The Menin-MLL1 interaction is a molecular dependency in NUP98-rearranged AML

Translocations involving the NUP98 gene produce NUP98-fusion proteins and are associated with a poor prognosis in acute myeloid leukemia (AML). MLL1 is a molecular dependency in NUP98-fusion leukemia, and therefore we investigated the efficacy of therapeutic blockade of the Menin-MLL1 interaction in NUP98-fusion leukemia models. Using mouse leukemia cell lines driven by NUP98-HOXA9 and NUP98-JARID1A fusion oncoproteins, we demonstrate that NUP98-fusion driven leukemia is sensitive to the Menin-MLL1 inhibitor VTP50469, with an IC50 similar to what we have previously reported for MLL-rearranged and NPM1c leukemia cells. Menin-MLL1 inhibition upregulates markers of differentiation such as CD11b and downregulates expression of pro-leukemogenic transcription factors such as Meis1 in NUP98-fusion transformed leukemia cells. We demonstrate that MLL1 and the NUP98 fusion protein itself are evicted from chromatin at a critical set of genes that are essential for maintenance of the malignant phenotype. In addition to these in vitro studies, we established patient-derived xenograft (PDX) models of NUP98-fusion driven AML to test the in vivo efficacy of Menin-MLL1 inhibition. Treatment with VTP50469 significantly prolongs survival of mice engrafted with NUP98-NSD1 and NUP98-JARID1A leukemias. Gene expression analysis revealed that Menin-MLL1 inhibition simultaneously suppresses a pro-leukemogenic gene expression program, including downregulation of the HOXA cluster, and upregulates tissue-specific markers of differentiation. These preclinical results suggest that Menin-MLL1 inhibition may represent a rational, targeted therapy for patients with NUP98-rearranged leukemias.

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In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.2605.2605 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2605-2605

Author(s):

Lars Bullinger ◽

Konstanze Dohner ◽

Richard F. Schlenk ◽

Frank G. Rucker ◽

Jonathan R. Pollack ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Serum Levels ◽

Leukemia Cell Lines ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

Abstract Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

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Clathrin Assembly Protein CALM Is Necessary for Leukemia Cell Proliferation and Erythroid Development Via Regulating Transferrin Receptor Endocytosis

Blood ◽

10.1182/blood.v118.21.244.244 ◽

2011 ◽

Vol 118 (21) ◽

pp. 244-244

Author(s):

Yuichi Ishikawa ◽

Manami Maeda ◽

Min Li ◽

Sung-Uk Lee ◽

Julie Teruya Feldstein ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemia Cell ◽

