Preclinical Studies of Cytotoxicity, Drug Synergy and Biological Correlates of Clofarabine Against Infant Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4342-4342
Author(s):  
Aarthi Jayanthan ◽  
Anjali Singh ◽  
Victor A Lewis ◽  
Aru Narendran
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Drug Combination ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Preclinical Studies ◽  
Drug Synergy ◽  
Mitochondrial Integrity ◽  
Combination Studies ◽  
Infant Leukemia

Abstract Abstract 4342 Preclinical Studies of Cytotoxicity, Drug Synergy and Biological Correlates of Clofarabine Against Infant Leukemia Cells. Infants with leukemia, specifically those who relapse on frontline therapy, are extremely difficult to cure and are candidates for novel therapies that induce remission, allowing them to proceed to transplantation. The unique molecular, growth and chemoresistance properties of infant acute lymphoblastic leukemia (iALL) allow for focused preclinical studies within this group. Clofarabine (Clolar), an anti-neoplastic purine nucleoside analog, has shown significant efficacy in older children with refractory lymphoblastic leukemia. Its safety profile has more recently been established in Phase I and II single-agent trials. In addition to its anti-metabolite action, clofarabine appears to disrupt the integrity of mitochondrial membranes and activates pathways of programmed cell death, adding to its theoretical potential to synergize with agents that interfere with mitochondrial integrity. Methods: Primary leukemic cells, cell lines derived from iALL patients and cell lines carrying the molecular abnormalities commonly found in iALL, were used in this study (n=10). Karyotypic abnormalities of these cells include: t(11;19) (q23;p13), t(9;11)(p21;q23) and t(4;11)(q21;q23). With respect to Flt-3 expression, cell lines demonstrating wild-type, internal tandem duplication (ITD) and over-expression phenotypes were also included in this panel. Primary infant AML samples (n=2) and the infant AML cell line TIB-202 (THP-1) containing the t(9;11)(p21;q23) rearrangement and MLL-AF9 fusion gene were also included. ALL cell lines derived from pediatric patients (n= 5) were evaluated in parallel. Stromal cells established from normal bone marrow specimens and peripheral blood mononuclear cells were evaluated under identical conditions for assessment of non-specific toxicity. An increasing concentration of clofarabine was added to leukemic and control cells (104 cells per well, in 96 well plates) cell lines. Over the following four days, cell growth inhibition was measured by the Alamar blue assay. For drug combination studies, leukemia cells were incubated with a panel of conventional and targeted therapeutic agents (n=12) alone or in combination with clofarabine (IC10 or IC25 concentrations). Growth inhibition under each condition was measured and combination indices were calculated according to established methods. Induction of apoptosis and the release of mitochondrial mediators were measured by Western blot analysis. Alteration in mitochondrial integrity was evaluated by immunocytochemistry for fluorescent labeled anti-mitochondrial Hsp70 and real-time imaging. Results and Discussion: Clofarabine inhibited growth of all iALL cells tested with IC50 values ranging from 0.1 μ M to 0.01 μ M. Primary iAML cells were found to be most sensitive to clofarabine. For iALL cell lines the highest IC50 value was found in Bel-1 cells, expressing a t(4;11)(q21;q23) karyotype. Drug combination studies showed significant synergy with 17-AAG (Hsp90 inhibitor, CI 0.7), sorafenib (CI 0.12), bortezomib (CI 0.3) and rapamycin (CI 0.2). No drug combinability was noted, with conventional alkylating agents and antimetabolites. Interestingly, the therapeutic opioid methadone (D,L-methadone hydrochloride), used extensively in the treatment of cancer pain and opioid addiction, showed significant synergy with clofarabine at low concentrations (CI 0.74, range 0.66 – 0.79 μ M). Incubation of cells with clofarabine (IC25) for 48 hours resulted in detectable activation of caspase 9 and cleavage of PARP. We demonstrate the ability of clofarabine to induce cytotoxicity against a panel of leukemia cells that carry the molecular aberrations and growth properties seen in iALL. We also present data on the biological correlates and synergistic effects of clofarabine with other anti-leukemic agents. Of particular interest is the synergy with methadone, which has been shown previously to affect mitochondrial activity in leukemia cells. Data presented in study provide key initial data to construct effective xenograft studies and to formulate a clofarabine based treatment protocol for iALL in the near future. Disclosures: Narendran: Genzyme Canada: Research Funding.

