Established Leukemia Cell Lines Research Articles

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

Detection of AML-specific mutations in pediatric patient plasma using extracellular vesicle-derived RNA.

Despite high remission rates, almost 25% of patients with AML will suffer relapse 3-5years after diagnosis. Therefore, in addition to existing diagnostic and MRD detection tools, there is still a need for the development of novel approaches that can provide information on the state of the disease. Extracellular vesicles (EVs), containing genetic material reflecting the status of the parental cell, have gained interest in recent years as potential diagnostic biomarkers in cancer. Therefore, isolation and characterization of blood and bone marrow plasma-derived EVs from pediatric AML patients could be an additional approach in AML diagnostics and disease monitoring. In this study, we attempt to establish a plasma EV-RNA-based method to detect leukemia-specific FLT3-ITD and NPM1 mutations using established leukemia cell lines and primary pediatric AML plasma samples. We were successfully able to detect FLT3-ITD and NPM1 mutations in the EV-RNA using GeneScan-based fragment-length analysis and real-time PCR assays, respectively, in samples before therapy. This was corresponding to the gDNA mutational analysis from leukemic blasts, and supports the potential of using EV-RNA as a diagnostic biomarker in pediatric AML.

Complete Sequences of the Human T-Cell Leukemia Virus Type 1 Proviral Genomes from Newly Established Adult T-Cell Leukemia Cell Lines in Oita Prefecture, Japan.

ABSTRACTWe report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan.

The U2AF homology motif kinase 1 (UHMK1) is upregulated upon hematopoietic cell differentiation

UHMK1 (KIS) is a nuclear serine/threonine kinase that possesses a U2AF homology motif and phosphorylates and regulates the activity of the splicing factors SF1 and SF3b155. Mutations in these components of the spliceosome machinery have been recently implicated in leukemogenesis. The fact that UHMK1 regulates these factors suggests that UHMK1 might be involved in RNA processing and perhaps leukemogenesis. Here we analyzed UHMK1 expression in normal hematopoietic and leukemic cells as well as its function in leukemia cell line.In the normal hematopoietic compartment, markedly higher levels of transcripts were observed in differentiated lymphocytes (CD4+, CD8+ and CD19+) compared to the progenitor enriched subpopulation (CD34+) or leukemia cell lines. UHMK1 expression was upregulated in megakaryocytic-, monocytic- and granulocytic-induced differentiation of established leukemia cell lines and in erythrocytic-induced differentiation of CD34+ cells. No aberrant expression was observed in patient samples of myelodysplastic syndrome (MDS), acute myeloid (AML) or lymphoblastic (ALL) leukemia. Nonetheless, in MDS patients, increased levels of UHMK1 expression positively impacted event free and overall survival.Lentivirus mediated UHMK1 knockdown did not affect proliferation, cell cycle progression, apoptosis or migration of U937 leukemia cells, although UHMK1 silencing strikingly increased clonogenicity of these cells. Thus, our results suggest that UHMK1 plays a role in hematopoietic cell differentiation and suppression of autonomous clonal growth of leukemia cells.

Characterization of purine-rich element binding protein B as a novel biomarker in acute myelogenous leukemia prognostication.

Acute myelogenous leukemia (AML) is an aggressive hematologic cancer characterized by infiltration of proliferative, clonal, abnormally differentiated cells of myeloid lineage in the bone marrow and blood. Malignant cells in AML often exhibit chromosomal and other genetic or epigenetic abnormalities that are useful in prognostic risk assessment. In this study, the relative expression and novel single-stranded DNA (ssDNA) binding function of purine-rich element binding proteins A and B (Purα and Purβ) were systematically evaluated in established leukemia cell lines and in lineage committed myeloid cells isolated from patients diagnosed with a hematologic malignancy. Western blotting revealed that Purα and Purβ are markedly elevated in CD33+ /CD66b+ cells from AML patients compared to healthy subjects and to patients with other types of myeloid cell disorders. Results of in silico database analysis of PURA and PURB mRNA expression during hematopoiesis in conjunction with the quantitative immunoassay of the ssDNA-binding activities of Purα and Purβ in transformed leukocyte cell lines pointed to Purβ as the more distinguishing biomarker of myeloid cell differentiation status. Purβ ssDNA-binding activity was significantly increased in myeloid cells from AML patients but not from individuals with other myeloid-related diseases. The highest levels of Purβ activity were detected in myeloid cells from primary AML patients and from AML patients displaying other risk factors forecasting a poor prognosis. Collectively, these findings suggest that the enhanced ssDNA-binding activity of Purβ in transformed myeloid cells may serve as a unique and measurable phenotypic trait for improving prognostic risk stratification in AML.

