovery of therapeutics that can eradicate these cells is essential to achieve complete patient recovery.We hypothesize that by using an in silico strategy and stem cell gene expression data we can identify bioactive compounds that target LSCs with minimal impact on normal hematopoietic stem cell (HSC) function. We had previously generated human LSC and HSC expression signatures from 16 primary AML and 4 independent pooled cord blood samples that were prognostic for patient survival (Eppert et al Nat Med 2011). By interrogating existing gene expression profiles of drug response with our LSC and HSC signatures, as well as additional novel signatures, we identified 92 molecules predicted to inhibit human LSC properties without harming HSCs. Nearly 60% of the positive hits were anti-infectives, psychotropics, hormones and steroids, anticancer or cardiovascular therapies. In support of the efficacy of this bioinformatic approach, we identified known chemotherapeutic agents such as azacitidine, daunorubicin and doxorubicin, although these were predicted to harm HSCs. We assessed the anti-LSC activity of one sub-group known to target a class of G-protein-coupled receptors (GPCR) to further validate the list of candidate compounds. Using a primary human AML sample (8227) with known LSC phenotype (CD34+), we tested 2 candidate anti-GPCR compounds (hits A and B) and observed that they were highly effective in targeting both bulk cells and LSCs. The IC50 for bulk cells was 3.4 µM and 4.2 µM for compounds A and B, respectively (A - 6 day treatment, B - 2 day treatment). The IC50 of the CD34+ cell fraction, which included stem and progenitor cells, was 5.9 µM and 4.1 µM, respectively. Thus, these compounds are effective at targeting both bulk and LSC-enriched AML cell populations. The efficacy of these predicted compounds against the primitive cells within the CD34+ population was assessed using colony formation assays on human AML. 8227 cells treated with compound B resulted in significantly fewer colonies compared to the DMSO control (3.8 ± 3.0 vs 13.3 ± 2.8; at 4.5 µM). To test whether our candidate compounds target leukemic cells and not normal blood cells, we examined 3 additional primary human AML samples and a cord blood sample. There was only a minor effect on normal cells, including stem and progenitor cells, but significant cell death in all leukemia samples (70% and more at ≥ 3 µM). Next, we will determine the effectiveness of our compounds in vivo using established xenografts of primary human AML samples and cord blood samples.In conclusion, using our in silico approach we successfully identified novel anti-LSC compounds that have efficacy in vitro with minimal toxicity on primary HSCs. Furthermore, as each predicted compound is associated with specific molecular pathways, our approach has improved our understanding of LSC biology by acting as a screen for LSC-related pathways. In the near future, we will continue investigating the 92 candidate compounds to identify, develop, and transition a novel anti-LSC compound into clinical use. DisclosuresNo relevant conflicts of interest to declare.

