Survivin Is Highly Expressed in AML Stem Cells and Predicts Poor Clinical Outcome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 238-238
Author(s):  
Bing Z Carter ◽  
Yihua Qiu ◽  
Xielin Huang ◽  
Lixia Diao ◽  
Nianxiang Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Cox Model ◽  
Survivin Expression ◽  
Leukemic Cells ◽  
White Blood Count ◽  
Expression Levels ◽  
Mutational Status

Abstract Abstract 238 Survivin, a member of the inhibitor of apoptosis (IAP) protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignant cells, including leukemic cells. We showed previously that targeting survivin expression in AML cells induces a cell proliferation defect and subsequent cell death involving the mitochondrial pathway. To assess its usefulness as a prognostic marker in AML, we profiled survivin expression in samples from 511 newly diagnosed AML patients using a validated reverse-phase protein array and correlated its protein expression levels with clinical outcomes. We found that survivin levels were higher in the bone marrow (BM) than in peripheral blood (PB) leukemic cells in 140-paired samples (p = 0.0001) consistent with its function in cell proliferation. Survivin levels did not correlate with cytogenetic groups, the mutational status of nucleophosmin or FLT-3, BM or PB blast counts, white blood count, or patient performance status. Interestingly, survivin levels were found to significantly predict shorter overall (P = 0.016) and event-free (P = 0.023) survival in multivariate Cox model analysis. Age, gender, white blast count, BM blasts, cytogenetics, and survivin levels were included in the final model. The groups expressing low and high survivin were defined by the median value. This was also true when data sets from BM and PB were analyzed separately. Further, we found that survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk AML blasts and total CD34+ cells (2.06 and 1.91 fold, respectively, P < 0.001) when we analyzed survivin expression in 37 samples of isolated CD34+38− AML cells (see Figure). These results suggest that survivin expression levels are prognostic in AML and that survivin is overexpressed in AML stem/progenitor cells, may play an important role in AML stem cells, and may thus be an important target in AML therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Survivin is highly expressed in CD34+38− leukemic stem/progenitor cells and predicts poor clinical outcomes in AML

Blood ◽  
2012 ◽  
Vol 120 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Bing Z. Carter ◽  
Yihua Qiu ◽  
Xuelin Huang ◽  
Lixia Diao ◽  
Nianxiang Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Progenitor Cells ◽  
Clinical Outcomes ◽  
Myeloid Leukemia ◽  
Cox Model ◽  
Survivin Expression ◽  
Leukemic Cells ◽  
Strongly Correlated ◽  
Apoptosis Protein ◽  
Sample Set

Abstract Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34+38− AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk blasts and total CD34+ AML cells (P < .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.

Role of hERG1 K+ Channels as a Positive Regulator In SDF-1alpha-Mediated Proliferation of Leukemia Cells and Leukemia Stem/Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4786-4786
Author(s):  
Fang Zheng ◽  
Huiyu Li ◽  
Fang Liu ◽  
Wen Du ◽  
Shiang Huang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Leukemic Cell ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
K Channels