The Novel Technique of Genetically Engineered Patient-Derived Xenografts (GEPDX) Reveals That the X-Linked Inhibitor of Apoptosis Protein (XIAP) Plays an Essential Role for Maintenance and Growth of Patients' Acute Lymphoblastic Leukemia In Vivo

Expression profiling and next generation sequencing have enabled a detailed knowledge on alterations present in tumors from individual patients. In contrast, only limited understanding exists on the role that each alteration plays for the existing tumor. Of direct clinical interest, genes are of special interest which harbor an essential function for tumor maintenance and growth as they represent putative targets for anti-cancer therapy.Characterizing gene functions is demanding regarding both techniques and resources. Questions on gene function are often studied in established tumor cell lines, although establishing cell lines from primary tumors is rarely successful in acute leukemias. Primary acute leukemia cells poorly grow in vitro and established acute leukemias cell lines rely on additional mutations enabling in vitro growth, making them doubtful models to study genes with essential function.To bridge the gap, we aimed at studying gene function in the complex environment of individual tumors. As primary acute leukemia cells are unable to grow in vitro, we used the orthotopic model of patient-derived xenograft (PDX) leukemia and amplified cells in mice.We established a novel technique to manipulate distinct signaling proteins in PDX cells using lentiviruses and knockdown. We expressed small hairpin RNA (shRNA) in the background of micro RNA 30 (miR30) under control of a Pol II promoter and 3 prime of dsRED as molecular marker. This approach closely links expression of the shRNA to the fluorochrome and resulted in a potent and stable knockdown. We expressed a control shRNA targeting Renilla luciferase and several shRNA sequences targeting XIAP. In order to discriminate different derivative cell populations within a single mouse, we co-expressed a second fluorochrome from a second plasmid so that green cells harbored a knockdown of XIAP, while blue cells harbored the control construct, thus allowing in vivo outcompete proliferation assays. We called our new approach "genetically engineered PDX (GEPDX)" models in parallel to genetically engineered mouse models (GEMM).We used our new technology to study the role of XIAP, the X-linked inhibitor of apoptosis for acute lymphoblastic leukemia (ALL), the single most frequent tumor in children. XIAP is frequently and highly overexpressed in hematological malignancies and its up-regulation was shown to be associated with inferior prognosis of patients in different tumors. Nevertheless XIAP's role for tumor maintenance remains unclear.In several preB- and T-cell ALL cell lines, potent and stable knockdown of XIAP did not alter cell proliferation in vitro or upon xeno-transplantation in vivo. Thus expression levels of XIAP seem irrelevant for spontaneous proliferation of established acute leukemia cell lines in vitro and in vivo.We next studied PDX ALL cells growing in mice as model closer related to patients. We generated GEPDX cells from two children with relapse of a B precursor ALL with either knockdown of XIAP or control knockdown together with the appropriate molecular color markers. When blue and green GEPDX cells harboring control or XIAP knockdown, respectively, were co-transplanted into mice at a 1:1 ratio in a competitive outcompete assay, control transfected cells significantly overgrew or even eliminated cells with XIAP knockdown in both samples studied. GEPDX cells with knockdown of XIAP showed a significant and dose-dependent growth disadvantage in vivo compared to control cells indicating that XIAP played an essential role for PDX cells growing in vivo.Thus, our novel technique of genetic engineering in PDX cells revealed an essential role of XIAP for tumor maintenance and growth in patients' tumor cells making XIAP an attractive therapeutic target in ALL. As established ALL cell lines were unable to unravel this important role of XIAP, GEPDX might be superior to cell lines for identifying genes with essential function. GEPDX represent a powerful new tool to characterize the complex environment of individual patients' tumor cells in vivo, the function of the many lesions and alterations described by expression profiling and next generation sequencing. DisclosuresNo relevant conflicts of interest to declare.

Microfluidic channel‐assisted screening of hematopoietic malignancies

A simple microfluidic fluorescence in situ hybridization (FISH) device allowing accurate analysis of interphase nuclei in 1 hr in narrow channels is presented. Photolithography and fluorosilicic acid etching were used to fabricate microfluidic channels (referred to as FISHing lines) that allowed analysis of 10 samples on a glass microscope slide 0.2 µl of sample volume was used to fill a micro-channel, which resembled a 250-fold reduction compared to conventional FISH. FISH signals were comparable to conventional FISH, with 50-fold less probe consumption and 10-fold less time. Cells were immobilized in single file in channels just exceeding the diameter of the cells, and were used for minimal residual disease (MRD) analysis. To test the micro-channels for application in FISH, MRD was simulated by mixing K562 cells (an established chronic myeloid leukemia cell line) carrying the BCR/ABL fusion gene across 1:1 to 1:1,000 Jurkat cells (an established acute lymphoblastic leukemia cell line). The limit of detection was seen to be 1:100 cells and 1:1,000 cells for FISHing lines and conventional FISH, respectively; however, the conventional method seemed to over-score the presence of K562 cells. This may in part be attributed to FISHing lines practically eliminating the chance of duplicate screening of cells and hastened the time of screening, enhancing scoring of all cells within the channels. This was compared to 1 in 500 cells on the slide being analyzed with the conventional FISH.