Leukemia Cells ◽

Leukemia Cell Lines ◽

Erythroid Development

Abstract Abstract 244 Clathrin assembly lymphoid myeloid leukemia (CALM) protein is implicated in clathrin dependent endocytosis (CDE) and the CALM gene is the target of the t(10;11)(p13;q14-21) CALM/AF10 translocation, which is observed in multiple types of acute leukemia. Although the translocation generally dictates poor prognosis, the molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. To determine the role of CALM and CDE in normal hematopoiesis and leukemogenesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/FMx1Cre+) Calm knockout (KO) mutants. Furthermore, we determined the impact of Calm loss on leukemia cell growth in vitro and in vivo employing a series of leukemia cell lines and leukemia mouse models. Hematopoietic-specific Calm knockout mice (CalmF/FMx1Cre+) exhibited a hypocromatic anemia with increased serum iron levels. We observed significant reduction in mature erythroblasts/erythrocytes (TER119+CD71-) with concomitant increase in immature erythroblasts (TER119+CD71+) in the spleen of CalmF/FMx1Cre+ mice. The frequencies of erythroblasts in S phase were lower and the proportions of apoptotic (cleaved PARP positive) erythroblasts were increased in CalmF/FMx1Cre+ mice. Surface transferrin receptor 1 (Tfr1, CD71) levels were significantly up-regulated in Calm-deficient hematopoietic progenitors, and uptake of Alexa647-conjugated transferrin was abrogated in Calm-deficient erythroblasts, revealed by immunofluorescence analysis. Freez-etch electron microscopy analysis showed a defective clathrin coated vesicle (CCV) formation in Calm-deficient erythroblasts, indicating that Calm is indispensable for iron-bound transferrin internalization by regulating CCV formation, thereby critical for erythroid differentiation and hemoglobinization. CALM was highly expressed in leukemia/lymphoma cell lines and primary acute myeloid leukemia samples, although its expression was limited to erythroblasts in normal hematopoietic lineage cells. Treatment of leukemia cell lines with Desferoxamine (DFO), an iron chelator, led to a significant increase in Calm mRNA levels, suggesting that Calm expression is regulated by intracellular iron levels. Since highly proliferative leukemia cells demand iron as a cofactor for ribonucleotide reductase (RNR), we hypothesized that Calm is required for leukemia cell proliferation by regulating iron-bound transferrin internalization. To determine the effect of Calm inactivation in leukemia cells, we transduced a series of leukemia cell lines with a lentivirus-based ShRNA vector (pLKO-GFP), which allowed shRNA-expressing cells to be traced by green fluorescent protein (GFP). Calm shRNA transduced cells, but not cells transduced with scrambled shRNA, showed a proliferative disadvantage compared to non-transduced cells. To determine the effect of Calm deletion in leukemia cells in vivo, the CALM/AF10 oncogene was retrovirally transduced into either wild type (WT) or CalmF/FMx1Cre+ bone marrow (BM) cells and the cells were subsequently transferred to lethally-irradiated recipient mice. The Calm gene was deleted in donor cells via pIpC injections one month after transplant (before leukemia development) and survival curves generated. The recipients transplanted with the BM cells from CalmF/FMx1Cre+ mice showed a significantly delayed onset of leukemia and longer survivals compared to control (p=0.001), indicating that Calm is necessary for the development of CALM/AF10-induced leukemia. We next assessed whether Calm is required for the “maintenance” of leukemia in vivo. Leukemia cells were harvested from the primary recipients transplanted with the CALM/AF10-transduced CalmF/FMx1Cre+ BM cells (in which the endogenous Calm genes were intact) and transferred to the secondary recipients. The leukemic secondary recipient mice were then injected with pIpC and survival curves generated. Calm inactivation significantly delayed leukemia progression by blocking leukemia cell proliferation. Taken together, our data indicate that Calm is essential for erythroid development and leukemia cell proliferation by regulating TFR1 internalization. Since Calm inactivation significantly blocked the leukemia cell proliferation in vitro and in vivo, our findings may provide new therapeutic strategies for acute myeloid leukemia. Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

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JQ1, a Potential Therapeutic Molecule for Myeloid Leukemia with PTEN Deficiency

Blood ◽

10.1182/blood.v128.22.5899.5899 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5899-5899 ◽

Cited By ~ 2

Author(s):

Nicholas J Baltz ◽

Natalia C Colorado ◽

Yan Yan ◽

Shelly Lensing ◽

Delli Robinson ◽

...

Keyword(s):