Abstract Abstract 4786 Background: Mounting evidence that leukemia stem cells (LSCs) occupy and receive important signals from specialized areas (“niches”) that alter the stromal microenvironment and disrupt normal hematopoiesis. The innovative therapeutic strategies focus on targeting of microenvironmental interactions in leukemia. Therefore, it is important to fully elaborate the mechanisms of microenvironment- mediated leukemogenesis. Stromal-cell derived factor-1alpha (SDF-1à) is the main cytokine produced by bone marrow stromal cells. The SDF-1à/CXCR4 axis specifically mediates homing and migration of leukemic blasts. While our previous work has shown that SDF-1à significantly increases hERG1 K+ tail current and a specific hERG1 K+ channels inhibitor significantly blocks SDF-1à- induced migration of leukemic cells. In fact, recent studies suggested that the human ether à-go-go-related gene (HERG) K+ channels are constitutively expressed in AML stem/progenitor cells, and regulate cell proliferation as well as clinical prognosis. Here we investigate the hypothesis that a new leukemic blast–stromal interaction is mediate by hERG1 K+ channels and SDF-1à. Methods: Proliferation assay, apoptosis and cell cycle analysis were used to analyze effects of E-4031(a specific hERG1 K+ channels inhibitor) in the presence of SDF-1à on leukemia cell lines HL-60. RT–PCR and western blot analysis were used to determine changes in herg1 expression and Wnt/β-catenin signaling pathway in response to SDF-1à in the presence and absence of E-4031. Primary leukemias obtained from the bone marrow of de novo AML patients (n=6) at diagnosis. Mononuclear cells were isolated from the samples using Ficoll-Paque density gradient separation, and cultured with SDF-1à in the presence and absence of E-4031. AML colony-forming cell (CFC) assays and flow cytometry were performed to assess the effects of E-4031 in the presence of SDF-1à on LSCs. Results: SDF-1a enhanced cell proliferation in a dose-dependent manner. The maximal increase by 1.6 times was obtained for 100ng/ml. While this effect was impaired by E-4031, which significantly impaired cell proliferation induced by SDF-1a with a concentration of 100ng/mL by (40.3±8.4)%. In addition, E-4031 inhibited SDF-1a-stimulated leukemic cell proliferation by inducing G0/G1 arrest. Cell apoptosis analysis revealed that either E-4031 or SDF-1a has direct effect on HL-60 cell apoptosis. Unexpected, there was no significant synergistic effect upon apoptosis. After exposures to 100ng/ml SDF-1à, hERG1 mRNA and protein levels increased significantly, by approximately 1.5-fold above control levels. Moreover, SDF-1a increased the expression of Wnt/β-catenin target genes, including β-catenin, cyclin-D1, and c-myc. Interestingly, this manner was abolished by E-4031. The presence of progenitor cells was evaluated by plating suspension cells cultured with SDF-1a in CFC assays. E-4031 decreased numbers of CFC in suspension to 77.3%. Upon expansion with SDF-1a, E-4031 resulted in a significant reduction in the number of progenitors to 31.8%. The effects on LSCs were determined on phenotypically described stem cells from AML. Treatment with 1μ M E-4031 for 48 hours inhibited the proliferation of LCSs compared with untreated controls, a mean viability of 11.8% for CD34+CD38- and 10.4% for CD34+CD38+. In contrast, a significant decrease in the viability of stem cells after E-4031 in the present of SDF-1a treatment, with only 9.6% for CD34+CD38- and 9.5% for CD34+CD38+. Conclusions: Initial studies provided evidence that the hERG1 K+ channels and SDF-1 emerged as mediators of stromal/leukemic cell interactions, which largely contribute to the proliferation mediated by the microenvironment. Likewise, other components of bone marrow microenvironment, such as Wnt/β-catenin signaling pathway, may modulate hERG1 K+ channels in leukemic cells. Taken together, these results provided rationale for studies of new molecular events involved in bone marrow microenvironment and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Early and Late Leukemic Stem Cells in Murine Leukemia Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1544-1544
Author(s):  
Fang Dong ◽  
Haitao Bai ◽  
Shanshan Zhang ◽  
Xiaofang Wang ◽  
Jinhong Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Leukemic Cells ◽  
Mouse Bone Marrow ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Murine Leukemia