Identification of a Neoplastic Stem Cell in Human Mast Cell Leukemia

Leukemic stem cells (LSCs) have recently been identified as an important target of therapy in various human leukemias and related blood cell disorders. Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal growth and accumulation of mast cells (MCs) in various organ systems, including the bone marrow (BM). Whereas patients with indolent SM (ISM) have a normal life-expectancy, patients with more advanced forms of SM have a poor prognosis. In these patients, neoplastic MCs are usually resistant against conventional drugs and various targeted drugs. MC leukemia (MCL) is the rare leukemic variant of advanced SM, defined by a rapidly devastating expansion of immature MCs in various hematopoietic organs and a poor prognosis with short survival times. Although MCL is considered a stem cell disease, little is known about the origin and phenotype of MCL-initiating LSCs. We examined the phenotypic and functional characteristics of putative LSCs in patients with aggressive SM (ASM, n=12) and MCL (n=6). Putative LSCs were identified and characterized phenotypically by flow cytometry. Highly enriched, sorted LSCs were injected into NOD-SCID-IL-2Rγ-/- mice exhibiting a 220 amino acid isoform of human membrane-bound hSCF (NSGSCF). We found that disease-initiating and propagating LSCs reside within a CD34+ fraction of the MCL clone. Whereas cell fractions containing CD34+ cells as well as highly enriched CD34+ cells produced engraftment in NSGSCF mice with a MCL-like disease (43-77% human MCL cells in mouse BM after 10-22 weeks), no substantial engraftment was produced by MC-rich but stem cell-depleted, KIT+/CD34─ cell fractions obtained from the same patients (<1% engraftment in mouse BM). In dilution experiments, engraftment of CD34+ cells was documented down to a minimum of 50 cells per mouse. The identity of engrafting MCL cells was confirmed by morphology, phenotyping and molecular studies demonstrating the presence of KIT mutations that were initially detected in the primary MCL samples used. Moreover, we were able to confirm long-term engraftment by successful serial transplantations into secondary recipient mice. In consecutive experiments, we were able to show that CD45+/CD34+/CD38─ cells also produce leukemic engraftment in NSGSCF mice. As assessed by flow cytometry, these CD34+/CD38─ MCL LSCs were found to express several stem cells markers, including aminopeptidase-N (CD13), leukosialin (CD43), Pgp-1 (CD44), the IL-3R alpha-chain (CD123), AC133 (CD133) and CXCR4 (CD184). In addition, in most patients examined, MCL LSCs were found to display IL-1RAP, a surface antigen that is otherwise expressed in CML LSCs but is not expressed in normal stem cells. In addition, MCL LSCs were found to express various cell surface targets, including CD33 and CD52. By contrast, MCL LSCs did not express CD2, CD25, CD26 and CLL-1. The more mature progenitor cell fractions (CD34+/CD38+) were found to stain positive for CD13, CD33, CD43, CD44, CD90, CD117, CD123, CD133 and CD184. Mature clonal MCs expressed a similar phenotype, including molecular markers and targets, such as CD13, CD30 CD33, CD52 and CD184. In patients with ISM and aggressive SM (ASM), the CD34+/CD38─ stem cells exhibited a similar surface marker profile compared to MCL, but expressed lower levels of CD133 and did not express IL-1RAP. In the validation phase of our study, we examined the effects of target-specific antibodies. As assessed by flow cytometry, the CD52-targeting antibody alemtuzumab was found to induce complement-dependent lysis of CD34+ and CD34+/CD38─ cells in all MCL samples analysed. Furthermore, pre-incubation of MCL cells with alemtuzumab prior to injection into NSGSCF mice resulted in a significantly reduced engraftment (2.7±4.1%) after 22 weeks. In conclusion, our data show that the MCL clone originates from a primitive hematopoietic stem cell that co-expresses CD34, CD123, CD133 and IL-1RAP but lacks CD25 and CD26. In addition, our data show that MCL LSC express a number of clinically relevant surface targets, including CD33, CD52 and CD117 (KIT). These observations may facilitate LSC detection and isolation in MCL and may lead to the development of novel LSC-eradicating treatment concepts in this highly aggressive and drug-resistant form of leukemia. DisclosuresValent:Novartis: Consultancy, Honoraria, Research Funding.

Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) support the growth and differentiation of normal hematopoietic stem cells (HSCs). Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs) in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML) were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6weeks. Four samples showed CD34+CD38− predominance, and four were predominantly CD34+CD38+. CD34+ CD38− predominant leukemia cells maintained the CD34+ CD38− phenotype and were viable for 6weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34+ CD38+ predominant leukemic cells maintained the CD34+CD38+ phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34+ blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell–cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs.

Phenotyping Of Leukemic Stem Cells In Ph+ ALL and Ph- ALL Reveals Unique Profiles Of Markers and Targets In Distinct Disease Variants