Abstract 1710: An anti-apoptotic Bcl-2 family protein index predicts the response of leukemic cells to the pan-Bcl-2 inhibitor S1.

Abstract Bcl-2-like proteins prevent cell death through expression level, post transcriptional modification, and protein-protein-interaction. Recent studies have shown that antiapoptotic Bcl-2-like members are overexpressed in many haematological malignancies. BH3 mimetics are promising anticancer agents by directly target these proteins and their interactions. S1 (8-oxo-3-thiomorpholin-4-yl-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile) is an authentic BH3 mimetic and a triple inhibitor of Bcl-2, Mcl-1, and Bcl-XL. In the present study, we found S1 exhibited a variant sensitivity with LD50 values ranging >2 logs (0.24-80.4 μM) in five established leukemia cell lines (Jurkat, K562, HL60, U937 and PTA-3920) and forty-one primary leukemic cells (including Acute myeloid leukemia (AML), Acute lymphoblastic leukemia (ALL), Chronic Myeloid Leukemia (CML), and Chronic lymphoblastic leukemia (CLL)). Through a semi-quantitative approach by analyzing western blotting bands via imaging software, it revealed that the index value phosphoralated Bcl-2 (pBcl-2) to Bcl-2 and Mcl-1 provided a highly significant linear correlation with S1 sensitivity (r = 0.69, P < 0.001 in primary cells, and r = 0.76, P = 0.05 in cell lines). In contrast, antiapoptotic Bcl-2-like protein levels, used individually or in combination could not predict S1 sensitivity. Phosphorylated Bcl-2 antagonized S1 by sequestering the Bak and Bim proteins that were released from Mcl-1 in leukemia cell lines. Furthermore, pBcl-2/Bak, pBcl-2/Bax, and pBcl-2/Bim complexes cannot be disrupted by S1. The shift of proapoptotic proteins from being complexed with Mcl-1 to being complexed with pBcl-2 is revealed for the first time, and it could be the mechanism underlying the index value described herein. This study defines the antiapoptotic role of pBcl-2 linked with the protein-protein interaction network within Bcl-2 family. Citation Format: Yubo Liu, Zhichao Zhang. An anti-apoptotic Bcl-2 family protein index predicts the response of leukemic cells to the pan-Bcl-2 inhibitor S1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1710. doi:10.1158/1538-7445.AM2013-1710 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Hematopoietic Cell Kinase (HCK) Is Highly Expressed in Myelodysplastic Syndrome (MDS) and Regulates Proliferation, Cell Cycle Progression and Hematopoietic Differentiation