AT9283, A Novel Aurora Kinase/Jak2 Inhibitor Demonstrates Activity against Refractory Infant Leukemia Cells: Studies On Growth Inhibition, Biological Correlates, Drug Synergy and Effects On Leukemia Stem-Like Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3078-3078
Author(s):  
Shamim Lotfi ◽  
Aarthi Jayanthan ◽  
Victor A. Lewis ◽  
Greg Guilcher ◽  
Matthew S Squires ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Aurora Kinase ◽  
Marrow Stromal Cells ◽  
Leukemia Cells ◽  
Combination Studies ◽  
Infant Leukemia

Abstract Abstract 3078 Poster Board III-15 Leukemia in children less than 1 year of age confers a poor prognosis, despite intensification of therapy. These leukemias possess unique biologic characteristics including the presence of mixed-lineage leukemia (MLL) gene rearrangement and high expression of Fms-like tyrosine kinase 3 (FLT3). AT9283, a potent inhibitor of Aurora A and B kinases, JAK2, JAK3, and mutant Abl Kinase, has demonstrated inhibition of multiple solid tumor cell lines in vitro and in mouse xenograft models. Aurora kinase inhibition has been shown to inhibit cancer cell growth by interfering with the mitotic apparatus. We investigated the activity of AT9283 against cell lines derived from refractory infant leukemia cells to identify its efficacy in a future treatment protocol. Method Five cell lines derived from infant leukemia cells were used (ALL: BEL1, KOPN8, KCCF2, B1 and AML: TIB202). We also included the cell line SEM that was derived from a 5 year old child with t (4;11) MLL-AF4 preB-ALL. Normal bone marrow stromal cells were used to evaluate cytotoxicity against non-malignant cells. AT9283 was provided by Astex Therapeutics Ltd. (Cambridge, UK). Approximately 1×104 cells per well were seeded in 96-well plates and incubated with increasing concentrations of AT9283, alone or in combination with a panel of conventional and novel therapeutic agents. After four days, cell survival was measured by Alamar blue assay and IC50 values and combination indices were calculated. Stem-like cells were quantified by the distribution of ALDH bright cells by Aldefluor assay (Stem cell technologies) and characterized by conventional clonogenic assays. Alterations in cell-signaling pathways and survival proteins were measured by Western blot analysis using total and phospho-specific antibodies. Results AT9283 inhibited the growth of all five cell lines with a 10 fold variation in IC50 within cell lines (IC50 range, 0.1 to 0.01 μM). There was a corresponding increase in the number of cells displaying a polyploid phenotype, an effect of aurora kinase inhibition. No significant cytotoxicity against bone marrow stromal cells was seen under the experimental conditions used in this study (IC50 > 10 μM). Changes in the activation and expression of a variety of intracellular proteins were noted, including the down regulation of activated ERK1/2, MYC and AKT within 10 minutes of exposure to the agent. An increase in the activated form of RAF and ATF2 was observed immediately after drug exposure. Importantly, a significant decrease in the level of constitutive pFLT-3 was demonstrated. A concurrent increase in cleaved PARP was also noted, indicating the initiation of apoptosis. In combination studies, the HDAC inhibitor Apicidin showed synergy across all cell lines (CI range: 0.07 to 0.62). A decrease in ALDH bright stem-like cells was observed in a dose dependent manner, up to 50% over 24 hours at IC50 concentrations. Conclusions Our in vitro studies show that AT9283 significantly decreases the growth and survival of infant leukemia cell lines. Importantly, AT9283 potently induces FLT3 de-phosphorylation, inhibiting a critical growth stimulatory pathway of infant ALL cells. We have identified changes in a number of signaling and apoptotic molecules that can provide a panel of markers for biological correlative analysis for drug activity in vivo. Also, the drug combination studies demonstrate the potential of HDAC inhibition to synergize with the activity of this agent. Finally, the effect on stem-like cells provides a rationale and critical preclinical data for the formulation of an effective clinical trial for the treatment of infants with refractory ALL. Disclosures Squires: AstexTherapeutics Ltd: Employment.

