scholarly journals Chidamide Combined with Decitabine Synergistically Induce Apoptosis of Acute Myeloid Leukemia By up-Regulating Perp gene In Vivo and in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 536-536
Author(s):  
Qing Li ◽  
Yunfan Yang ◽  
Yu Wu
Keyword(s):  
Cell Lines ◽  
Drug Combination ◽  
Transcriptome Sequencing ◽  
Myeloid Leukemia ◽  
Protein Levels ◽  
Apoptotic Protein ◽  
Induced Apoptosis ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells. The long term survival of AML is not satisfactory, so new treatment should be explored. Here, we show that chidamide(CH), a histone deacetylase inhibitor, combined with decitabine(DE) induces apoptosis of AML cell lines and primary refractory/relapsed AML cells by up-regulating PERP. This may provide a new option for AML treatment. Methods and results: We first examined the half-inhibitory concentrations (IC50) of chidamide and decitabine against THP-1, MV4-11, HL60 and Kasumi-1 cell lines using MTT (Fig1 A-D). And the drug combination is performed according to the IC50. In the double-drug combination experiment, we used MTT to detect the effect of drugs on the proliferation of the four cell lines (Fig1 E-H), used calcusyn 2.0 software to calculate the synergistic effect (Fig2), flow cytometry to detect apoptosis (Fig3 A-D), and western blot to detect the pro-apoptotic protein (C-CASPASE 3 and C-CASPASE 9) and anti-apoptotic proteins (CASPASE 8, BCL-2 and BCL-XL) (Fig3 E-H). We found that chidamide combined with decitabine synergistically inhibited proliferation of AML cell lines, induced apoptosis, up-regulated pro-apoptotic protein levels and down-regulated anti-apoptotic protein levels. To investigate this combination therapeutic effect in vivo, we selected 5 refractory/relapsed AML patients, extracted primary AML cells, and used ATP chemiluminescence kit for drug sensitivity test. The results confirmed that four of the five patients with AML showed sensitivity to combinations (Fig4). To further explore the mechanism of action of CH combination with DE, we performed transcriptome sequencing (Fig5). Analysis of the sequencing results, the gene PERP, which shows the significant difference in the apoptotic pathway, was further examined. The PERP is a new member of the PMP-22/GAS3 family as an apoptosis-associated target of TP53. RT-QPCR and WB verified the role of PERP in apoptosis in DE and CH combination (Fig6). The results showed that the combination could up-regulate the PERP gene than the single drug. When we explored the role of the PERP gene in AML cell lines, we knocked down the PERP gene by lentivirus and detected cell proliferation after infection. Pretreated AML cell lines by lentivirus-infection (Fig7A-F), then we tested for proliferation (Fig7G-I) (Fig8A-C), apoptosis (Fig8D-E), and pro-apoptotic protein expression (Fig8G-I). The results showed that knocking down the PERP gene promoted the proliferation of AML cell lines and attenuated the sensitivity of AML cell lines to chemotherapeutic drugs. We also compare the mRNA level of PERP between 35 AML patients and 20 normal and found that the PERP mRNA of AML patients was significantly lower than the normal (Fig9). MV4-11 cells were exposed to CH and DE alone or in combination, and proteomic sequencing was performed to examine the effect of the drug on cellular protein. The result indicates to some extent that CH contributes more to the combined effect. And the drug causes changes in multiple pathways in the cell (Fig10). Conclusion: Our experiments revealed that CH combined with DE may have therapeutic effects on AML and, to some extent, reveal the mechanism of dual drug combination. Legends to figures Fig1. 50% inhibitory concentration (IC50) values of chidamide and decitabine alone treated AML cell lines. Fig2. Chidamide acts synergistically in AML with DE. Fig3. Chidamide in combination with decitabine significantly induced apoptosis in AML cell lines. Fig4. The sensitivity of relapsed or refractory AML primary cells to chidamide and decitabine alone or in combination. Fig5. Gene expression analysis showed an obvious difference based on treatment. Fig6. Verify transcriptome sequencing results by real-time QPCR and by western blotting with or without drug treatment. Fig7. The effect of down-regulation of PERP by Lentivirus-mediated RNAi on AML cells proliferation. Fig8. PERP knockdown causes AML cells to develop resistance to combination drugs. Fig9.The level of PERP mRNA in peripheral blood mononuclear cells of AML and normal humans. Fig10. Proteomics sequencing results show the differentially expressed protein and a cluster analysis of the functions or pathways enriched by differentially expressed proteins in GO and KEGG pathways compared to single agents. Disclosures No relevant conflicts of interest to declare.

