Disulfiram, an Clinically Used Anti-Alcoholism Drug Has the Potential to Target Leukemia Stem and Progenitor Cells in a Copper-Dependent Manner

Abstract Abstract 4088 Backgrounds Acute myeloid leukemia(AML) is a hierarchical disease initiating from a rare population of cells known as leukemia stem cells (LSCs), which are typically enriched in CD34+CD38- cells and presumed responsible for the relapse and refractory of AML. Moreover, current regimens may not effectively discriminate between normal and malignant cells. For this reason, it is important to identify therapies that can specifically target the LSC population without affecting normal cells. Disulfiram (DS) is an anti-alcoholism drug that has recently been indicated to show cytotoxic to multiple cancers including acute myeloid leukemia (AML) and the antineoplastic activity was enhanced in the present of copper (Cu). In the present study, we investigated the effect of DS/Cu on LSCs and further explored its mechanism. Methods and Results CD34+CD38- leukemia stem cell (LSC) enriched subpopulations were sorted from both KG1a cell lines and primary AML bone marrow or peripheral blood mononuclear cells (n=6) by fluoresce-activated cell sorting (FACS) analysis. Using MTT cell proliferation assay and Annexin-V/PI staining assay, We demonstrated that DS/Cu inhibited proliferation and induced apoptosis in CD34+CD38−KG1a cells (IC50= 0.788± 0.451 μM at 24h). With the increasing concentrations of DS (DS=0.05, 0.5, 5, 50μM), the apoptotic proportion increased from 7.2% to 89.5% at 24h. Apoptosis was also observed in CD34+CD38- primary AML cells and the exposure to DS/Cu (DS=0.01, 0.1, 1μM;Cu=0.5μM clearly inhibited the growth of AML-colony-forming units (CFUs) for both CD34+CD38-LSC enriched subpopulations (AML-CFUs decreased from 34.2% to 0% in KG1a cells), but was relatively sparing to normal hematopoietic progenitors. Further more, using flow cytometric analysis, western blot and RT-PCR, we identified that the change in redox status and redox-dependent signaling events play a crucial role in DS/Cu-induced apoptosis. We showed that DS/Cu(DS= 0.625,1.25,2.5,5μM, Cu=1μM) increased reactive oxygen species (ROS) and activated its downstream apoptosis-related SAPK/JNK pathway in association with blockade translocation of Nrf2 and expression of Nrf2-regulated genes in CD34+CD38−KG1a cells. Notably, blockade of ROS by glutathione precursor N-acetylcysteine (NAC)(10mM) strongly diminished DS/Cu mediated lethality and restored Nrf2 nuclear translocation and blocked JNK activation. Additionally, consistent with the ROS accumulation, we also seen that translocation of RelA/p65 and the expression of NF-κb-related gene, associated with abnormal apoptotic response of LSCs, were significantly inhibited by DS/Cu. Conclusion Taken together, we concluded that DS/Cu might selectively eradicate LSCs by induction of oxidatibe stress and blockade the NF-κb pathway and offers a potential therapeutic option in AML. Disclosures: No relevant conflicts of interest to declare.

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FLT3 Inhibitors Induce p53 Instability Due to Increased Interaction between p53 and MDM2 By Inhibiting STAT5 Phosphorylation in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-149406 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3333-3333

Author(s):

Han Zhong Pei ◽

Xiaomei Zhuang ◽

Ming Yang ◽

Yao Guo ◽

Zhiguang Chang ◽

...