Abstract Acute leukemia is uncontrolled proliferation of leukemic stem cells (LSCs). Murine models of leukemia suggest that LSCs arise from lineage-committed progenitor cells. However, whether LSCs also directly arise from long-term (LT) and short-term (ST) hematopoietic stem cells (HSCs) and other hematopoietic progenitor cells (HPCs) and whether differentiation potentials influence the leukemia types are poorly understood. In this study, we used two murine leukemia models (AML with MLL-AF9 fusion protein and T-ALL with Notch-1 intracellular domain, ICN-1). Two HSC and three HPC populations were sorted from B6 (CD45.1) mouse bone marrow (BM) by flow cytometry: HSC1, CD150+CD41-CD34-Lin-Sca-1+c-Kit+ (LSK) cells; HSC2, CD150-CD41-CD34-LSK cells; HPC1, CD150+CD41+CD34-LSK cells; HPC2, CD150+CD41+CD34+LSK cells; HPC3, CD150-CD41-CD34+LSK cells. HSC1, HSC2, HPC1, and HPC3 are enriched in LT-HSCs, ST-HSCs, repopulating common myeloid progenitors, and lymphoid-primed multipotent progenitors, respectively. 400-600 cells were sorted from each population, pre-stimulated for 24 hrs, and transduced with MLL-AF9 or ICN-1 retrovirus for another 24 hrs. Cells were mixed with 3-5 x 105 BM cells (CD45.2), and injected into the lethally irradiated mice (CD45.2). The recipient mice were monitored by detection of GFP+ cells in the peripheral blood (PB). When the recipient mice showed > 50% GFP+ cells in the PB or became ill, mice were killed and analyzed for leukemia. In the MLL-AF9 AML model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 6 weeks after transplantation. Leukemic cells in PB and BM appeared positive for Mac-1/Gr-1, but negative for CD3 and B220. We detected a new LSC population in the BM: Lin-Mac-1+c-Kit+Sca-1+CD150-CD16/32+ cells. Both CD34-negative and CD34-positive cells were detected in this population. We named this population as "early LSCs" because it was phenotypically similar to ST-HSCs and/or LMPPs in normal mouse BM cells except Mac-1 expression. Early LSCs differed from previously identified LSCs (L-GMP) because they are Sca-1-positive. We now consider L-GMP as one of "late LSC" types. Early LSCs formed leukemic colonies in vitro and initiated the AML in serial transplantation. These results show the similar AML develops regardless of HSC and HPC types transduced. Both early and late LSCs likely play a role in establishment of AML. In the ICN-1 T-ALL model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 4 weeks after transplantation. Most leukemic cells in PB and BM appeared positive for CD4/CD8, but negative for Mac-1/Gr-1 and B220. We did not detect early LSCs in BM. To identify late LSCs in BM, we injected the CD3+CD8+CD4- and CD3+CD8+CD4+ cells into the sub-lethally irradiated mice. T-ALL developed from both populations (median latency, 23 vs 35 days). These results show the similar T-ALL develops regardless of HSC and HPC types transduced. In this case, late LSCs can be directly generated without early LSCs from HSC or HPC populations. Taken together, we found that the similar types of leukemia develop regardless of different types of initiating cells in both models. We also found new LSC populations, early LSCs in the AML model and late LSCs in the T-ALL model. Both early and late LSCs were able to re-initiate leukemia after secondary transplantation. In conclusion, highly purified murine HSCs and HPCs were used for the first time to initiate leukemia. Both MLL-AF9 AML and ICN-1 T-ALL models suggest that gene mutations at all differentiation stages from HSCs to HPCs potentially induce the similar types of leukemia. Of note is that distinct lineage differentiation potentials of HSCs and HPCs do not affect leukemia types. More importantly, early LSCs may serve as the earliest event in leukemogenesis in AML. In the case of ALL, gene mutations seem carried over until reaching the developmental stage of late LSCs. Our results suggest that both early and late LSCs should be eradicated to achieve complete remission and prevent relapse. Disclosures No relevant conflicts of interest to declare.

Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2733-2740 ◽  
Author(s):  
Baijun Fang ◽  
Chunmei Zheng ◽  
Lianming Liao ◽  
Qin Han ◽  
Zhao Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Blood Cells ◽  
Fusion Gene ◽  
Fetal Liver ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Leukemic Cells

AbstractOverwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly, the BCR/ABL fusion gene, which is present in chronic myelogenous leukemia (CML), was also detected in the endothelial cells of patients with CML, suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1–positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome–positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells, thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells.

scholarly journals Targeting Survivin Expression Induces Cell Proliferation Defect and SubsequentCell Death Involving Mitochondrial Pathway in Myeloid Leukemic Cells