AbstractAbstract 3813The pathogenesis of MDS is complex and remains elusive. The molecular characterization of this disease has been a tool to obtain a better understanding. Our group recently identified new possible target genes involved in MDS pathophysiology through the microarray analysis of CD34+ cells from MDS patients. One of the interesting overexpressed genes found was the HCK gene, a Src kinase family member. This family integrates signaling from a variety of cell-surface receptors, such as RTK, EPO receptor, chemokine receptor, MET and CXCR4, to regulate diverse cellular responses including proliferation, differentiation, apoptosis and migration. HCK is restrictedly expressed in hematopoietic cells and overexpressed in some types of solid cancer and in acute lymphoid leukemia; however, functional role of HCK in MDS and myeloid cells is still unknown. Here, using qPCR we investigated the HCK mRNA expression in CD34+ and total bone marrow (BM) cells from 54 MDS patients (FAB 39 low-risk and 15 high-risk), receiving no treatment, and 24 normal donors. We evaluated the role of HCK, studying its effect on proliferation, cell cycle and apoptosis by silencing this gene in U937 myeloid cell line. To inhibit HCK, specific shRNA expressing lentiviral vectors targeting the HCK gene or no specific sequence were used. Cell growth was measured by MTT colorimetric assay. Annexin-PI was used to evaluate apoptosis and cell cycle was analyzed by flow cytometry. These assays were carried out in lentiviral transduced cells treated, or not, with rapamycin(10 or 100nM) and LY294002(50nM). We further analyzed the HCK expression during granulocytic and erythroid differentiation of established leukemia cell lines models. Granulocytic differentiation was induced in NB4 or HL60 cells by treatment with ATRA(10−6 M) for 4 days. Erythroid differentiation was induced in KU812 cells by treatment with HE(50μM) and HU(100μM) for 4 days. We observed a significant increase in HCK mRNA expression of MDS CD34+ cells (P=0.002) and BM cells (P=0.03) compared to normal cells. HCK mRNA expression in low-risk MDS cells was higher compared to high-risk MDS cells. In U937 cells, HCK inhibition resulted in a significant decrease in proliferation compared to control cells (P<0.0001). Interestingly, the combination of HCK inhibition/rapamycin or LY treatment, inhibitors of PI3K/mTOR pathway, showed no reduction in cell proliferation compared to HCK inhibition alone. HCK inhibition induced cell cycle delay in G0/G1 phase and a consequent decrease in cell percentages in the S phase. The combination of HCK inhibition/rapamycin or LY treatment showed a significant decrease in cell number in G0/G1 phase (P=0.007) and increase in S phase (P=0.01) compared to HCK inhibited alone. Apoptosis was unaffected by HCK knockdown. HCK expression was significantly increased in NB4 or HL60 cells after granulocytic differentiation and in KU812 cells after erythroid differentiation (P<0.01). Furthermore, HCK mRNA was significantly up-regulated during erythroid differentiation of MDS low-risk CD34+ cells but not in normal CD34+ cells. Considering that some transmembrane receptor (CXCR4, MET, chemokine) expressions are altered in MDS patients and their downstream pathway is mediated by Scr family/PI3K, our results suggest that HCK overexpression in MDS may be related to alteration in the expression of some of these cell-surface receptors. Loss of HCK expression may contribute to disease progression by skewing the balance between proliferation and differentiation, perhaps giving malignant cells a proliferative advantage. Further, HCK inhibition in U937 cells potently decreases proliferation through PI3K/mTOR pathway since the combination of HCK inhibition/rapamycin or LY treatment showed no alteration in the proliferation. In addition, HCK protein might have role in cell cycle control by modulating cell cycle gene expression in G0/G1 phase check point. Moreover, granulocyte and erythroid differentiation results in an HCK overexpression probably because this gene is an adaptor protein that is essential for intermediating downstream signaling of activated cell-surface receptors. Our findings also indicate that HCK plays a role in myeloid cell abnormalities, through different cell activities, including proliferation, cell cycle and differentiation, suggesting that HCK represents a possible therapeutic target in MDS. Support: FAPESP, CNPq. Disclosures:No relevant conflicts of interest to declare.

Resistance of leukemic stem-like cells in AML cell line KG1a to natural killer cell-mediated cytotoxicity

Leukemic stem cells (LSCs) play the central role in the relapse and refractory of acute myeloid leukemia (AML) and highlight the critical need for the new therapeutic strategies to directly target the LSC population. However, relatively little is known about the unique molecular mechanisms of drug and natural killer cells (NK)-killing resistance of LSCs because of very small number of LSCs in bone marrow. In this study, we investigated whether established leukemia cell line contains LSCs. We showed that KG1a leukemia cell line contained leukemic stem-like cells, which have been phenotypically restricted within the CD34+CD38− fractions. CD34+CD38− cells could generate CD34+CD38+ cells in culture medium and had renewal function. Moreover, CD34+CD38− cells had self-renewal potential. We found that leukemic stem-like cells from KG1a cells were resistant to chemotherapy and NK-mediated cytotoxicity. NKG2D ligands involve in protecting LSCs from NK-mediated attack. Taken together, our studies provide a novel cell model for leukemic stem cells research. Our data also shed light on mechanism of double resistant to chemotherapy and NK cell immunotherapy, which was helpful for developing novel effective strategies for LSCs.

Src Activation Plays an Important Key Role in Lymphomagenesis Induced by FGFR1 Fusion Kinases

Chromosomal translocations and activation of the fibroblast growth factor (FGF) receptor 1 (FGFR1) are a feature of stem cell leukemia-lymphoma syndrome (SCLL), an aggressive malignancy characterized by rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. It has been suggested that FGFR1 proteins lose their ability to recruit Src kinase, an important mediator of FGFR1 signaling, as a result of the translocations that delete the extended FGFR substrate-2 (FRS2) interacting domain that Src binds. In this study, we report evidence that refutes this hypothesis and reinforces the notion that Src is a critical mediator of signaling from the FGFR1 chimeric fusion genes generated by translocation in SCLL. Src was constitutively active in BaF3 cells expressing exogenous FGFR1 chimeric kinases cultured in vitro as well as in T-cell or B-cell lymphomas they induced in vivo. Residual components of the FRS2-binding site retained in chimeric kinases that were generated by translocation were sufficient to interact with FRS2 and activate Src. The Src kinase inhibitor dasatinib killed transformed BaF3 cells and other established murine leukemia cell lines expressing chimeric FGFR1 kinases, significantly extending the survival of mice with SCLL syndrome. Our results indicated that Src kinase is pathogenically activated in lymphomagenesis induced by FGFR1 fusion genes, implying that Src kinase inhibitors may offer a useful option to treatment of FGFR1-associated myeloproliferative/lymphoma disorders.