Gene Expression ◽

Western Blot ◽

Colony Formation ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

K562 Cells ◽

Leukemia Cells ◽

Jak Inhibitors ◽

Facs Analysis ◽

Leukemia Cell Lines

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy that continues to have high relapse and treatment-related mortality rates, despite recent advances in clinical management and therapy. Janus kinase (JAK) inhibitors inhibit the activity of the JAK/STAT pathway and have demonstrated some clinical responses in AML patients. However, survival analysis suggests that more than half of AML patients do not benefit from treatment with JAK inhibitors. Furthermore, PTEN deficiency is frequently found in patients in the late stages of cancer, which causes hyperactivated AKT and MAPK pathways. However, emerging data suggests that leukemia cells with PTEN deficiency are resistant to MAPK inhibitors. Over the past decade, it has been demonstrated that dysregulated epigenetics play an important role in myeloid leukemogenesis. The bromodomain and extraterminal domain (BET) family includes adaptor proteins Brd2, Brd3, Brd4, and Brdt that regulate gene expression via binding to acetylated chromatin and subsequently activating RNA Polymerase II driven transcriptional elongation, resulting in the promotion of gene expression. BRD4 is a BET protein required for disease maintenance in AML. JQ1 is a small molecule that interferes with transcriptional regulators, such as BRD4, by preventing them from interacting with acetylated regions of the genome and thus inhibiting the transcriptional activation of BRD4 target genes. Prior research in lymphocytic leukemia cell lines suggests that JQ1 also decreases STAT5-dependent gene transcriptional activities. We hypothesize that the inhibition of BET proteins may correct the over-activated transcriptional activities in myeloid leukemia cells and induce disease regression. We tested our hypothesis in PTEN deficient myeloid leukemia cell lines, TF-1a and K562, and used human cord blood mononuclear cells (CB) for normal cell comparison. Methods: 1) To test whether JQ1 can inhibit colony formation, we seeded cells on 0.3% agar and McCoys' 5A medium supplemented with nutrients and 15% fetal bovine serum, without cytokines, and added JQ1 diluents to the cultures at concentrations of 32.5-1000nM overnight after the cultures were established. 2) To test whether JQ1 can inhibit leukemia cell proliferation, we cultured cells in liquid medium with JQ1 for 48-72 hours, and quantified the viable cells using alamarBlue® assay. 3) To investigate whether JAK/STAT5 activity is altered by JQ1 in leukemia cells, we quantified phosphorylated STAT5 (pSTAT5) in cells via flow cytometry and western blot. We treated the cells with JQ1 at various concentrations for 2 hours and then stimulated the cells for 15 minutes in medium with 0.5% BSA and 10ng/mL GM-CSF prior to staining the cells with anti-pStat5 (pY694) antibody conjugated with Alexa Fluor® 647 for FACS analysis or lysing the cells for western blot analysis. Results: In the colony formation assay, we found that TF-1a cells were more sensitive to JQ1 than the CB cells and K562, with an IC50 of 62.5-125 nM for TF-1a cells (p<0.0001), and 250-500nM for both CB and K562 cells, respectively. Proliferation assay results also supported that TF1a cells are sensitive to JQ1 with an IC50 of 125-250nM, whereas neither CB nor K562 reached the IC50 in the tested concentration range. This suggests that the IC50 of JQ1 for TF1a cells is achievable at concentrations that are mostly nontoxic to normal CB cells, but K562 cells are not sensitive to JQ1. FACS analysis revealed that pSTAT5 is constitutively activated in K562 cells but not in TF-1a cells. Interestingly, the levels of pSTAT5 in both TF-1a and K562 cells were not altered by JQ1 treatment at tested concentrations, which was confirmed by western blot. Conclusions: Our data suggest: 1) JQ1 and other bromodomain inhibitors could be potential therapeutic molecules for selected myeloid leukemias; 2) JQ1 inhibition on colony formation and proliferation in TF-1a cells is not pSTAT5 related. Further studies are underway to test whether JQ1 is effective in primary mouse leukemia cells with Pten deficiency. Disclosures No relevant conflicts of interest to declare.

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RNA Binding Protein Rbmx Is Required in Acute Myeloid Leukemia By Regulating the Transcriptional Activity of the Heterochromatin Protein HP1α

Blood ◽

10.1182/blood-2018-99-112543 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 883-883

Author(s):

Camila Prieto ◽

Diu Nguyen ◽

Ly P Vu ◽

Alexendar Perez ◽

Saroj Gourkanti ◽

...

Keyword(s):