Keyword(s):

Drug Resistance ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mononuclear Cells ◽

Nuclear Translocation ◽

Conflicts Of Interest ◽

P53 Protein ◽

Downstream Signaling ◽

Flt3 Inhibitors ◽

Acute Myeloid

Abstract Frequently mutated in Acute myeloid leukemia (AML), FLT3 is considered as one of the favorable targets for treatment. The FLT3 internal tandem duplication (ITD) mutation enhances kinase activity and causes hyperactivation of downstream signal transduction. Several small molecule FLT3 inhibitors have developed, but their clinical efficacy is limited due to generation of drug resistance. In this study, we define a new mechanism of drug resistance toward tyrosine kinase inhibitors (TKIs). Initially, we found a rapid decrease in the protein level of tumor suppressor p53 in FLT3-ITD-positive MV4-11 and MOLM13 cells and peripheral blood mononuclear cells (PBMCs) from FLT3-ITD AML patients upon treatment with TKIs including sorafenib, sunitinib and quizartinib. The decrease is not caused by changes in mRNA expression as revealed by qPCR analyses but rather by accelerated protease degradation because the p53 protein was stabilized by proteasome inhibitor MG132. Furthermore, treatment of cells with RG7388, a potent disruptor of p53 and MDM2 interaction, prevented the TKI-induced p53 loss. Since MDM2 is the most important E3 ligase responsible for ubiquitination of p53, the data suggest that TKIs may lead to the degradation of p53 by promoting ubiquitination. Indeed, ubiquitination assays verified that TKIs promoted K48 poly-ubiquitination of p53. Previous studies have demonstrated that activations of FLT3 downstream signaling components such as ERKs and Akt reduce p53 protein stability through ubiquitination by activating MDM2. It is somewhat unexpected that inhibition of FLT3-ITD and its downstream signaling pathways also resulted in decreased p53 stability due to increased ubiquitination. We treated FLT3-ITD-containing cells with specific ERK, AKT and STAT5 inhibitors. Interestingly, while inhibition of ERKs and AKT had no significant effect on the stability of p53, STAT5 inhibition resulted in a reduced level of p53 accompanied by increased K48 poly-ubiquitination. We further analyzed the interaction of p53 with MDM2 in AML cells by using immunoprecipitation. The results showed that the p53-MDM2 interaction was significantly enhanced after treatment with TKIs and STAT5 inhibitors, which was diminished in the presence of RG7388. Subcellular fractionation revealed the presence of p53 and STAT5 in both nucleus and cytoplasm. Treatment of cells with TKIs resulted in a decreased level of p53 and STAT5 in the nucleus, and immunoprecipitation of nuclear proteins with a p53 antibody revealed a reduced association of p53 with STAT5. Taken together, the data suggest that FLT3 inhibitors inhibited nuclear translocation of STAT5 and reduced its interaction of p53 thereby facilitating p53/MDM2 interaction and subsequent ubiquitination and degradation of p53. This study reveals a novel mechanism by which drug resistance to TKIs may occur and further support the use of MDM2/p53 interaction inhibitors in combination with TKIs for treatment of AML. Disclosures No relevant conflicts of interest to declare.

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The pan-Bcl-2 inhibitor obatoclax promotes differentiation and apoptosis of acute myeloid leukemia cells

Investigational New Drugs ◽

10.1007/s10637-020-00931-4 ◽

2020 ◽

Vol 38 (6) ◽

pp. 1664-1676

Author(s):

Małgorzata Opydo-Chanek ◽

Iwona Cichoń ◽

Agnieszka Rak ◽

Elżbieta Kołaczkowska ◽

Lidia Mazur

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Treatment Strategies ◽

Leukemic Cell ◽

Leukemic Cells ◽

Dependent Manner ◽

Apoptotic Pathway ◽

Acute Myeloid Leukemia Cells ◽

Induced Apoptosis ◽

Acute Myeloid

Summary One of the key features of acute myeloid leukemia (AML) is the arrest of differentiation at the early progenitor stage of myelopoiesis. Therefore, the identification of new agents that could overcome this differentiation block and force leukemic cells to enter the apoptotic pathway is essential for the development of new treatment strategies in AML. Regarding this, herein we report the pro-differentiation activity of the pan-Bcl-2 inhibitor, obatoclax. Obatoclax promoted differentiation of human AML HL-60 cells and triggered their apoptosis in a dose- and time-dependent manner. Importantly, obatoclax-induced apoptosis was associated with leukemic cell differentiation. Moreover, decreased expression of Bcl-2 protein was observed in obatoclax-treated HL-60 cells. Furthermore, differentiation of these cells was accompanied by the loss of their proliferative capacity, as shown by G0/G1 cell cycle arrest. Taken together, these findings indicate that the anti-AML effects of obatoclax involve not only the induction of apoptosis but also differentiation of leukemic cells. Therefore, obatoclax represents a promising treatment for AML that warrants further exploration.