Downregulation of IRS2 in Myelodysplastic Syndrome (MDS) May Be Related to Impaired Cell Differentiation,

Abstract 3814Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia. Abnormalities in cell differentiation are probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling from upstream activators to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R), the Erythropoietin receptor (EPOR) and Thrombopoietin receptor (MPL). Recently, we reported that IRS2 was downregulated in MDS cells compared to normal cells, and in high risk compared to low-risk MDS. Additionally, IRS2 was significantly upregulated during erythroid differentiation of normal CD34+ cells, but not in MDS CD34+ cells. In view of the role of IRS2 in EPO, IGF1 and TPO signaling, and possibly in erythroid, granulocytic, and megakaryocytic differentiation, we hypothesized that IRS2 might be related to the impaired hematopoiesis of MDS cells. The aim of the present study was to correlate the mRNA expression levels of IRS2 in bone marrow from MDS patients with laboratorial and clinical data. We also elucidated the expression levels of IRS2 during megakaryocytic, granulocytic and erythroid differentiation of established leukemia cell line models. We studied 12 healthy donors and 32 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification). Gene expression was evaluated by Q-PCR in total cells from bone marrow samples and cell lines. IRS2 protein expression and phosphorylation was evaluated by Western blot and immunoprecipitation. K562 cells were induced to megakaryocytic differentiation by treatment with 20nM PMA for 4 days. NB4 cells were induced to granulocytic differentiation by treatment with 10−6 M ATRA for 4 days. KU812 cells were induced to erythroid differentiation by treatment with 50μM HE and 100 μM HU for 4 days. Spearman correlation analysis showed that IRS2 expression demonstrated a significant positive correlation with peripheral blood neutrophils (r =0.43, P =0.01) and platelets counts (r =0.36, P =0.04), and bone marrow granulocytes percentages (r =0.40, P =0.02). In contrast, a significant negative correlation was observed between IRS2 expression with bone marrow erythroblasts (r =-0.360, P =0.04) and myeloid precursor percentages (grouping myeloblast, promyelocytes, myelocytes, metamyelocytes and blasts) (r =-0.60, P <0.01). There was no correlation between IRS2 expression with age, hemoglobin, number of erythrocytes in the peripheral blood, and number of dysplasias in bone marrow. Finally, since IRS2 is involved with TPO, IGF1 and EPO signaling, and IRS2 expression was correlated with platelet, granulocyte and erythroblast numbers in MDS patients, we monitored IRS2 expression during hematopoietic cell differentiation. Interestingly, a significant increase in IRS2 expression and phosphorylation was observed in K562, NB4 and KU812 after megakaryocytic, granulocytic and erythroid differentiation, respectively. In summary, the lower expression of IRS2 in MDS and its upregulation during differentiation may suggest that defective IRS2 expression in MDS contributes to disease progression and impaired hematopoietic cell differentiation, a hallmark of the disease. Disclosures:No relevant conflicts of interest to declare.

Identification and Purification of Leukemic Stem-Like Cells in Acute Myeloid Leukemia Cell Line

Acute myeloid leukemia (AML) is initiated and maintained by a rare population of (leukemic stem cells) LSCs. LSCs play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population for ultimately curing leukemia which is it is important to identify and study LSCs. However, relatively little is known about the unique molecular mechanisms of survival and self-renewal of LSCs because of very small number of LSCs in bone marrow. In this study, we investigated whether established leukemia cell lines contain LSCs. We showed that leukemia cell line contain leukemic stem-like cells which have been phenotypically restricted within the CD34+CD38− fraction. We demonstrated that CD34+CD38− cells could generate CD34+CD38+ cells in culture medium and had proliferation function. Moreover, CD34+CD38− cells had self-renewal potential both in vitro soft agar colonies formation assay and in vivo NOD/SCID mouse xenotransplant model serial transplantation. Furthermore, CD34+CD38− cells isolated from leukemia cell line were found resistant to conventional chemotherapy and NK cells-mediated cytotoxicity and these were related to up-regulation of ABCG2 and MRP-1 and antiapoptotic proteins of Bcl2. Down-regulation of NKG2D ligand also played a critical role in NK cytotoxicity resistance. Taken together, our studies provide a novel cell model for leukemic stem cells research. Our data also shed light on mechanism of double resistant to resistant to chemotherapy and NK cell immunotherapy, which was helpful for developing novel effective strategies for LSCs.