Colony Formation ◽

Myeloid Leukemia ◽

Rna Binding ◽

Flow Analysis ◽

Leukemia Cell ◽

Leukemia Cells ◽

Heterochromatin Protein ◽

Leukemia Cell Lines ◽

Myeloid Leukemia Cell

Abstract Acute myeloid leukemia (AML) is characterized by a block in the development of myeloid cells, often due to dysregulation of genes involved in key processes including self-renewal, proliferation, and differentiation. Somatic mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to be important in hematological malignancies. For example, our group and others have recently determined that increased expression of MUSASHI-2 and SYNCRIP drives aggressive leukemia. To discover novel RBP regulators of leukemia, we performed an in vivo pooled shRNA screen of 127 MSI2 direct protein interactors and associated genes (Vu et al. Nat Gen. 2017). In this screen, shRNAs specific to the RBP RBMX (RNA binding motif protein, X-linked) were selectively depleted in murine MLL-AF9 driven leukemia. RBMX has been implicated in regulating alternative splicing, chromatin cohesion, and DNA-damage response, but its function in hematopoiesis and leukemia is not known. We confirmed that depletion of RBMX with shRNAs in murine MLL-AF9 leukemia cells resulted in reduced myeloid colony formation, increased apoptosis, and increased differentiation as determined by flow analysis of myeloid cell surface markers Gr-1 and Mac-1 (mean of 61-65% shRNA versus mean of 12.95% control). Furthermore, RBMX is highly expressed among human myeloid leukemia cell lines (n=10/11) and primary AML patient samples (n=2/4). Depletion of RBMX with shRNAs led to a dramatic decrease in cell proliferation and 3-fold induction of apoptosis in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX depletion in AML cells induced myeloid differentiation and significantly delayed leukemogenesis cells in vivo (median survival of 51.5 days in control versus median 'not reached' in shRNA1 and shRNA2). To determine if there is a differential requirement of RBMX in survival of leukemia cells compared to normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBMX with shRNAs in normal murine bone marrow c-Kit+ cells and found no significant changes in colony formation. Depleting RBMX with shRNAs in human cord blood derived CD34+ HSPCs resulted in reduced colony formation but no increase in apoptosis. Thus, these data suggest that there is a differential requirement for RBMX in myeloid leukemia cells compared to normal cells. To uncover the mechanism of RBMX function, we performed RNA-sequencing of human AML cells (MOLM-13) depleted for RBMX. Gene set enrichment analysis demonstrated a loss of cell cycle and DNA repair associated programs in RBMX depleted cells. Complex chromosomal karyotyping analysis of these cells revealed increased metaphases with breaks and gaps (mean of 30.67% shRNA versus mean of 13.33% control) and irregular chromatin compaction (mean of 47.67%shRNA versus mean of 20% control), while cell cycle analysis showed significantly increased S-phase arrest as determined by flow analysis of Hoechst stained cells (mean of 37-40% shRNA versus of 24.18% control). Reanalysis of RBMX transcriptome-wide binding sites in 293T cells revealed that RBMX directly binds to heterochromatin protein HP1α transcripts (Liu et al. Nucleic Acids Res. 2017). HP1α, also called CBX5, is a key heterochromatin protein that binds to histone H3-K9 tri-methylation marks to promote heterochromatin formation, which is critical in chromatin condensation and chromosome segregation. HP1α has also been determined to be required for MLL leukemia stem cell maintenance. We demonstrated that RBMX depletion resulted in a significant decrease of HP1α mRNA expression without affecting its mRNA stability in AML cells. We confirmed that RBMX depletion reduced the protein abundance of HP1α. Moreover, overexpression of HP1α rescued the effect of RBMX depletion on cell growth and apoptosis. Our study finds that RBMX binds to HP1α mRNA and regulates the transcriptional activity of the HP1α locus, which then maintains proper chromatin compaction in leukemia cells. Overall, we determine that RBMX function is critical for myeloid leukemia survival and has potential as a novel therapeutic target in AML. Disclosures No relevant conflicts of interest to declare.

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Characterization of Double BCR-ABL–positive Cell Lines Established from a Patient with Blasic Crisis of Chronic Myeloid Leukemia Who Showed Clinical Resistant to Imatinib

Blood ◽

10.1182/blood.v112.11.4244.4244 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4244-4244

Author(s):

Tsuyoshi Nakamaki ◽

Norimichi Hattori ◽

Hidetoshi Nakashima ◽

Takashi Maeda ◽

Hirotsugu Ariizumi ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Kinase Domain ◽