The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 915-915
Author(s):  
Stuart A Rushworth ◽  
Lyubov Zaitseva ◽  
Megan Y Murray ◽  
Matthew J Lawes ◽  
David J MacEwan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking ◽  
Acute Myeloid

Abstract Introduction Despite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. Methods To investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts. Results We initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1. Conclusions These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. Disclosures No relevant conflicts of interest to declare.

Selective Abrogation of STAT5 Expression Impairs the Proliferation of Acute Myeloid Leukemia Cells with Various Cytogenetic Alterations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5024-5024
Author(s):  
Youngsoo Kim ◽  
Tianyuan Zhou ◽  
Shuling Guo ◽  
Andy Siwkowski ◽  
Donna Witchell ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Significant Inhibition ◽  
Acute Myeloid

Abstract STAT5 is a key common downstream mediator of multiple signaling pathways which are often dysregulated in various hematologic malignancies, including acute myeloid leukemia (AML). Due to the heterogeneity and high relapse rate of the disease, the treatment options for AML are currently limited. Although the approach of treating the disease by inhibiting upstream kinases such as FLT3 within these signaling pathways appeared promising, the clinical efficacy of these drugs as mono-therapy have been disappointing. We hypothesized that this lack of efficacy might be due to the residual STAT5 activity that is present even in the presence of these inhibitors in vivo. Therefore, abrogating the expression of the final regulator of these pathways, STAT5, might be a much more efficient way of blocking signaling, thus inhibiting the proliferation and survival of AML cells. In this study, we first investigated the role of STAT5 in the proliferation of AML cells by selectively suppressing the expression of the gene using 2nd-Generation antisense oligonucleotides (ASOs). Suppression of STAT5 following ASO treatment (>80% over control ASO) led to a significant inhibition of cell proliferation (50~70% over control ASO), a decrease in colony formation, and a modest induction of apoptosis in a range of AML lines including KG-1α, MV-4-11, and MOLM-13. STAT5 ASO treatment was highly specific for the STAT5 target and produced predictable effects on gene expression, as demonstrated by the downregulation of Pim-1 and cyclin D1, well-known STAT5 regulated genes. No changes in the expression levels of Bcl-XL, STAT1, and STAT3 were observed. Furthermore, relative anti-proliferative activity within the various AML lines correlated well with the relative levels of STAT5 activity. Interestingly, there was a strong correlation between the extent of STAT5/Pim-1 downregulation and the degree of anti-proliferation, suggesting a possible role of Pim-1 as a downstream effector of STAT5 ASO anti-proliferative activity. Studies comparing the relative effects of the STAT5 ASO inhibitor with the potent multi kinase inhibitor CEP701 in various AML cell lines demonstrated potent anti-proliferative activity for the STAT5 inhibitor in the cell lines including KG-1α that display resistance to the multi kinase inhibitor. Taken together, these results suggest that a STAT5 ASO therapeutic approach may have utility for the treatment of AML and related hematologic disorders.

scholarly journals Concurrent Inhibition of Pim-1 and FLT3 Kinases in FLT3-ITD Acute Myeloid Leukemia Post-Translationally Downregulates the Anti-Apoptotic Protein Mcl-1 through Downregulation of the Mcl-1 Deubiquitinase USP9X