Characterization of Leukemic Stem Cells in a Newly Established Acute Myeloid Leukemia (AML) Cell Line 2006-5

AML cell line 2006-5 was established by serial culture of an AML patients' PBMC (M5).Compared to primary AML cells, this line expresses CD13, CD33, CD15, CD4, CD86, CD54 but lost CD34, CD64, CD123 and HLA-Dr. A group of CD13−/CD33− cells was detected consisting of 0.2% of total cells, but no significant CD34, CD90 or CD123 expression cells could be detected by FACS. After depleting of CD13+/CD33+ cells by magnetic beads, the CD13−/CD33− cells were enriched to 95%. Significant CD34+, CD90+ or CD123+ cells (0.2–0.5%) appeared in the CD13−/CD33− enriched fraction of AML cells. Leukemia colony forming (LCF) cell assay analyzed at day 14 demonstrated CD34+, CD90+ or CD123+ cells selected by magnetic beads formed typical compact large colonies of more than 1000 cells representing 40–60% of total colonies (Fig left panel). The CD13−/CD33− enriched fraction formed medium colonies with a dense center surrounded by a halo of migrating cells (80–90% of total colonies) (Fig right). Untreated 2006-5 cells formed small diffuse colonies (70–80%). Re-plating of compact colonies selected from CD34+, CD90+ or CD123+ cell LCF assays formed the above 3 type of colonies in one culture dish. Although a daughter line can be generated from a single colony formed by CD13-CD33− cells, no compact colonies could be observed in re-plating experiment. After 14 days of culture, immature erythroid cell clusters were observed in the area surrounding compact colonies suggesting that the progenitors of the colony forming leukemia cells were at the GEMM stage. Small colonies of untreated cells gradually became apoptotic and necrotic after 10 days. These results support the theory that AML is organized as a hierarchy similar to the normal hematopoietic system. CD34+, CD90+ or CD123+ cells with very low frequency of 1–2 in 1×105 2006-5 line cells demonstrated high proliferation and differentiation potential, and may represent leukemia stem cells. CD13−/CD33− cells with lower proliferation rate may belong to more differentiated leukemia progenitors without self renew potential. If additional experiments support the notion that cells forming compact colonies are leukemia stem cells, they may serve as a useful resource to test the biology of leukemia and its therapy. [Display omitted] [Display omitted]

Small Molecule Inhibition of Cdc7, a Key Cell Cycle Regulator and Novel Therapeutic Target, Successfully Inhibits Leukemia Cell Growth in Vitro and in Vivo

Cdc7 is a heterodimeric serine/threonine protein kinase that is a key regulator in the process of initiation of DNA replication and the G1 to S phase transition. Both the kinase and its known substrates are over-expressed in the majority of human cancers. As a result of the recent progress in the areas of pharmacogenetics and high throughput screening technology, identifying specific small molecule inhibitors of cell cycle regulated protein kinases has provided a means not only to study these signal transduction pathways but also to identify potential novel therapeutic agents. To this end, we have developed an assay for Cdc7 kinase inhibitory activity using a highthroughput screening (HTS) approach, screening over 250,000 natural and synthetic small molecules. As a result, we have identified and confirmed seventeen compounds, representing nine different chemical scaffolds, with Cdc7 kinase inhibitory activity. Based on potency, we selected the lead compound (CKI-7) which was further characterized using kinase profiling, microarray experiments, and standard cell based cytotoxicity assays. These latter studies demonstrated that CKI-7 induced cytotoxicity of established leukemia and lymphoma cell lines in culture with inhibitory concentrations (IC50s) in the low nanomolar range. Significantly, CKI-7 likewise induced cytotoxicity of MDR1 overexpressing cell lines with similar IC50s, demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. We additonally demonstrate that CKI-7 induces cytotoxicity of patient-derived primary acute leukemia tumor cells (both chemotherapy naïve and relapsed/refractory samples) in vitro at similarly low nanomolar concentrations. In vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilzing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Standard cell cycle synchronization studies established that exposure to CKI-7 results in cell cycle dependent caspase 3 activation and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a substrate biomarker assay. In conclusion, our data confirm that Cdc7 is a new promising target for cancer therapy, and that CKI-7, a selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.

Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of JAK2V617F

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

New Pinostilbene Analogues Overcome Anthracycline Resistance in Acute Lymphoplastic Leukemia Ex Vivo.