Acute lymphoblastic leukemia (ALL) is a life-threatening hematopoietic neoplasm characterized by abnormal growth and accumulation of lymphatic blast cells in various hematopoietic tissues. In a substantial number of patients, the Philadelphia (Ph) chromosome and the related oncoprotein BCR/ABL, are detectable. Despite recent advances in the management and therapy of patients with ALL, including the use of BCR/ABL1 tyrosine kinase inhibitors (TKI), the prognosis is still poor. Therefore, several attempts have been made to improve targeted treatment approaches in ALL. One strategy is to identify markers and targets expressed on leukemic stem cells (LSC) in these patients and to apply targeted drugs in order to eliminate LSC. In patients with Ph+ ALL, the leukemia-initiating cell-population is considered to reside within a CD34+/CD38- fraction of the clone. In the present study, we examined the expression of various stem cell markers and target antigens in CD34+/CD38- stem cells and in more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL (n=12), Ph- ALL (n=13), Ph+ CML (n=20), and in control bone marrow (BM) samples (unexplained cytopenia, n=10). Surface expression of target antigens was analyzed by multicolor flow cytometry, and mRNA expression levels by qPCR. As assessed by flow cytometry, CD34+/CD38- cells were found to co-express CD19, the stem cell-homing receptor CD44, the Campath-1 antigen (CD52), AC133 (CD133), FLT3 (CD135), and CXCR4 (CD184) in all ALL patients examined. In a majority of the ALL patients tested (14/25), LSC also expressed Siglec-3 (CD33). In CML, LSC were found to express a similar profile of antigens, including CD33, CD44, CD52, CD133, CD135, and CXCR4, but these cells did not express CD19. In control BM samples, CD34+/CD38- cells expressed a similar phenotype, but the levels of CD33 and CD52 were lower compared to LSC in ALL and CML. The IL-1RAP was found to be expressed on LSC in patients with Ph+ CML and Ph+ ALL, but not on LSC in Ph- ALL or in normal BM stem cells. By contrast, the SCF receptor KIT (CD117) was found to be expressed on LSC in Ph+ CML but was hardly detectable on LSC in patients with Ph+ ALL or Ph- ALL. The IL-2RA (CD25) and the SDF-1-degrading surface enzyme dipeptidyl-peptidase IV (DPPIV=CD26) were expressed on LSC in patients with CML and in all patients with Ph+ ALL exhibiting BCR/ABL-p210, whereas in Ph+ ALL with BCR/ABL-p190, LSC variably expressed CD25, and did not express CD26. In patients with Ph- ALL and in the normal BM, CD34+/CD38- cells did not express CD25 or CD26. The target receptor CD20 was detectable on ALL LSC in 7/18 patients examined. All target receptors tested were also detectable on more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL and Ph- ALL. In consecutive studies, expression of target antigens was confirmed at the mRNA level by qPCR analyses of highly enriched ALL LSC. Finally, we were able to show that the CD52-targeting drug alemtuzumab induces rapid lysis of CD34+/CD38- ALL LSC in all patients examined (Figure). In summary, our data show that LSC in Ph+ ALL and Ph- ALL express a unique phenotype, including clinically relevant cytokine receptors and cell surface target antigens, including the Campath-1 antigen, CD52. In Ph+ ALL with BCR/ABL-p210, the phenotype of ALL LSC largely resembles the phenotype of LSC in Ph+ CML, confirming the close relationship and similar pathogenesis of these two types of leukemias. [Display omitted] Ficoll-isolated MNC of 4 patients with Ph+ ALL were incubated in control medium (Co) or in various concentrations of alemtuzumab (10-300 µg/ml) in RPMI 1640 medium in the presence of 30% human serum at 37°C for 1 hour. After washing, cells were stained with fluorochrome-conjugated mAb against CD34, CD38 and CD45 for 15 minutes. DAPI-staining was used to evaluate the percentage of viable cells. Cells were analysed using a FACSCanto II and FlowJo software. Results show the numbers of viable CD34+/CD38- cells and are expressed as percent of control (Co). Values represent the mean±S.D. of four independent experiments. Asterisk (*): p<0.05 compared to control. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Acute myeloid leukemia following exposure to benzene more closely resembles de novo than therapy related‐disease

Benzene (Bz) is widely regarded as a prototype environmental leukemogen and individuals chronically exposed are at risk for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). It is widely assumed that initiation and pathogenesis of AML following Bz exposure (Bz-AML) is similar or identical to therapy-related AML (t-AML), in which clonal cytogenetic abnormalities, including aneuploidy, are initiating events. However, this assumption is not supported by studies reporting actual disease outcomes together with cytogenetic analyses. Here, using clinically relevant cytogenetic, hematologic, and epidemiological methods, we directly show for 722 consecutive AML cases that the pattern of clonal cytogenetic abnormalities encountered in Bz-exposed cases (n = 78) more closely resembles de novo-AML than t-AML. The prevalence of aneuploidy in Bz-exposed- and de novo-AML cases was identical (23%), and no significant increases in -5/5q- (RR = 0.79) (95% CI: 0.29-2.12) or -7/7q- (RR = 1.27) (95% CI: 0.55-2.92) abnormalities were observed between Bz- vs de novo-AML, respectively. Previous studies have suggested a role for autoimmunity in Bz related MDS including immune mediated inflammatory features and positive responses to immunosuppressive therapy which are indistinguishable from those reported in MDS with low risk of progression to AML. These observations are more consistent with an epigenetic model for initiation of Bz-AML in which altered homeostatic regulation in the bone marrow niche, not direct cytogenetic injury, predominates in the initial development of the leukemic stem cell phenotype, a mechanism biologically distinct from previous models of clonal cytogenetic injury. These findings are important for further understanding the biological basis of AML, particularly in environmental and occupational settings.