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 35-35 ◽  
Author(s):  
Patrick R. Baldwin ◽  
Shivani Kapoor ◽  
Karthika Natarajan ◽  
Rossana Trotta ◽  
Adriana Tron ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Proteasomal Degradation ◽  
Protein Levels ◽  
Flt3 Inhibitor ◽  
Apoptotic Protein ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

Abstract Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival following both chemotherapy and allogeneic hematopoietic stem cell transplantation. Inhibitors of FLT3 signaling have shown activity in clinical trials in FLT3-ITD AML, but efficacy has generally been limited and transient. Concurrent inhibition of other targets in FLT3-ITD signaling pathways is being explored as an approach to increasing the depth and duration of responses to FLT3 inhibitors. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3, thereby promoting FLT3 signaling in a positive feedback loop in cells with FLT3-ITD. Pim kinase inhibitors are in clinical trials. We previously showed that combinations of clinically active Pim kinase and FLT3 inhibitors at pharmacologically relevant concentrations enhance apoptosis and decrease clonogenic growth of FLT3-ITD AML cell lines and primary patient cells in vitro and suppress growth of FLT3-ITD AML cells in vivo, in relation to treatment with FLT3 or Pim inhibitors alone. Here we studied the mechanistic effects of concurrent Pim kinase and FLT3 inhibition, demonstrating a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. Ba/F3-ITD cells, transfected with FLT3-ITD, were cultured with the pan-Pim kinase inhibitor AZD1208 at 1 μM, a concentration chosen based on in vitro and phase I clinical trial data, and/or the FLT3 inhibitor quizartinib at 1 nM, its IC50 concentration, and expression of the anti-apoptotic proteins Mcl-1, Bcl2 and Bcl-xL and the pro-apoptotic proteins BAD/S112 p-BAD, BAK, BAX and Bim was measured by western blot analysis. Mcl-1 expression decreased in a time-dependent manner with AZD1208 and quizartinib co-treatment, but not with treatment with either inhibitor alone, while levels of the other proteins did not change. Mcl-1 downregulation with Pim kinase and FLT3 inhibitor combination treatment was then confirmed in the human FLT3-ITD AML cell lines MV4-11 and MOLM-14. Mcl-1 expression is regulated at multiple levels, and we next sought to determine the mechanism(s) by which it is downregulated by concurrent Pim and FLT3 inhibition. While Mcl-1 protein levels decreased, Mcl-1 mRNA levels did not change, indicating post-transcriptional regulation. Additionally, levels of miR-29b, a negative regulator of Mcl-1 translation,decreased similarly in Ba/F3-ITD cells treated with AZD1208 and quizartinib, compared to quizartinib alone. Polysome profiling showed decreased total mRNA translation, but no selective reduction in Mcl-1 translation. In contrast, the progressive decrease in Mcl-1 protein expression with AZD1208 and quizartinib co-treatment was abrogated by addition of the proteasome inhibitor MG-132, demonstrating that Mcl-1 protein is downregulated by enhanced Mcl-1 proteasomal degradation. This mechanism was further confirmed by demonstration of an increase in ubiquitinated Mcl-1 prior to Mcl-1 downregulation in cells co-treated with AZD1208 and quizartinib, but not with each inhibitor alone or with DMSO control. The deubiquitinase USP9X decreases Mcl-1 ubiquitination and consequent proteasomal degradation, and we found that USP9X expression is downregulated prior to the increase in ubiquitinated Mcl-1 and the subsequent decrease in Mcl-1 protein levels during AZD1208 and quizartinib co-treatment, but was not altered by treatment with either inhibitor alone. In contrast, expression of the ubiquitin E3 ligases Mule/ARF-BP1, SCFβ-TrCP and Trim17, which mediate Mcl ubiquitination, did not change prior to Mcl-1 downregulation. Preclinical studies in our laboratory and others have shown in vitro and in vivo efficacy of combination treatment with Pim kinase and FLT3 inhibitors in FLT3-ITD AML, suggesting clinical promise of this approach. Here we show that, mechanistically, concurrent Pim kinase and FLT3 inhibition causes a post-translational decrease in expression of the anti-apoptotic protein Mcl-1 via enhanced proteasomal degradation, preceded by downregulation of the Mcl-1 deubiquitinase USP9X and an increase in ubiquitinated Mcl-1, a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. Disclosures Tron: AstraZeneca: Employment; AstraZeneca: Employment. Huszar:AstraZeneca: Employment.