Cell Cycle ◽  
2003 ◽  
Vol 2 (5) ◽  
pp. 486-491 ◽  
Author(s):  
Bing Z. Carter ◽  
Rui-Yu Wang ◽  
Wendy D Schober ◽  
Michele Milella ◽  
David Chism ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Survivin Expression ◽  
Mitochondrial Pathway ◽  
Leukemic Cells

Chemical Derivatives of the Anti-Leukemia Stem Cell Compound 4-Benzyl-2-Methyl-1,2,4-Thiadiazolidine-3,5-Dione (TDZD-8) with Improved Activity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3764-3764
Author(s):  
Monica L. Guzman ◽  
Shama Nasim ◽  
Marlene Balys ◽  
Cheryl A Corbett ◽  
Peter A Crooks ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Water Solubility ◽  
Conflicts Of Interest ◽  
Membrane Integrity ◽  
Hematopoietic Cells ◽  
Hematologic Malignancies ◽  
Leukemic Cells ◽  
Chemical Derivatives ◽  
High Concentrations ◽  
Rapid Kinetics

Abstract Abstract 3764 Poster Board III-700 We have recently described the novel and unique anti-leukemic properties of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). Indeed, TDZD-8 was shown to eradicate leukemia at the bulk, stem, and progenitor level with rapid kinetics (typically < 2 h) with minimal toxicity to normal hematopoietic cells. Moreover, the cytotoxic activity of this drug is observed only in hematologic malignancies. The precise mechanism of TDZD-8 is not fully appreciated, but the compound has been shown to inhibit NFkappaB, GSK3beta, protein kinase C, FMS-like tyrosine kinase (Flt3), AKT and KDR. In addition, TDZD-8 rapidly depletes free thiols and appears to disrupt membrane integrity. Interestingly, TDZD-8 results in rapid mitochondrial swelling, followed by vacuole formation and accumulation. Despite possessing potent and specific anti-leukemia activity, the clinical utility of TDZD-8 is limited by the need for high concentrations (20 microM) and poor solubility. Therefore, to improve the pharmacological properties of TDZD-8, we initiated efforts to generate derivatives with greater anti-leukemia activity at lower concentrations and with greater water-solubility. To this end, an extensive structure-activity relationship study was carried out to examine the effects on anti-leukemic activity resulting from introduction of substituents at the C-2 and C-4- positions of the thiadiazolidine ring of TDZD-8, and of replacement of the TDZD ring with isosteric scaffolds. In total, we have screened more than 60 new analogs. Preliminary screens were performed using two different AML cell lines, evaluating the LD50 for each of the analogs relative to the parental compound. We found that introducing substitutuents into the main TDZD ring resulted in loss of anti-leukemic activity of the compounds. Moreover, substitution in the benzyl/phenyl ring does not affect anti-leukemia activity of the TDZD analogs. Importantly, we found that N-2 halogenoethyl analogs, exhibit exceptional activity against leukemic cells. Of the halogenoethyl analogs evaluated, the iodoethyl analog TD-361 was the most active compound with an LD50 of 0.49 microM in MV-411 cells. Compounds exhibiting increased anti-leukemia activity were subjected to further testing in phenotypically-defined AML stem/progenitor cells. All analogs demonstrated efficacy in primary AML cells at lower concentrations than TDZD-8. Moreover, these analogs still maintained the rapid kinetics observed with TDZD-8. Finally, we performed colony assays to determine the effect of new analogs on progenitor cells from normal and leukemic cells. These studies demonstrated that the more active TDZD analogs retained the selective ability of TDZD-8 to abate AML progenitor cells without harming normal hematopoietic cells. In summary, we have identified the critical chemical moieties for the observed activity of TDZD-8, and have also discovered analogs with improved anti-leukemia activity. Going forward, the most active derivatives are being optimized for water-solubility and will then be evaluated using primary human AML specimens engrafted into immune deficient mice. Based on evidence to date, we propose that the TDZD family of compounds may represent a new class of drugs for the treatment of leukemia and related hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Predominant Complement Mediated Lysis of B-CLL Cells by Therapeutic MAbs Rituximab and Campath-1H in Whole Blood Assays.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3445-3445 ◽  
Author(s):  
Josee Golay ◽  
Luca Bologna ◽  
Elisa Gotti ◽  
Alessandro Rambaldi ◽  
Renato Bassan ◽  
...  
Keyword(s):  
Target Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Human Igg ◽  
Blood Samples ◽  
Expression Levels ◽  
Cd20 Expression