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Ezh2 Plays a Critical Role in the Progression of MLL-AF9-Induced Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.57.57 ◽

2011 ◽

Vol 118 (21) ◽

pp. 57-57

Author(s):

Satomi Tanaka ◽

Goro Sashida ◽

Satoru Miyagi ◽

Koutaro Yokote ◽

Chiaki Nakaseko ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Target Genes ◽

Nuclear Translocation ◽

Conflicts Of Interest ◽

Critical Role ◽

Histone H3 ◽

Chip Sequencing ◽

Acute Myeloid

Abstract Abstract 57 The polycomb group proteins function in gene silencing through histone modifications. They have been characterized as a general regulator of stem cells, but also play a critical role in cancer. EZH2 is a catalytic component of the polycomb repressive complex 2 (PRC2) and tri-methylates histone H3 at lysine 27 to transcriptionally repress the target genes. Although EZH2 is over-expressed in various cancers including hematological malignancies, it remains unknown how EZH2 contributes to the initiation and/or progression of acute myeloid leukemia (AML). To understand the role of EZH2 in AML, we transformed granulocyte macrophage progenitors (GMPs) from Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 fusion gene. Then, Ezh2 was deleted by inducing nuclear translocation of Cre by adding tamoxifen to culture. We found that proliferation of Ezh2δ/δ transformed cells was severely compromised upon deletion of Ezh2 (Ezh2δ/δ) in liquid culture. They gave rise to a significantly reduced number of colonies in replating assays. Of note, while Ezh2+/+ cells formed compact colonies composed of immature myeloblasts, Ezh2δ/δ cells formed dispersed colonies composed of differentiated myeloid cells. We next transplanted Cre-ERT;Ezh2+/+ and Cre-ERT;Ezh2flox/flox GMPs transformed by MLL-AF9 into recipient mice. All the recipient mice developed AML by 3 weeks after transplantation. At 3 weeks after transplantation, we depleted Ezh2 by intraperitoneal injection of tamoxifen. Deletion of Ezh2 significantly prolonged the survival of the recipient mice (60 days vs. 76 days, p<0.0001), although all the mice eventually died of leukemia. Nonetheless, as was detected in vitro, Ezh2δ/δ AML cells in BM were apparently differentiated in morphology compared with the control. Ezh2δ/δ AML cells in BM gave rise to 10-fold fewer colonies in methylcellulose medium compared with Ezh2+/+ AML cells, and again showed an obvious tendency of differentiation. These observations imply that Ezh2 is critical for the progression of MLL-AF9 AML and maintains the immature state of AML cells. To elucidate the mechanism how Ezh2 promotes the progression of MLL-AF9-induced AML, we examined the genome-wide distribution of tri-methylation of histone H3 at lysine 27 (H3K27me3) by ChIP-sequencing and microarray-based expression analysis. ChIP-sequencing using Ezh2+/+ and Ezh2δ/δ BM AML cells identified 3525 and 89 genes exhibiting a ≧ 10-fold enrichment in H3K27me3 levels in Ezh2+/+ and Ezh2δ/δ AML cells, respectively, confirming a drastic reduction in the levels of global H3K27me3 in the absence of Ezh2. Microarray analysis using lineage marker (except for Mac1)−Sca-1−c-Kit+FcγRII/IIIhi BM AML cells revealed 252 upregulated and 154 downregulated genes (≧ 2-fold) in Ezh2δ/δ AML cells compared with Ezh2+/+ AML cells. Of interest, the absence of Ezh2 did not affect the transcriptional activation of the major target genes of MLL-AF9, including HoxA9 and Meis1. Because Ezh2 functions as transcriptional repressor, de-repressed genes could be direct targets of Ezh2. Based on these data, we are now engaged in further comprehensive analysis to narrow down the direct target genes of Ezh2 responsible for the progression of AML. Collectively, our findings suggest that Ezh2 is the major enzyme for H3K27me3 in AML and contributes to the progression of AML by regulating transcription a cohort of genes that are supposedly relevant to the self-renewal capacity and perturbed differentiation of AML stem cells. Disclosures: No relevant conflicts of interest to declare.