scholarly journals ALKBH5 Functions As an Oncogene in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3910-3910
Author(s):  
Chao Shen ◽  
Yue Sheng ◽  
Rui Su ◽  
Xiaolan Deng ◽  
Sean Robinson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Tp53 Mutation ◽  
Mouse Bone Marrow ◽  
Mutation Status ◽  
Acute Myeloid

Abstract N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) has been shown to play important roles in diverse cellular and pathological processes (Deng X, et al. Cell Res. 2018;28:507-517). ALKBH5, recently identified as a m6A demethylase, was reported to promote tumorigenesis and proliferation in glioblastoma stem-like cells (GSCs) (Zhang, S. et al. Cancer Cell. 2017;31:591-606) and breast cancer stem cells (BCSCs) (Zhang, C et al. PNAS. 2016;113: E2047-E2056). While ALKBH5 is well-recognized to function as an oncogene in solid tumors, it was reported that shallow/deep deletion of ALKBH5 is associated poor prognosis in patients with acute myeloid leukemia (AML), and is frequently co-existing with TP53 mutation (Kwok, C. T et al. J Hematol Oncol. 2017; 10(1): 39), implying that ALKBH5 may function as a tumor suppressor in AML. Thus, a systematic investigation of the definitive role of ALKBH5 in AML is warranted. To this end, we performed series of in vitro and in vivo experiments to determine the function of ALKBH5 in AML. For the in vitro experiments, we used three lentiviral shRNAs (shALKBH5-A, shALKBH5-D and shALKBH5-E) to deplete ALKBH5 expression in three human AML cell lines with different TP53 mutation status: NOMO-1 (TP53-mutant), MV4;11 (TP53-WT) and MA9.3 cells (TP53-WT). Somewhat surprisingly, ALKBH5 depletion significantly (p<0.05) inhibited AML cell proliferation/growth in all three AML cells lines, regardless of the status of TP53 mutation. We next conducted colony forming assays and found that ALKBH5 knockdown significantly (p<0.01) impaired the colony forming ability to 18% ~45% of the control group level in all three AML cell lines. We further showed that ALKBH5 depletion caused a significant increase in apoptosis (with a 1.5 ~ 4 fold increase; p<0.001) in all three AML cell lines, which is consistent with the previous report that knockout of ALKBH5 caused severe apoptosis of mouse testis cells (Zheng G et al. Mol Cell. 2013; 49:18-29). In contrast, ALKBH5 knockdown did not significantly affect cell cycles. To further confirm ALKBH5's role in AML development in vivo. We utilized Xenografted AML model as well as mouse bone marrow transplantation (BMT) model. Consistent with the in vitro results, we found that NSGS mice xeno-transplanted with MV4;11-ALKBH5-knockdown cells survived significantly longer than those with MV4;11 control cells (p<0.001). Moreover, we have also conducted mouse bone marrow transplantation (BMT) assays with MLL-AF9-transduced mouse bone marrow lineage negative (Lin-) progenitor cells collected from mice carrying Alkbh5 wild-type (Alkbh5+/+), or heterozygous (Alkbh5+/-) or homozygous (Alkbh5-/-) deletion. Consistent with the xeno-transplanted mouse model results, our BMT assays also showed that Alkbh5 depletion significantly inhibited leukemogenesis and prolonged survival in BMT recipient mice (median survival of ALKBH5wt/wt +MA9 vs. ALKBH5+/- +MA9 or ALKBH5-/- +MA9: 32 days vs. 64 days or 68 days; p<0.005). Taken together, our in vitro and in vivo functional studies data indicate ALKBH5 also functions as an oncogene in AML regardless of TP53 mutation status, similar to its role in solid tumors. We are currently conducting as series of studies to reveal the molecular mechanism(s) underlying the oncogenic role of ALKBH5 in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1003
Author(s):  
Valentina Gaidano ◽  
Mohammad Houshmand ◽  
Nicoletta Vitale ◽  
Giovanna Carrà ◽  
Alessandro Morotti ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Salvage Pathway ◽  
Pyrimidine Salvage ◽  
Induced Apoptosis ◽  
Acute Myeloid