Abl Kinase ◽

Leukemia Cell Lines ◽

Clinical Resistance

Abstract Pervious in vitro studies have shown that molecular alterations of BCR-ABL-positive leukemia cells such as amplification of BCR-ABL gene and/or mutation(s) of abl kinase domain cause resistant to imatinib. However recent study showed that alterations of imatinib bioavailability might be a important factor to cause clinical resistant in BCR-ABL-positive leukemia patients, showing a differences between in vivo and in vitro sensitivity to imatinib of BCR-ABL-positive cells. To analyze mechanism(s) of clinical resistance to imatinib and to overcome the resistance, we have sequentially established and characterized two leukemia cell lines from a patient with myeloid blastic crisis of chronic myeloid leukemia (CML) who showed progressively resistant to imatinib. Case report and establishment of cell lines: a 59-years-old women developed blastic crisis preceded by four years of chronic phase of CML. Increased blasts in crisis was positive for CD13, 33 and showed double Ph-chromosome in addition to complexed chromosomal alterations such as, add(3)(p13), add(3)(q11), add(5)(q11), der(19)(3;19) (p21;q13). After repeated courses of combination chemotherapy including, 600mg of imatinib was administered orally in combination with chemotherapeutic drugs. For a brief period Imatinib showed clinical effects and slowed the increase of BCR-ABL-positive cells, however myeloblast progressively increased in peripheral blood in spite of daily administration of imatinib and she died four months treatment with imatinib. Two myeloid leukemia cell lines, NS-1 and NS-2 were established, after obtaining informed consent, from peripheral blood at day 65 and day 95 after initiation of imatinib administration, respectively. Cell surface phenotype and karyotype of these cell lines were identical to original blasts. NS-1 and NS-2 cell lines were characterized compared with BCR/ABL-positive K562 erythroleukemia cell line as a control Quantitative analysis by real-time polymerase chain reaction showed that copy number of BCR-ABL transcript were 2.2 × 105 and 1.6 × 10 5/μg RNA in NS-1 and NS-2 respectively, showing slightly lower than those (5.8 × 105) in K562 cell line. Although nucleotide sequence analysis showed that a point mutation in abl kinase domain resulted in amino acid substitution pro310ser in NS-1 cell line, no additional mutation was found in NS-2 cell line. Western blot analysis showed levels of both 210 KD BCR-ABL protein and BCR-ABL phosphorylation were similar in NS-1, NS-2 and K562 cells. Although two hours incubation with 10 mM imatinibin vitro did not show any detectable difference in levels of phosphorylation of BCR-ABL protein between NS-1 and NS-2 cell lines, sensitivity to imatinib measured by MTT assay showed that IC50 was 0.1 mM, 0.5 mM and 1.0mMin NS-1, NS-2 and K562 cell lines respectively. The measured IC50 of both NH-1 and NH-2 cell lines were much lower than reported plasma concentrations achieved by oral administration of 600 mg of imatinib (above 10 μM). The present results suggest difference between in vivo and in vitro sensitivity to imatinib indicate that alteration of bioavailability of imatinib possibly involved in clinical resistance to this drug, accumulations of BCR-ABL gene amplification and/or mutation are not necessarily a major reason of progressive clinical resistance to imatinib in BCR-ABL positive leukemia.

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The CXCR4 Antagonist AMD3100 Has Dual Effects, Initially Enhancing and Later Inhibiting Survival and Proliferation, in Myeloid Leukemia Cells in Vitro.

Blood ◽

10.1182/blood.v114.22.1721.1721 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1721-1721

Author(s):

Ha-Yon Kim ◽

Ji-Young Hwang ◽

Seong-Woo Kim ◽

Gak-Won Yun ◽

Young-Joon Yang ◽

...

Keyword(s):

Cell Lines ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Leukemia Cells ◽

Dependent Manner ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Leukemia Cell Lines ◽