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Activity of the Hypoxia-Activated Prodrug, TH-302, in Human Acute Myeloid Leukemia Models

Blood ◽

10.1182/blood.v120.21.3611.3611 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3611-3611

Author(s):

Scott Portwood ◽

Deepika Lal ◽

Yung-Chun Hsu ◽

Rodrigo Vargas ◽

Meir Wetzler ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Chemotherapeutic Agents ◽

Scid Mice ◽

Hypoxic Conditions ◽

Acute Myeloid

Abstract Abstract 3611 Recent evidence has demonstrated the bone marrow (BM) microenvironment, the principal site of acute myeloid leukemia (AML) initiation and expansion, is characterized by intrinsically low oxygen tension. Theoretically, such microenvironmental changes may lead to the selective outgrowth of AML clones which are “better adapted” to survive within a severely hypoxic microenvironment and/or may confer resistance to chemotherapeutic agents, similar to solid tumor cells. We report here that human AML cells (HL60, ML-2) cultured under chronic hypoxic conditions mimicking the marrow microenvironment (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic cells, as determined by flow cytometric analysis, western blot analysis, and cell viability assays. Similar results were noted in primary AML samples treated with cytarabine under normoxic and hypoxic conditions in colony formation assays (n=3 samples, p=0.01). In order to improve upon chemotherapy outcomes, we investigated the effects of TH-302, a hypoxia-activated bromo-isophosphoramidate mustard prodrug, which is currently undergoing clinical trial evaluation in multiple tumor types. Treatment of AML cell lines (HL60, HEL) and primary AML samples with TH-302 (at doses ranging from 0.1– 5 mM, p values ranging from <0.05–0.0001) resulted in dose- and hypoxic-dependent inhibition of AML proliferation and apoptosis. In vivo TH-302 treatment significantly decreased disease burden, as measured by total animal bioluminescence, and prolonged overall survival in two systemic human AML xenograft models (HEL-luciferase, HL60-luciferase) (Figure 1). Immunohistochemical studies demonstrated that TH-302 treatment reduced numbers of hypoxic (pimonidazole-positive) cells within the leukemic marrow microenvironment. Because prior data in animal models has shown that AML progression within the marrow is associated with expansion of hypoxic BM areas, we examined the effects of TH-302 treatment on systemic AML growth when initiated early (prior to AML inoculation) or late (several days following AML engraftment) in the disease process. TH-302 was equally effective at both time points. Although anti-vascular therapy has been shown to enhance tumor hypoxia in other cancer types, we noted no synergistic or additive in vivo effects when TH-302 therapy was combined with sorafenib, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in our models. TH-302 therapy administered for two weeks in non-leukemic and leukemia-engrafted mice was not associated with hematologic toxicities. In summary, our results demonstrate the anti-leukemic activity of TH-302 in preclinical AML models and suggest that the efficacy of this and other drugs for AML therapy may be uniquely affected by the BM microenvironment. Further clinical development of TH-302 and other hypoxia-targeted drugs for AML therapy are warranted. Based on our data, higher TH-302 doses and/or chronic drug administration may be needed for optimal in vivo anti-leukemic activity. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Disclosures: No relevant conflicts of interest to declare.