scholarly journals Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 665
Author(s):  
Margot S.F. Roeten ◽  
Johan van Meerloo ◽  
Zinia J. Kwidama ◽  
Giovanna ter Huizen ◽  
Wouter H. Segerink ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Proteasome Subunit

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Preclinical evaluation of the ETS inhibitor TK216 against relapsed and refractory childhood leukemia.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 10033-10033
Author(s):  
Ritul Sharma ◽  
Satbir Thakur ◽  
Mohit Jain ◽  
Chunfen Zhang ◽  
Anne-Marie Langevin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Drug Combination ◽  
Childhood Leukemia ◽  
Sensitive Cell Line ◽  
Pediatric Leukemia ◽  
Drug Synergy ◽  
Molecular Abnormalities ◽  
Anti Cancer ◽  
Combination Studies

10033 Background: Although survival rates have improved in the recent past, relapse and refractory disease remain a significant cause of death in children with leukemia. This calls for an urgent need for the development of novel therapies that could effectively treat leukemias in children. The E26 transformation specific (ETS) family of transcription factors regulate various normal cellular functions but are abnormally expressed in various cancers, including leukemia. TK216 is an ETS inhibitor, that has shown pre-clinical activity and clinical efficacy in solid tumors. In this study, we explore the feasibility of using TK216 as a therapeutic agent for the treatment of high risk refractory pediatric leukemia. Methods: A panel of pediatric leukemia derived cell lines and primary blast cells representing a spectrum of molecular abnormalities seen in pediatric leukemia were treated in vitro with TK216 to determine cytotoxicity. Normal lymphocytes were used as controls and cell viability was determined 72 hours post-treatment by Alamar blue assay. The induction of tumor cell apoptosis and target modulation were detected by Western blotting. Alterations in the cell cycle were assessed by FACS analysis with PI staining. Drug combination studies were carried out with established anti-leukemic agents to identify synergy for greater therapeutic efficiency. Results: TK216 decreased cell viability in leukemia cells compared to normal lymphocyte controls in a dose-dependent manner with variations in sensitivity noted with inherent molecular abnormalities. The IC50 values observed ranged from 0.22 µM for the most sensitive cell line, MV4-11 to 0.95 µM for least sensitive cell line, SUP-B15. Apoptosis induction upon TK216 treatment was confirmed by PARP cleavage and caspase 3 activation. Cell cycle analysis demonstrated increased sub-G1 population of cells after TK216 treatment. A strong correlation between sub-G1 population and sensitivity of the cell line towards TK216 (47% in MV4-11 vs 3.72% in SUP-B15) was observed. Screening of a panel of 200 FDA approved anti-cancer agents in drug combination studies identified potential agents for drug synergy. Significant drug synergy was noted with TK216 in combination with the epigenetic modifier 5-azacytidine and the Bcl-2 inhibitor, Venetoclax. [Combination Index for Venetoclax and TK216, mean = 0.65 for MV4-11 and 0.33 for SUP-B15]. Conclusions: Data from our study demonstrate that the ETS inhibitor TK216 induces apoptosis and cell cycle arrest in pediatric leukemia cells at physiologically relevant concentrations. Our combination studies identified distinct anti-cancer agents that could be used for developing effective drug combination regimens with TK216. Overall, our findings provide essential preclinical data for the consideration of TK216 in early phase clinical trials for the treatment of selected high-risk and refractory childhood leukemia.