Number Of Cells

Abstract Abstract 1721 Poster Board I-747 AMD3100, a small bicyclam antagonist for chemokine receptor CXCR4, induces the peripheral mobilization of hematopoietic stem cells. It also induces the segregation of leukemia cells in the bone marrow microenvironment, which should enhance the chemosensitivity of the cells. Based on these observations, AMD3100 is being considered for clinical use. However, AMD3100 activates G-protein coupled with CXCR4 and acts as a partial CXCR4 agonist. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloid leukemia cells. As demonstrated previously, both AMD3100 and T140, another CXCR4 antagonist, markedly inhibited stromal cell-derived factor-1 (SDF-1)-induced chemotaxis and induced the internalization of CXCR4 in myeloid leukemia cell lines (U937, HL-60, MO7e, KG1a, and K562 cells) and CD34+ primary human acute myeloid leukemia (AML) cells. SDF-1 alone did not stimulate the proliferation of these leukemia cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100, but not T140, stimulated the proliferation of all five leukemia cell lines and primary AML cells in a dose-dependent manner in serum-free conditions for up to 5 days (∼ 2-fold increases at a concentration of 10-5M), which was abrogated by pretreating the cells with pertussis toxin. AMD3100 binds to CXCR7, another SDF-1 receptor, and all of the cells examined in this study expressed CXCR4 on the cell surface to some extent. The proliferation-enhancing effects of AMD3100 were not changed by knocking-down CXCR7 using the siRNA technique, whereas knocking-down CXCR4 significantly delayed the enhanced proliferation induced by AMD3100. Neither AMD3100 nor T140 induced the phosphorylation of Akt, Stat3, MAPK p44/p42, or MAPK p38, which are involved in SDF-1 signaling. In extended cultures of these cells for up to 14 days, AMD3100, but not T140, induced a marked decrease in the number of cells, compared to the control, after incubation for 5-7 days. Adding SDF-1 at the beginning and middle of the incubation did not affect the early increase or later decrease in the number of cells. AMD3100 reduced the apoptosis of these cells to a modest degree over the first 5-7 days and then markedly increased it. Consistent with the proliferation assay, AMD3100 increased the number of leukemia cell colonies during the early period of the assay, while it markedly decreased the number and size of the colonies in the later period of the assay. In conclusion, AMD3100 exerts dual effects, initially enhancing and subsequently inhibiting the survival and proliferation, in myeloid leukemia cells in vitro. Disclosures No relevant conflicts of interest to declare.

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Disulfiram, an clinically used anti-alcoholism drug has the potential to target acute myeloid leukemia cells in a copper-dependent manner in vivo

Blood ◽

10.1182/blood.v122.21.5040.5040 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5040-5040

Author(s):

Bing Xu ◽

Rongwei Li ◽

Huijuan Dong ◽

Feili Chen ◽

Yuejian Liu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Specific Antigen ◽

Leukemia Cell ◽

Leukemia Cells ◽

Control Group ◽

Acute Myeloid

Abstract Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p < 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

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The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Blood ◽

10.1182/blood.v128.22.3523.3523 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3523-3523

Author(s):

Danielle Garshott ◽

Nicole Melong ◽

Tania T. Sarker ◽

Yue Xi ◽

Amy Brownell ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Leukemic Cell ◽

K562 Cells ◽

Leukemia Cells ◽

Wild Type ◽

Leukemia Cell Lines ◽

Proliferation Assays

Abstract Background: Acute leukemias are the most common cancers in childhood. Despite multi-agent chemotherapy protocols and the introduction of novel molecularly targeted therapies which have resulted in improved survival over the last few decades, relapsed acute lymphoblastic leukemia remains the second most common pediatric cancer diagnosis. In addition, morbidities from current chemotherapy regimens are unacceptably high. Abundant evidence point to a major role for mediators of the unfolded protein response (UPR) in normal and leukemic white blood cell biology. We have demonstrated that activation of the UPR is a productive approach to inhibit the proliferation of solid tumor cell lines in vitro and to reducing xenograft burden in vivo. The UPR consists of genetically distinct mechanisms that serve to clear misfolded proteins from the endoplasmic reticulum (ER) and enhance protein folding, or induce apoptosis if the initiating stress is prolonged or robust. ML291 is a novel UPR-inducing sulfonamidebenzamide, identified through cell-based high throughput screening and iterative SAR-guided chemical synthesis, that overwhelms the adaptive capacity of the UPR and induces apoptosis in a variety of solid cancer models. Objective: To determine the ability of ML291 to activate the UPR and induce apoptosis in a panel of leukemia cell lines, and to use CHOP-null K562 cells to elucidate the relative contribution of the UPR. We hypothesized that ML291 might activate the PERK/eIF2a/CHOP (apoptotic) arm of the UPR and reduce leukemic cell burden in vitro and in vivo. Methods: MTT and luciferase-based proliferation assays, flow cytometry and RT-qPCR were used to evaluate cell growth, UPR activation and apoptosis in a panel of leukemia cell lines that included AML, ALL and CML in cells exposed to ML291. CRISPR-Cas9 genome editing was used to delete CHOP in K562 (human myeloid leukemia) cells. Deletion was validated by immunoblot analysis and these cells were subjected to the same proliferation and gene analyses described above. The in vivo response to ML291 therapy was evaluated in an established zebrafish xenograft assay (Corkery et al. BJH 2011) in which embryos were xenotransplanted with wild type or CHOP knockdown K562 cells and embryos bathed in ML291. Results: Immunoblot and RT-qPCR analysis revealed an accumulation of proteins and increased gene expression for downstream UPR genes, including CHOP, GRP78/BiP, GADD34 and XBP1 in leukemia cells following ML291 treatment, indicating the activation of the UPR. Increased expression of the apoptotic genes, NOXA, PUMA and DR5 was also observed post-treatment with ML291; and dose response proliferation assays performed after 24 hours revealed IC50 concentrations of 1 - 30µM across cell lines. CHOP deleted K562 cells were protected from cell death when cultured with increasing concentrations of ML291, and were significantly less able to translocate phosphatidylserine across the cell membrane and activate the caspase cascade. When zebrafish embryos xenotransplanted with K562-wild type or -CHOP-null cells were bathed in water containing 5mM ML291 for three days, there was a significant reduction in leukemia cell burden exclusively in theK562 wild type xenografts. Conclusion: Collectively these data indicate that intact PERK/eIF2a/CHOP signaling is required for efficient leukemic cell apoptosis in response to ML291 in vitro and in vivo, and support the hypothesis that small molecule enforcement of the UPR might be a productive therapeutic approach in leukemia. Disclosures No relevant conflicts of interest to declare.