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Unraveling The Leukemic Nature Of hMICL and CD123 Expressing Cells In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.2626.2626 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2626-2626 ◽

Cited By ~ 2

Author(s):

Line Nederby ◽

Peter Hokland ◽

Gordon Brown ◽

Maria Hansen ◽

Charlotte Guldborg Nyvold ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Capillary Electrophoresis ◽

Myeloid Leukemia ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Fragment Analysis ◽

Marker Combination ◽

Allelic Burden ◽

Acute Myeloid

Abstract Flow cytometry constitutes a cornerstone in the diagnosis and follow up of acute myeloid leukemia (AML) and it is based on the identification of leukemia-associated immunophenotypes (LAIPs). We have recently demonstrated that the C-type lectin hMICL in combination with CD123 constitute a highly stable and reliable LAIP marker combination at diagnosis and relapse. In addition, we have shown that an hMICL/CD123-based assay is an effective tool for the monitoration of minimal residual disease (MRD) in AML. To what extent hMICL/CD123 marking identifies early leukemic cells is, however, not established. We hypothesized that this could be addressed by studying molecular aberrations in leukemic cell subsets according to their expression of hMICL and CD123. Employing cell sorting and mutational analyses, we here establish the leukemic origin of hMICL and CD123 expressing cell populations. Analyzing diagnostic AML samples with homozygous FLT3-ITD aberrations allowed for verification of pure malignant clones. Five patients with FLT3-ITD allelic burden of >50% (range 77-93%, median 85%) as measured by DNA fragment analysis by capillary electrophoresis on mononuclear cells (MNC) were identified in our local database of 600 cases. We found that 5/5 patients displayed a normal karyotype and carried NPM1 mutations (NPM1 allelic burden 42-48%, median 46%). In contrast, mutations in FLT3-D835, IDH1-R132, c-KIT-D816V or indel mutations in CEBPA and WT-1 exon 7 were absent. From samples of cryopreserved mononuclear cells (bone marrow (n=4) and peripheral blood (n=1)), CD45low/SSClow blast cell subsets with the following immunophenotypes were sorted by FACS: CD34+/hMICL+/CD123+, CD34+/hMICL+/CD123-, CD34+/hMICL-/CD123+, and CD34+/hMICL-/CD123-. In one case of CD34 negative AML the sorted subsets were CD34-/hMICL+/CD123+, CD34-/hMICL+/CD123-, CD34-/hMICL-/CD123+, and CD34-/hMICL-/CD123-. Sorted cell subsets were analyzed for FLT3-ITD and NPM1 mutations using fragment analysis by capillary electrophoresis. The results of the fragment analyses are tabulated in the table below. In all cases the hMICL and CD123 expressing subsets of interest closely approximated 100% FLT3-ITD allelic burden. In contrast, hMICL-/CD123- cells approximated only a 50% FLT3-ITD allelic burden. Of note, an extended search in our AML database, revealed only 9 of 600 patients to have an FLT3-ITD allelic burden >50% (range 52-94%, median 81%) hence indicating a state of either homo- or hemizygosity. Interestingly, with the exception of one case carrying a chromosome 13 duplication, each of these 9 patients also harbored a mutation in the NPM1 gene as the only other known aberration. In conclusion using AML patients with high FLT3-ITD allelic burdens we have been able to show that blasts expressing hMICL and/or CD123 at diagnosis are indeed malignant thus further substantiating the use of these antigens in AML MRD detection. Additionally, a direct relationship between NPM1 and FLT3-ITD homo-/hemizygosity may be suggested in the evolution of the malignant clone.Phenotype of sorted cell subsetNumber of patientsFLT3-ITD allelic burden (%) Min-max (median)NPM1 allelic burden (%) Min-max (median)MNC577-93 (85)42-48 (46)CD45low/SSClow/CD34+/hMICL+/CD123+495-100 (98)48-50 (49)CD45low/SSClow/CD34+/hMICL+/CD123-1*9248CD45low/SSClow/CD34+/hMICL-/CD123+497-100 (99)47-51 (48.5)CD45low/SSClow/CD34+/hMICL-/CD123-436-68 (47)16-38 (25)CD45low/SSClow/CD34-/hMICL+/CD123+110046CD45low/SSClow/CD34-/hMICL+/CD123-19448CD45low/SSClow/CD34-/hMICL-/CD123+110047CD45low/SSClow/CD34-/hMICL-/CD123-17735*Subset only present in one of four patients Disclosures: No relevant conflicts of interest to declare.