Metabolic Profile Of Leukemia Cells Influences Treatment Efficacy Of L‑Asparaginase

2019 ◽  
Author(s):  
Katerina Hlozkova ◽  
Alena Pecinova ◽  
David Pajuelo Reguera ◽  
Marketa Simcikova ◽  
Lenka Hovorkova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Metabolic Profile ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Lower Sensitivity ◽  
Leukemia Cell Lines

Abstract Background Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.Methods Altogether, 19 leukemia cell lines and primary leukemia cells from 11 patients were used. Glycolytic function and mitochondrial respiration were measured using Seahorse bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.Results We characterized the basal metabolic state of the cells derived from different leukemia subtypes using cell lines and primary samples and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile, which is mainly driven by their hematopoietic lineage of origin from which they derived. However, majority of lymphoid leukemia cell lines and patients with lower sensitivity to L-asparaginase clustered regardless their hematopoietic phenotype together with myeloid leukemias. Furthermore, we observed a correlation of specific metabolic parameters with sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in other tested cytostatic drugs.Conclusions These data support the prominent role of the cell metabolism in the treatment effect of L-asparaginase. Based on these findings metabolic profile could identify leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization.

Abstract 5422: Drug combination studies in a panel of 30 cell lines established from patient-derived tumor xenograft

2010 ◽  
Author(s):  
Gerhard Kelter ◽  
Victoria Smith ◽  
Thomas Metz ◽  
Heinz-Herbert Fiebig ◽  
Thomas Beckers
Keyword(s):  
Cell Lines ◽  
Drug Combination ◽  
Tumor Xenograft ◽  
Combination Studies

scholarly journals Comparative cellular pharmacology of daunorubicin and idarubicin in human multidrug-resistant leukemia cells

Blood ◽  
1992 ◽  
Vol 79 (12) ◽  
pp. 3267-3273 ◽  
Author(s):  
E Berman ◽  
M McBride
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Fold Increase ◽  
Multidrug Resistant ◽  
Fresh Medium ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Cellular Pharmacology

Abstract We examined the effect of daunorubicin (DNR), the new anthracycline derivative idarubicin (IDR), and verapamil on two leukemia cell lines that displayed the multidrug resistant (MDR) phenotype and used laser flow cytometry to quantitate intracellular anthracycline content. The vinblastine-resistant human lymphoblastic leukemia cell line CEM-VBL demonstrated minimal DNR uptake; simultaneous incubation with verapamil and DNR increased intracellular DNR uptake fourfold. IDR uptake was 10 times more rapid in these cells and simultaneous incubation with IDR and verapamil resulted in only a 1.2-fold increase of intracellular IDR. Similar results were observed in the vincristine-resistant human myeloid leukemia cell line HL-60/RV+. Intracellular retention of DNR and IDR was also measured in each cell line. In CEM-BVL cells, 38% of the original DNR concentration remained after a 2-hour resuspension in fresh medium compared with 71% of the original IDR concentration. In HL- 60/RV+ cells, 36% of the DNR concentration remained compared with 51% of the IDR concentration. After incubation of CEM-VBL and HL-60/RV+ cells with DNR for 1 hour followed by resuspension in fresh medium plus verapamil, intracellular DNA retention increased 5- and 5.2-fold, respectively. However, incubation of these cells for 1 hour with IDR followed by resuspension in fresh medium plus verapamil resulted in only a 1.6- and 2.4-fold increase in intracellular IDR retention. Lastly, clonogenic experiments were performed to correlate intracellular anthracycline content with cytotoxicity. DNR alone had a minimal effect on the clonogenic growth of CEM-VBL cells, whereas the combination of DNR plus verapamil resulted in approximately 80% growth inhibition. However, incubation of these cells with IDR alone resulted in greater than 95% growth inhibition. These results suggest that IDR may be more effective than DNR in leukemia cells that display the MDR phenotype.

scholarly journals Unbiased High-Throughput Drug Combination Pilot Screening Identifies Synergistic Drug Combinations Effective against Patient-Derived and Drug-Resistant Melanoma Cell Lines

2020 ◽  
pp. 247255522097091
Author(s):  
David A. Close ◽  
John M. Kirkwood ◽  
Ronald J. Fecek ◽  
Walter J. Storkus ◽  
Paul A. Johnston
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Melanoma Cell ◽  
High Throughput ◽  
Drug Combination ◽  
High Throughput Screening ◽  
Dna Methyltransferase ◽  
Cross Resistance ◽  
Braf Mutations ◽  
Melanoma Cell Lines