Anthracylines play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and relapsed ALL in childhood, however resistance to anthracyclines leads to a poor prognosis. In the present study, we have synthesized two new pinostilbene analogues, i.e.trans-3,4′-dihydroxy-5-methoxystilbene and(E)-resveratrol 4′-O-ß-D-glucopyranoside, which are able to overcome anthracycline resistance in childhood acute lymphoblastic leukemia (ALL) ex vivo and induce apoptosis in established leukemia cell lines via mitochondrial pathway.Apoptosis induction has been investigated by flowcytometric measurement of DNA-fragmentation [LC 50: 10 μM for (1) and (2)], mitochondrial membrane potential reduction and phosphatedylserin-staining on cell membrane surface. Cell death by necrosis could be excluded by a lactatdehydrogenase-release assay. For the first time we analysed the antileukemic and chemopreventive potentials of the pinostilbene analogues (1) and (2) ex vivo in a significant number of primary lymphoblasts of patients suffering from childhood ALL.Patients: Primary lymphoblasts isolated from 22 children with de novo ALL (median: 6.8 years; range 0,6–16.9 years) and relapsed ALL (median:7.2 years) were tested for ex vivo drug response with the anthracyclines daunorubicin (10 μmol/l) and doxorubicin (10 μmol/l) and two new pinostilbene analogues (1) and (2) (10μmol/l), according to their LC50 values in established cell lines. We could demonstrate in these primary cells that the pinostilbenes (1) and (2) were even more effective as compared with the anthracyclines. Out of 22 patients 14 were female (14 de novo ALL) and 8 were male (6 de novo ALL, 2 relapsed ALL). Within these cell populations following immunologic subgroups were found: c-ALL, pre-B-ALL, pro-B-ALL, T-ALL and pre-T-ALL.Results: Daunorubicin induced apoptosis in 6 out of 22 lymphoblast populations (response rate 27,3 %). A similar response rate was observed after treatment with doxorubicin: only 5 of 22 lymphoblast populations responded (22,7%). Nevertheless, far higher response rates were observed for (1) with 11/15 (73,3 %; p<0.005) and for (2) with 15/17 (88,2%; p<0.0002, all p-values by t-test). Interestingly, treatment of daunorubicin-resistant lymphoblasts resulted in significant apoptosis induction in 6 out of 10 cell populations after treatment with compound (1) (response rate 60 %) and in 6/6 after treatment with compound (2) (response rate 100%). Furthermore, pinostilbene (2) showed significant synergistic activity with daunorubicin in 2 of 3 lymphoblast populations. We clearly demonstrated that the ex vivo treatment of lymphoblasts from children with de novo and relapsed ALL with the new pinostilbenes (1) and (2) induced significantly higher response rates than daunorubicin or doxorubicin treatment. In conclusion, the high ex vivo sensitivity of anthracycline resistant leukemia cells to pinostilbene treatment reveals the great proapoptotic and chemopreventive potential of this new class of antileukemic agents.

Human Plasmacytoid Dendritic Cell like Leukemia Cell Line (PMDC05) Established from a Patient with CD4+CD56+ Acute Leukemia.

Human dendritic cell precursors are commonly divided into two distinct subsets: myeloid DC and Plasmacytoid DC (pDC). The pDC, which show plasma cell like morphology, have been defined as the population that produce a large amount of type I interferon in response to viruses. The surface phenotypes of human pDCs are defined as CD4+, DC11c−, CD45RA+, IL3Rα (CD123)+, CD1c (BDCA-1)−, CD303 ((BDCA-2)+ and lineage negative. On the other hand, leukemia/lymphoma cells in CD4+CD56+ leukemia/lymphoma have been proposed to be of pDC lineage. CD4+CD56+ pDC leukemia/lymphoma are a rare hematological malignancy, totally only about 100 cases in the world by the literatures. We established a pDC like leukemia cell line (PMDC05) from leukemia cells of a patient with CD4+CD56+ acute leukemia. PMDC05 showed a complex hypoploid chromosomal abnormalities (44, XY) including add(5)(q22), add(15)(q26) and del(15)(q11q15), which is identical to original leukemia cells. Abnormalities including 5q and 15q are reported as the frequent aberrations in CD4+CD56+ pDC leukemia/lymphoma. PMDC05, which morphology was similar to plasma cells, was positive for CD4, CD56, CD123, CD33, CD86, HLA-ABC, HLA-DR, CD1a, CD40, and CD83 but negative for linage markers. Cytokine receptors for GM-CSF, IL3Rα and IL-6Rα were positive on PMDC05. The expression of Trail and Flt-3L was positive. By the culture with IL-3, CPG-A/B, GM-CSF, molecules associated with antigen presentation such as CD1a and CD40 were up-regulated. Besides, the addition of LPS increased the expression of CD40, CD80 and CD83 on PMDC05. PMDC05 by itself possessed a potent antigen presenting ability to naïve T cells and the treatment of PMDC05 with IL-3, CPG-A/B, or GM-CSF enhanced the antigen presenting ability to naïve T cells. TLR7, TLR 8 and TLR 9 as well as TLR1, TLR2, TLR4 were demonstrated to be expressed on PMDC05 by RT-PCR and RQ-PCR showed that the expression of TLR7 and TLR9 was most characteristic. λ-like 14.1 and preTα was also demonstrated to be expressed on PMDC05 by RT/RQ-PCR. PMDC05 possessed an ability to uptake the antigens like FITC-dextran and lucifer yellow. Although IFN-α was not identified to be secreted from PMDC05 by the stimulation of influenza virus, IFN-γ and TNF-α was demonstrated to be secreted to the similar level in pDC, which was examined simultaneously with PMDC05 by CBA assay. These data demonstrated that newly established leukemia cell line PMDC05 is involved in pDC lineage and PMDC05 provides invaluable tools not only for the elucidation of pathophysiology of CD4+CD56+ leukemia/lymphoma but also for the investigation of differntiation and regulation of pDC. In addition, PMDC05 could be applied for generating tumor-specific CTL clone, which may be used for anti-tumor cellular immunotherapy.