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Small Molecule Inhibition of SIRT Activity: A Novel Therapeutic Approach for Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.84.84 ◽

2011 ◽

Vol 118 (21) ◽

pp. 84-84

Author(s):

Steffan T Nawrocki ◽

Claudia M Espitia ◽

Kevin R. Kelly ◽

William G. Bornmann ◽

Jennifer S Carew

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Small Molecule ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Clonogenic Survival ◽

Altered Gene ◽

Induced Apoptosis ◽

Acute Myeloid ◽

Novel Therapeutic

Abstract Abstract 84 New therapeutic strategies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML), which is an extremely aggressive disease with very few long-term survivors. The sirtuin deacetylases (SIRTs) are critical regulators of genes that are essential for longevity, cell growth, tumor suppression, and apoptosis. Elevated SIRT expression has been reported in several types of cancer and may promote pathogenesis and drug resistance by increasing the lifespan and survival capacity of malignant cells. Our preliminary analysis of SIRT expression indicated that SIRT1 was consistently expressed at significantly higher levels in AML cell lines and primary AML blasts as compared with normal controls. In order to investigate the potential role of SIRT1 as a regulator of AML pathogenesis, we utilized shRNA to stably knockdown its expression in MV4-11 and KG-1 AML cells. Cells with targeted SIRT1 knockdown displayed an altered gene expression profile as compared with non-targeted controls. Moreover, antagonizing SIRT1 expression significantly impeded the progression of AML in a xenograft mouse model. A number of deacetylase inhibitors have been clinically evaluated for cancer therapy. However, disrupting SIRT function as an anticancer strategy remains to be rigorously investigated as none of these previously studied drugs significantly inhibit the activity of this class of NAD+-dependent deacteylases. Tenovin-6 is a novel small molecule SIRT inhibitor. We investigated the efficacy and pharmacodynamic effects of tenovin-6 in AML cell lines, primary blasts from patients with AML, and mouse models. Treatment with tenovin-6 induced apoptosis and dramatically diminished AML clonogenic survival. Tenovin-6 promoted a dose-dependent increase in the acetylated levels of the SIRT-regulated gene p53 in AML cells and triggered the induction of several p53 transcriptional targets including p21 and PUMA. Targeted knockdown of PUMA with shRNA significantly reduced the pro-apoptotic effects of tenovin-6, indicating that it is a critical mediator of its anti-leukemic activity. Notably, administration of tenovin-6 to mice implanted with AML cells was well-tolerated and led to a highly significant reduction in disease burden and increase in overall survival. Our collective findings demonstrate that SIRT1 is a promising novel therapeutic target in AML. Further investigation aimed to elucidate the safety, efficacy, and mechanism of action of tenovin-6 is warranted. Disclosures: No relevant conflicts of interest to declare.

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Treatment With G-CSF Reduces Acute Myeloid Leukemia (AML) Blasts Viability In Presence Of Bone Marrow Stroma

Blood ◽

10.1182/blood.v122.21.1422.1422 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1422-1422

Author(s):

Meritxell Nomdedeu ◽

Marta Pratcorona ◽

Marina Díaz-Beyá ◽

Xavier Calvo ◽

Mari Carmen Lara-Castillo ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Viability ◽

Clinical Benefit ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Cell Counting ◽