We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for BRAF and three with V600E- BRAF mutations. We conducted a pilot drug combination (DC) high-throughput screening (HTS) of 45 pairwise 4×4 DC matrices prepared from 10 drugs in the PDMCL assays: two B-Raf inhibitors (BRAFi), a MEK inhibitor (MEKi), and a methylation agent approved for melanoma; cytotoxic topoisomerase II and DNA methyltransferase chemotherapies; and drugs targeting the base excision DNA repair enzyme APE1 (apurinic/apyrimidinic endonuclease-1/redox effector factor-1), SRC family tyrosine kinases, the heat shock protein 90 (HSP90) molecular chaperone, and histone deacetylases. Pairwise DCs between dasatinib and three drugs approved for melanoma therapy—dabrafenib, vemurafenib, or trametinib—were flagged as synergistic in PDMCLs. Exposure to fixed DC ratios of the SRC inhibitor dasatinib with the BRAFis or MEKis interacted synergistically to increase PDMCL sensitivity to growth inhibition and enhance cytotoxicity independently of PDMCL BRAF status. These DCs synergistically inhibited the growth of mouse melanoma cell lines that either were dabrafenib-sensitive or had acquired resistance to dabrafenib with cross resistance to vemurafenib, trametinib, and dasatinib. Dasatinib DCs with dabrafenib, vemurafenib, or trametinib activated apoptosis and increased cell death in melanoma cells independently of their BRAF status or their drug resistance phenotypes. These preclinical in vitro studies provide a data-driven rationale for the further investigation of DCs between dasatinib and BRAFis or MEKis as candidates for melanoma combination therapies with the potential to improve outcomes and/or prevent or delay the emergence of disease resistance.

Identification of Sensitizing Targets to the Hypomethylating Agent 5-Azacytidine in Acute Myeloid Leukemia Cells Using RNAi-Based Functional Genomics Screening

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2665-2665
Author(s):  
Raoul Tibes ◽  
Ashish Choudhary ◽  
Amanda Henrichs ◽  
Sadia Guled ◽  
Irma Monzon ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Statistical Significance ◽  
Molecular Targets ◽  
Leukemia Cells ◽  
Hypomethylating Agents ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract • Hypomethylating agents like 5-Azacytidine (5Aza) have become an effective therapy for myelodysplastic syndromes (MDS) and show promise in acute myeloid leukemia (AML). In AML, complimentary mechanisms including epigenetic silencing of growth controlling genes, i.e. tumor suppressors, and activation of kinases contribute to malignant transformation. In order to enhance the therapeutic potential of epigenetic therapies, we developed a high-throughput RNA interference (HT-RNAi) platform for large-scale transient gene silencing in acute myeloid leukemia cells. This assay allows for the first time to individually silence hundreds or thousands of genes in combination with 5Aza to identify molecular targets whose inhibition enhances the anti-leukemic effect of hypomethylating agents. As part of assay development for HT-RNAi, ten AML cell lines were used to determine the median inhibitory concentration (IC50) of 5Aza for each AML cell lines. Furthermore, the ten cell lines were tested with a panel of cationic lipid transfection reagents at varying weight to volume (wt:vol) ratios to determine the optimal siRNA transfection conditions. Results from these studies identified two AML cell lines TF1 and ML4, which were advanced into kinome-epigenetic RNAi screens. Using a lipid-based method, cells were reverse transfected for 48hrs with 2 different siRNA sequences per gene targeting a total of 572 kinases. After 48hrs, 5Aza at the calculated IC25 was added for an additional 72 hrs and cell proliferation was measured using a luminescence-based assay. Data was background corrected and analyzed using the B-score method to report the strength and statistical significance of growth inhibition compared to controls. A B-score of <−2 indicates statistical significance with p<0.05 (>95% confidence); a B-score <−1.5 provides >87% confidence and was used as lowest cutoff given that screens are focused and contain validated siRNA to kinases. Analysis of two independent RNAi kinome screens, one in TF1 and the other in ML4, in combination with 5Aza, identified six and eleven kinases respectively whose silencing by two different siRNA sequences (2× coverage) potentiated the effects of 5Aza at B-score <−1.5. In ML4 cells 2 kinases were highly significant with a B-score for both siRNA <−2. Six kinases were common targets in both cell lines with significant growth inhibition at a B-score for both siRNA of at least <−1.5 making these kinases potential important modifiers of response to 5Aza. In summary, initial kinome RNAi screens in myeloid cells identified specific kinases as potential sensitizing targets to hypomethylating agents. Moreover, functional genomic RNAi screens provide a fast and attractive approach to identify molecular targets in AML for the rational development of combination therapies with hypomethylating agents as well as other drug classes.