Human Plasmacytoid Dendritic Cell Line (PMDC05) Established from a Patient with CD4+CD56+ Acute Leukemia.

Human dendritic cell (DCs) precursors are commonly divided into two distinct subsets: myeloid DCs (mDC) and Plasmacytoid DCs (pDCs). The pDCs, which show plasma cell like morphology, have been defined as the population that produce a large amount of type I interferon in response to viruses. The surface phenotypes of human pDCs are defined as CD4+, DC11c−, CD45RA+, IL3Rα (CD123)+ and lineage negative. But the developmental pathways and the regulations of pDCs have not been fully understood. On the other hand, CD4+CD56+ malignant cells in leukemia/lymphoma have been proposed to be of pDC lineage. CD4+CD56+ pDC leukemia/lymphoma are a rare hematological malignancy, totally only about 100 cases in the world by the literatures. In the recent report, these newly described CD4+CD56+ leukemic pDCs share common phenotypic and functional features with their normal counterparts. We encountered a patient with CD4+CD56+ acute leukemia in December 2004. The leukemia cells have been cultured in IMDM with 10 % FBS and a leukemia cell-derived cell line (PMDC05) was established. The effects of various cytokines on the differentiation and the function of PMDC05 were assayed by using IL-3, IL-4, IL-6, GM-CSF and CD40L alone or in combination. To evaluate the effects of CD40L, PMDC05 were cultured over adherent cell layer of 90 Gy irradiated CD40L cDNA-transfected NIH-3T3 cells at cell ration of 5:1 for 2 days. For investigation of the response of PMDC05 against the danger signals through toll like receptors, inactivated influenza viruses (A/H1N1 and A/H3N2) and GpG ODN 2006 were used. Antigen presenting ability of PMDC05 was evaluated by mixed leukocyte culture consisting of 50 Gy irradiated-PMDC05 cultured with various cytokines as stimulator cells and normal peripheral blood non-adherent cells and naïve cells as responder cells. PMDC05 maintained plasma cell like morphology with abundant cytoplasm and some cells showed small dendrites. PMDC05 showed a complex hypoploid chromosomal abnormalities (44, XY) including add(5)(q22), add(15)(q26) and del(15)(q11q15), which are identical to original leukemia cells. Abnormalities including 5q and 15q are reported as the frequent aberrations in CD4+CD56+ pDC leukemia/lymphoma. The surface phenotypes of PMDC05 were negative for CD3, CD14, CD16, CD19 and CD11c and highly positive for CD4, CD45RA, CD56, CD123, CD86, and HLA-DR. Moreover BDCA4 that is specific antigen of human blood pDC is markedly expressed on PMDC05. No TCR or IgH gene rearrangement was detected. Stimulation of PMDC05 with IL-3/CD40L, virus RNA or CpG, which are known as the potent exogenous signals for maturation of normal pDCs, showed to induce the high expression of the maturation markers such as CD83/CD40 and the production of INF-α. PMDC05 were demonstrated to possess a potent antigen presenting ability to allogeneic CD4+ cells in mixed leukocyte culture. The antigen presenting ability was remarkably enhanced in PMDC05 cultured with IL-3/CD40L for 2 days. These data demonstrated that newly established leukemia cell line PMDC05 is involved in pDC lineage and PMDC05 provides invaluable tools not only for the elucidation of pathophysiology and innovation of therapy in CD4+CD56+ leukemia/lymphoma but for the investigation of human pDCs.

Vesicular stomatitis virus: a potential therapeutic virus for the treatment of hematologic malignancy.

Certain strains of vesicular stomatitis virus (VSV) have been shown to be oncolytic in a wide variety of solid tumors. In the present study, we tested the leukemolytic properties of VSV using established leukemia cell lines and primary patient material. VSV efficiently killed essentially all leukemic cell lines. In contrast, however, normal clonogenic bone marrow progenitor cells and peripheral blood cells were remarkably refractory to infection by VSV. By exploiting this large difference in susceptibility to infection we successfully purged contaminating leukemic cells from cultures of peripheral blood progenitor cells (PBPC) using VSV. VSV was also able to infect and kill leukemic cells in primary samples taken from patients with multiple myeloma (MM). This study demonstrates the potential utility of VSV in the treatment, both ex vivo and in vivo, of hematologic malignancies.