Chibby drives β catenin cytoplasmic accumulation leading to activation of the unfolded protein response in BCR-ABL1 + cells

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL fusion protein. However, the phenotype of leukemic stem cells (LSC) is sustained by β catenin rather than by the BCR-ABL TK. β catenin activity in CML is contingent upon its stabilization proceeding from the BCR-ABL-induced phosphorylation at critical residues for interaction with the Adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3 (GSK3) destruction complex or GSK3 inactivating mutations. Here we studied the impact of β catenin antagonist Chibby (CBY) on β catenin signaling in BCR-ABL1+ cells. CBY is a small conserved protein which interacts with β catenin and impairs β catenin-mediated transcriptional activation through two distinct molecular mechanisms: 1) competition with T cell factor (TCF) or lymphoid enhancer factor (LEF) for β catenin binding; and 2) nuclear export of β catenin via interaction with 14-3-3. We found that its enforced expression in K562 cell line promoted β catenin cytoplasmic translocation resulting in inhibition of target gene transcription. Moreover, cytoplasmic accumulation of β catenin activated the endoplasmic reticulum (ER) stress-associated pathway known as unfolded protein response (UPR). CBY-driven cytoplasmic accumulation of β catenin is also a component of BCR-ABL1+ cell response to the TK inhibitor Imatinib (IM). It evoked the UPR activation leading to the induction of BCL2-interacting mediator of cell death (BIM) by UPR sensors. BIM, in turn, contributed to the execution phase of apoptosis in the activation of ER resident caspase 12 and mobilization of Ca2+ stores.

Modulation of Leukemic Stem Cell Self-Renewal and Cell Fate Decisions by Inhibition of Hedgehog Signalling in Human Acute Lymphoblastic Leukemia (ALL).

AbstractAbstract 2578The Hedgehog (Hh) pathway plays a functional role in embryonic development and promotes tumorigenesis in a diversity of cancers. Constitutive activation of Smo, an essential component of the Hh pathway, augments stem cell number and accelerates disease in BCR-ABL positive CML, whereas loss of Smo depletes CML stem cells by inhibition of self-renewal. Phase I clinical trials using Hh inhibitors have started in BCR-ABL pos ALL and CML. The role of Hh signalling on stem cell behaviour in BCR-ABL neg ALL has not yet been examined. The phenotype of leukemic stem cells (LSCs) and the target cells for transformation in ALL are controversial, but only a small subpopulation of cells seem to act as LSCs. These cells may be the most relevant targets for treatment regimens for compounds that interfere with self-renewal programs and that provide promising therapeutic options for improving treatment of adult ALL.Aims of the study are characterization of different genetically and phenotipically defined ALLs, using our twelve patient derived long term cultures (LTCs), according to their biologic behaviour including leukemia initiating capacity, assessment of the impact of Hh inhibition on proliferation, apoptosis and clonogenic capacity and LSC function and dissection of the role of different components of the Hh signalling pathway on cell fate decisions by means of single cell video microscopy. These studies are anticipated to yield information on the therapeutic potential of modulation of Hh signalling in both BCR-ABL pos and neg ALL and the potential value of combination therapy.As models of BCR-ABL pos and neg leukemias we used serum-, cytokine- and stroma-free long term cultures of primary ALL blasts. Clonogenic growth of ALL cells was assessed in semi solid methylcellulose based media. Cell subpopulations were isolated on the basis of CD20, CD34 and CD38 expression via FACS based sorting (BD FACS Aria). Cell proliferation was measured using XTT assays and Annexin V and 7 AAD FACS staining were used to quantitate apoptosis. Quantitative RT PCR of Hh signalling pathway components using predeveloped Taqman assays (Applied). Single cell video tracking to determine cell fate decisions was performed as previously described (Rieger et all, Science 2009), adapted to facilitate the analysis of ALL LTCs. Two novel Smo inhibitors being currently in clinical testing, LDE225 and BMS833923 were kindly provided by Novartis and Bristol Myers Squibb.Results: The expression pattern of surface markers varied profoundly between the different LTCs studied. In preliminary experiments designed to identify functionally distinct subpopulations of long term cultured ALL blasts, cells were isolated to greater than 90% purity based on CD20, CD34 and CD38 expression. With the exception of CD34 positive cells, the surface marker distribution rapidly reverted to an identical pattern as determined prior to culture in three cell lines studied. In two ALL LTCs, CD34 expression was associated with slower proliferation. All three cell lines displayed clonogenic capacity ranging from 0,25% to 8% and are able to engraft in NSG mice.Analysis of Hh Signalling in ALL LTCs by RT PCR demonstrated expression of Shh, Ptch, Smo, and the transcription factors Gli 1 + 3, indicating active Hh signalling in ALL. Interestingly the transcription factor Gli 2 was not expressed, the functional relevance of which remains as yet unclear. The Hh inhibitors LDE225 and BMS833923 (0,01μM to 5μM) showed no dose dependent effect on inhibition of proliferation or induction of apoptosis in ALL LTCs.In conclusion we found evidence of Hh activation in both BCR-ABL pos and neg LTC ALL cells. No impact of Smo inhibition on proliferation and apoptosis was observed in response to the Smo inhibitors LDE225 and BMS833923, consistent with the hypothesis that Hh signalling in these cells may affect primarily self-renewal mechanisms. Single cell imaging of ALL LTCs has been successfully established for up to nine days of culture and will be applied to the testing of Hh modulation on cell fate decisions. Disclosures:No relevant conflicts of interest to declare.