Evaluation of CD9 as a Candidate Leukemia Stem Cell Marker In Childhood Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3241-3241
Author(s):  
Noriko Satake ◽  
Astra Chang ◽  
Bridget McLaughlin ◽  
Sara Bauman ◽  
James Chan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Raman Spectroscopy ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Heterogeneous Group ◽  
Leukemia Cells ◽  
Nsg Mice ◽  
Primary Leukemia

Abstract Abstract 3241 Leukemia cells are believed to arise from leukemia stem cells (LSC). It is also known that LSC are responsible for relapse in certain types of leukemia, such as acute myeloid leukemia (AML). However, the existence and role of LSC in acute lymphoblastic leukemia (ALL) is unclear. CD9 was reported to be a marker for LSC in B-ALL using cell lines (Nishida H. et al., 2009). CD9 is a tetraspanin and is believed to be involved in cell adhesion, motility, and signaling events. It is also involved in metastasis; however, the mechanisms are unknown. Since childhood ALL is a heterogeneous group of diseases and cell lines can be different from primary leukemia cells, we tested the role of CD9 as a candidate LSC marker using primary precursor B (preB) ALL cells from pediatric patients. Two methods, Raman spectroscopy and serial transplantation of sorted leukemia cells in NOD/SCID/IL2R g null (NSG) mice, were used to confirm LSC. Raman spectroscopy is a laser-based technique for the single cell analysis of intrinsic molecular vibrations reflecting cellular biochemical information. It can provide a quantitative assessment of the levels of DNA, RNA, proteins, lipids, and carbohydrates in the cell, as well as molecular-level conformational changes. Previous studies by our group showed that unique Raman fingerprints were identified in normal blood cells, ALL cells, and stem cells, including hematopoietic stem cells and embryonic stem cells. Four preB ALL samples were stained for CD9 and sorted by flow cytometry. ALL samples were obtained from patients at diagnosis or freshly harvested from NSG mice engrafted with primary leukemia samples. All samples showed heterogeneous expression of CD9. CD9 high-positive cells and negative cells were flow sorted. Raman spectra of freshly sorted CD9 high-positive and negative cells were obtained. 10 to 20 cells were analyzed in each sample. CD34 positive cells, which were isolated from normal donors, were also analyzed by Raman spectroscopy as a control. No unique Raman fingerprints were identified to separate CD9 high-positive cells from negative cells using Principal Component Analysis (PCA). Furthermore, CD9 high-positive and negative cells from three preB ALL samples were transplanted into NSG mice via intra-bone marrow injection. Equal cell numbers (5×105 to 1.5×106 cells) were used for positive and negative samples in each injection. The majority of the mice from both groups (transplanted with CD9 high-positive or negative cells) developed leukemia 3 to 4 months after injection. Leukemia phenotype was confirmed to be the same as the original leukemia. In conclusion, although CD9 was shown to be a marker for LSC in B-ALL cell lines, it does not appear to be an LSC marker in primary preB ALL. Since childhood preB ALL is a heterogeneous group of diseases, larger cohorts are necessary to confirm our findings. Raman spectroscopy may be a useful screening tool for analysis of cellular intrinsic markers. Disclosures: No relevant conflicts of interest to declare.

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