Abstract Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit (<0.1%) in most cases analysed, suggesting that ADCC in the circulation is not a major mechanism of lysis in this disease subtype. Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.

The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1209-1209
Author(s):  
Chun Shik Park ◽  
Koramit Suppipat ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

Distinct Roles for the NF-κB Pathway In Myeloid and Lymphoid Transformation and Leukemogenesis by BCR-ABL.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1225-1225
Author(s):  
Mo-Ying Hsieh ◽  
Richard A. Van Etten
Keyword(s):  
Stem Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Myeloproliferative Neoplasm ◽  
Dominant Negative ◽  
Lymphoid Cells ◽  
Leukemic Cells ◽  
Mutant Form ◽  
B Lymphoid

Abstract Abstract 1225 The BCR-ABL tyrosine kinase, product of the t(9;22) Ph chromosome, activates multiple signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have shown that NF-κB is activated in BCR-ABL-expressing cell lines and contributes to transformation of primary B-lymphoid cells by BCR-ABL (Reuther et al., Genes Dev. 1998;12:968), but the mechanism of activation has not been defined (Kirchner et al., Exp. Hematol. 2003;31:504), and importance of NF-kB to myeloid and lymphoid leukemogenesis by BCR-ABL is unknown. To interrogate the role of NF-κB in BCR-ABL-mediated transformation, we utilized a super-repressor mutant form of IκBα (IκBαSR), which has been used to block NF-κB nuclear localization and transactivation by constitutively sequestering NF-κB in the cytoplasm. Using retrovirus co-expressing BCR-ABL and IκBαSR, we found that IκBαSR blocked nuclear p65/RelA expression and inhibited the IL-3 independent growth of Ba/F3 cells and primary B-lymphoid cells transformed by BCR-ABL. The effect of NF-κB inhibition was primarily on proliferation rather than on cell survival, as there was no increase in apoptosis in cells expressing IκBαSR. When primary bone marrow cells were transduced and transplanted under conditions favoring induction of B-ALL or CML-like myeloproliferative neoplasm in recipient mice, co-expression of IκBαSR significantly attenuated disease development and prolonged survival of diseased mice. Molecular analysis of these leukemias demonstrated that NF-κB inhibition decreased the frequency of leukemia-initiating (“stem”) cells in the CML model, but not in the B-ALL model, and was associated with decreased expression of c-Myc, an NF-κB target. To clarify the mechanism of activation of NF-κB in BCR-ABL-expressing cells, we targeted two upstream kinases that negatively regulate IκBα, IKKα/IKK1 or IKKβ/IKK2. To accomplish this, we engineered retroviruses co-expressing BCR-ABL and kinase-inactive, dominant-negative mutants of IKK1 (IKK1KM) or IKK2 (IKK2KM). Co-expression of either IKK mutant inhibited both B-lymphoid transformation and leukemogenesis by BCR-ABL, as well as induction of CML-like MPN, with IKK1 inhibition more effective than IKK2. Together, these results demonstrate that NF-κB is activated in part through the canonical IKK pathway in BCR-ABL-expressing leukemia cells, and that NF-κB signaling plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL. In CML, NF-κB may play a role for in generation and/or maintenance of leukemic stem cells. These results validate IKKs as targets for therapy in Ph+ leukemias, and motivate the evaluation of small molecule IKK inhibitors in these diseases. Disclosures: No relevant conflicts of interest to declare.

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