The Levels of a G-CSF-Inducible pSTAT3+pSTAT5+ Subpopulation of MDS Progenitors with Leukemic Stem Cell Phenotype Predict the Response to Azacytidine

AbstractAbstract 3795Both basal and cytokine-induced phosho-STAT3 and pSTAT5 levels are altered in leukemias and profiling the STAT signaling network at the single cell level provides prognostic information (Irish et al, 2004). As cell signaling interacts with the epigenome (Mohammad et al, 2010) and numerous genes involved in signaling are aberrantly methylated in MDS (Figueroa et al, 2010) we investigated the alterations of STAT3/5 signaling in MDS progenitors during azacitidine therapy.Bone marrow samples of 58 high-risk MDS and AML/MDS patients were obtained before (d0) and 15 days (d15) after azacytidine initiation and at the indicated time points. According to the IWG response criteria, patients were divided into 4 groups: response (CR and PR, n=11 and n=5, respectively), hematologic improvement (HI, n=8), stable disease (SD, n=11) and failure (F, n=23). Mononuclear cells were either left untreated or stimulated with G-CSF and GM-CSF for 15′ and then stained intracellularly with the indicated antibodies. The hematopoietic hierarchy and G-CSF receptor (CFS3R) transcripts by qPCR were assessed on immunomagnetically purified Lin-CD34+ and CD34+ cells, respectively. Statistical comparisons were done by ANOVA and unpaired or paired t-tests as appropriate. STAT signaling profiles (SPs) in CD34+ cells were grouped using complete linkage hierarchical clustering and correlated with treatment response, karyotype and transfusion rate by using χ2 or Fisher Exact tests.Unsupervised clustering of pretreatment SPs in CD34+ cells identified 2 clusters (Fig 1A) mainly differing in the degree of potentiated STAT3/5 phosphorylation. Patients in cluster I displayed weak potentiated expression of pSTAT3/5 and had marginally better response to azacytidine compared to the ones in cluster II (p=0.06), whereas there were no differences among the two groups regarding the MDS subtype (p=0.41), karyotype (p=0.45) and transfusion rate (p=0.33). We further identified a G-CSF-inducible pSTAT3/5 double positive (DP) subpopulation of MDS CD34+ cells, whose levels both at d0 and d15 of the 1st cycle were inversely associated with response (Fig 1B), whereas its kinetics were following the disease course and response to azacytidine (Fig 1C,D). The DP subset was enriched in GMPs compared to the G-CSF-unresponsive pSTAT3/5 double negative (DN) subpopulation, suggesting a higher leukemia initiating cell activity (Goardon et al, 2011), whereas the DN subset was marginally enriched in MEPs (Fig 2). Also, compared to the DN subset, the DP population exhibited decreased Ki67 expression and increased Bcl2 and p53 levels (Fig 3A), indicating quiescence and increased antiapoptotic and tumor suppressive properties, respectively. Of note, the levels of the above molecules in the two subsets remained unaltered both at d0 and d15, indicating that these subsets represent genuine cellular entities with solid properties. To determine if CSF3R expression and/or its modulation by azacitidine are responsible for the alterations of the DP population, we checked CSF3R levels in isolated CD34+ cells from patients with either absence or full expression of the DP subset because CSF3R is downregulated after G-CSF stimulation. The two groups showed identical protein and mRNA levels of CSF3R at both d0 and d15 (Fig 3B,C).In summary, we identified a G-CSF-inducible pSTAT3/5 DP subpopulation with leukemic stem cell properties, which is potentially involved in MDS biology and epigenetically modulated, as implied by its kinetics during hypomethylating therapy. More important, given the strong association of pretreatment levels of the DP subset with the response to azacitidine, the latter subset may serve both as a treatment target and response biomarker. [Display omitted] [Display omitted] [Display omitted] Disclosures:Kotsianidis:Genesis-Pharma: Honoraria, Research Funding.