Dependent Manner ◽

Dose Dependent ◽

Acute Myeloid

Abstract Background The simultaneous administration of G-CSF and chemotherapy as a priming strategy has resulted in a clinical benefit in determined subsets of patients diagnosed with acute myeloid leukemia (AML) (Löwenberg et al, NEJM 2003; Pabst T, et al, Blood 2012). However, the mechanism responsible for this anti-leukemic effect is not fully characterized. We hypothesize that the clinical benefit may occur at least partially by the effect of G-CSF on leukemic stem cells (LSC). Objective The main goal of this project was to determine the effect of G-CSF on primary AML samples in vitro, especially on LSCs. Methods and patients Peripheral blood mononuclear cells (PBMC) from 10 AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stroma cells. Cell viability (7-AAD -eBioscience- cell death exclusion and volumetric cell counting) and surface phenotype was determined by flow cytometry (FACSVerse, BD) 72 hours after treatment. Data were analyzed using the FlowJo (Trastar) software. For clonogenicity assays, AML primary samples were treated for 18 hours with G-CSF at increasing concentrations and cultured in H4034 Optimum MethoCult (StemCell Technologies) for 14 days. Colonies were counted based on cellularity and morphology criteria. Results G-CSF treatment showed no effect on cell viability of the bulk leukemic population or on the CD34 + immature subpopulation. A dose-dependent increase in CXCR4 surface expression was observed, reaching a 1.4-fold of change at the highest concentration of G-CSF (100 μg/mL). In contrast, treatment of leukemia cells with G-CSF in the presence of stroma cells reduced the overall cell viability. Thus, a 32% decrease of cell viability was measured at the highest concentration used (p = 0.0006), while no significant changes in the frequency of each leukemic subpopulations were observed. Clonogenic capacity was significantly reduced in a dose-dependent manner upon treatment with G-CSF, achieving a 41% reduction at the highest G-CSF concentration (100 μg/mL). Conclusions G-CSF reduces the viability of leukemic cells when these cells are in co-culture with the HS-5 stroma cell line, suggesting that the presence of stroma cells is required for the cytotoxical effect of G-CSF on the blast population. Interestingly, G-CSF treatment decreased the clonogenic capacity of AML samples, therefore suggesting that G-CSF exerts its effect at least partially on LSCs. Our findings support the design of studies to explore new strategies of chemotherapy priming in AML patients. Disclosures: No relevant conflicts of interest to declare.

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The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts

Blood ◽

10.1182/blood.v97.5.1413 ◽

2001 ◽

Vol 97 (5) ◽

pp. 1413-1421 ◽

Cited By ~ 125

Author(s):

Beverly D. Smolich ◽

Helene A. Yuen ◽

Kristina A. West ◽

Francis J. Giles ◽

Maher Albitar ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Growth Factor ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Dependent Manner ◽

Induced Apoptosis ◽

Dose Dependent ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

SU5416 and SU6668 are potent antiangiogenic small-molecule inhibitors of receptor tyrosine kinases, including those of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor, c-kit, is structurally related to these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemic blasts derived from 60% to 80% of acute myeloid leukemia (AML) patients. The c-kit kinase inhibitory activity of SU5416 and SU6668 was evaluated in MO7E cells, a human myeloid leukemia cell line. Tyrosine autophosphorylation of the receptor, induced by SCF, was inhibited in these cells by SU5416 and SU6668 in a dose-dependent manner (inhibitory concentration of 50% [IC50] 0.1-1 μM). Inhibition of extracellular signal–regulated kinase 1/2 (ERK1/2) phosphorylation, a signaling event downstream of c-kit activation, was also inhibited in a dose-dependent manner. Both compounds also inhibited SCF-induced proliferation of MO7E cells (IC50 0.1 μM for SU5416; 0.29 μM for SU6668). Furthermore, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner as measured by the increase in activated caspase-3 and the enhanced cleavage of its substrate poly(ADP-ribose) polymerase. These findings with MO7E cells were extended to leukemic blasts from c-kit+ patients. In patient blasts, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies indicate that SU5416 and SU6668 inhibit biologic functions of c-kit in addition to exhibiting antiangiogenic properties and suggest that the combination of these activities may provide a novel therapeutic approach for the treatment of AML.