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A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Blood ◽

10.1182/blood.v122.21.3962.3962 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3962-3962

Author(s):

Laura M Bystrom ◽

Hongliang Zong ◽

Hsiao-Ting Hsu ◽

Neng Yang ◽

Noa Greenberg ◽

...

Keyword(s):

Cell Lines ◽

Iron Metabolism ◽

Leukemia Cell ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Survival Pathways ◽

Leukemia Cell Lines ◽

Acute Myelogenous

Abstract Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse. We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status. Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds. Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry. Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron. The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures: Rivella: Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

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Bryostatin 1, a unique biologic response modifier: anti-leukemic activity in vitro

Blood ◽

10.1182/blood.v75.6.1319.1319 ◽

1990 ◽

Vol 75 (6) ◽

pp. 1319-1323 ◽

Cited By ~ 69

Author(s):

RJ Jones ◽

SJ Sharkis ◽

CB Miller ◽

EK Rowinsky ◽

PJ Burke ◽

...

Keyword(s):

Leukemia Cell ◽

Leukemia Cells ◽

Bryostatin 1 ◽

Hematopoietic Progenitors ◽

P388 Leukemia ◽

Leukemia Cell Lines ◽

Potent Inhibition ◽

Biologic Response

Abstract Bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, has demonstrated both antineoplastic activity against the murine P388 leukemia line in vivo and stimulatory activity against mouse and human hematopoietic progenitors. We studied the effects of bryostatin 1 on the growth of human leukemias in vitro. Bryostatin 1 inhibited 1 to 4 logs of clonogenic leukemia cell growth from three of four leukemia cell lines. Bryostatin 1 also inhibited, by at least 1 log, the proliferation of clonogenic acute nonlymphocytic leukemia (ANLL) cells from 10 to 12 patients with newly diagnosed or relapsed ANLL. Maximal inhibition of leukemic growth occurred at 10(-9) to 10(- 7) mol/L bryostatin 1. Interestingly, bryostatin 1 also inhibited the growth of hematopoietic progenitors from eight patients with myelodysplastic syndromes (MDS). Leukemia cells exposed to bryostatin 1 for up to 96 hours and then washed, demonstrated no substantial inhibition of clonogenic growth, indicating that the anti-leukemic effect of bryostatin 1 is cytostatic. The phorbol ester 12–0- tetradecanoylphorbol-13-acetate (TPA) produced more potent inhibition of clonogenic leukemia growth, and this inhibition was blocked by bryostatin 1. Thus, the anti-leukemic activity of bryostatin 1 may be mediated through activation of protein kinase C. Bryostatin 1 inhibits clonogenic leukemia cells at concentrations that stimulate normal hematopoietic progenitors. The differential effects of bryostatin 1 on normal and abnormal hematopoiesis suggest that bryostatin 1 may have value in the treatment of leukemias and MDS.

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