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Microrna-9 Promotes Proliferation of Leukemia Cells in Adult CD34 Positive Acute Myeloid Leukemia with Normal Karyotype By Down-Regulation of Hes1

Blood ◽

10.1182/blood.v126.23.1229.1229 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1229-1229

Author(s):

Chen Tian ◽

Guoguang Zheng ◽

M. James You ◽

Yizhuo Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Normal Karyotype ◽

Hematopoietic Stem ◽

Cell Counts ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies sustained by a small population of leukemic stem cells (LSCs) that can resist treatment and act as barriers to cure. Previously, we observed that Hes1 and p21 expression was down-regulated in AML cell lines compared to that of normal bone marrow mononuclear cells. However, the activation status of Hes1-p21 pathway and its regulation in LSCs as well as normal hematopoietic stem cells (HSCs) in AML has not been elucidated. In this study, the Hes1-p21 pathway in LSCs and leukemic progenitors (LPs) was studied in adult CD34+ AML with normal karyotype and no genetic mutations and the upstream miRNA regulators were screened. Our results showed that the level of either Hes1 or p21 was lower in LSCs or LPs than that of HSCs whereas the level of miR-9 was higher in LSCs or LPs than HSCs. An inverse correlation was observed in the expression of Hes1 and miR-9. Furthermore, we validated miR-9 as one of the regulators of Hes1 by reporter gene analysis. Knockdown of miR-9 by lentivirus infection suppressed the proliferation of AML cells by the induction of G0 arrest and apoptosis in vitro. Moreover, knockdown of miR-9 resulted in decreased circulating leukemic cell counts in peripheral blood and bone marrow, attenuated splenomegaly, and prolonged survival in a xenotransplant mouse model. Our results indicate that the miR-9-Hes1-p21 pathway plays an important role in supporting AML cell growth and survival, and that miR-9 has a potential to be a therapeutic target for suppressing AML. Disclosures No relevant conflicts of interest to declare.

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Anti-Leukemic Activity of Shikonin: Role of ERP57 in Shikonin Induced Apoptosis in Acute Myeloid Leukemia

Cellular Physiology and Biochemistry ◽

10.1159/000445652 ◽

2016 ◽

Vol 39 (2) ◽

pp. 604-616 ◽

Cited By ~ 12

Author(s):

Rachana Trivedi ◽

Gerhard A. Müller ◽

Manohar S. Rathore ◽

Durga P. Mishra ◽

Hassan Dihazi

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Expression ◽

Er Stress ◽

Myeloid Leukemia ◽

Therapeutic Option ◽

Stress Protein ◽

Acute Myeloid Leukemia Cells ◽

Induced Apoptosis ◽

Acute Myeloid

Background/Aims: ER-Stress and activation of unfolded protein response belong to the major factors involved in chemoresistance in cancer cells. In this study we investigated the effect of shikonin on the survival of acute myeloid leukemia cells and the role of ER-stress protein ERP57, a protein disulfide isomerase, in improvement of chemotherapy. Methods: Using MTT assay we studied cytotoxic effects of shikonin on HL-60 cells. The flow cytometry was adopted to examine the shikonin induced mode of cell death in HL-60 cells. The overall protein expression alteration resulting from shikonin treatment was investigated using proteomics methods. Western blotting was performed to quantify the alteration in protein expression in HL-60 after shikonin treatment. Silencing and overexpression studies were carried out to highlight the therapeutic role of ERP57 in shikonin effect on AML cells. Results: Shikonin induces apoptosis in HL-60 cells without significant effect on Primary cells from healthy volunteers. The apoptotic effect was dose and time dependent and was accompanied by strong alteration in cell proteome. Among the proteins targeted by shikonin, ERP57 was significantly downregulated in HL-60 after treatment. Compared to healthy control ERP57 was found to be highly expressed in AML cell line HL60 and was downregulated after shikonin treatment. Overexpression of ERP57 protected HL-60 from shikonin induced apoptosis, whereas knockdown of ERP57 expression resulted in increase in shikonin induced apoptosis. Conclusions: Our results demonstrate that ERP57 plays a crucial role in resistance towards shikonin induced apoptosis in AML cells. Targeting of ERP57 might offer a new therapeutic option for the treatment of acute myeloid leukemia.

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