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

scholarly journals Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

2021 ◽  
Author(s):  
Yudi Miao ◽  
Behnam Mahdavi ◽  
Mohammad Zangeneh
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Aqueous Extract ◽  
Myeloid Leukemia ◽  
Antioxidant Properties ◽  
Leukemia Cell ◽  
Sem Images ◽  
Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

scholarly journals The Salt-Inducible Kinase inhibitor YKL-05-099 suppresses MEF2C function and acute myeloid leukemia progressionin vivo

2019 ◽  
Author(s):  
Yusuke Tarumoto ◽  
Shan Lin ◽  
Jinhua Wang ◽  
Joseph P. Milazzo ◽  
Yali Xu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Disease Progression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Clinical Investigation ◽  
Genetic Targeting ◽  
Acute Myeloid

AbstractLineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a Salt-Inducible Kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF MEF2C. In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. Here, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells underin vitroandin vivoconditions. Similar phenotypes were obtained when exposing cells to YKL-05-099, which caused cell cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele ofSIK3, we found that the anti-proliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated two different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progressionin vivoand extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-positive AML and provide a rationale for developing drug-like inhibitors of SIK3 for definitive pre-clinical investigation and for studies in human patients with leukemia.Key PointsAML cells are uniquely sensitive to genetic or chemical inhibition of Salt-Inducible Kinase 3in vitroandin vivo.A SIK inhibitor YKL-05-099 suppresses MEF2C function and AMLin vivo.

scholarly journals The C-terminus of CBFβ-SMMHC is required to induce embryonic hematopoietic defects and leukemogenesis

Blood ◽  
2013 ◽  
Vol 121 (4) ◽  
pp. 638-642 ◽  
Author(s):  
Yasuhiko Kamikubo ◽  
R. Katherine Hyde ◽  
Ling Zhao ◽  
Lemlem Alemu ◽  
Cecilia Rivas ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Myeloid Leukemia ◽  
Single Copy ◽  
Full Length ◽  
Myeloproliferative Disease ◽  
Chromosome 16 ◽  
C Terminus ◽  
Acute Myeloid

Abstract The C-terminus of CBFβ-SMMHC, the fusion protein produced by a chromosome 16 inversion in acute myeloid leukemia subtype M4Eo, contains domains for self-multimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBFβ-SMMHC. To test the role of the fusion protein's C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBFβ-SMMHC (CBFβ-SMMHCΔC95). Embryos with a single copy of CBFβ-SMMHCΔC95 were viable and showed no defects in hematopoiesis, whereas embryos homozygous for the CBFβ-SMMHCΔC95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBFβ-SMMHC. Importantly, unlike mice expressing full-length CBFβ-SMMHC, none of the mice expressing CBFβ-SMMHCΔC95 developed leukemia, even after treatment with a mutagen, although some of the older mice developed a nontransplantable myeloproliferative disease. Our data indicate that the CBFβ-SMMHC's C-terminus is essential to induce embryonic hematopoietic defects and leukemogenesis.

Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia

Blood ◽  
2021 ◽  
Author(s):  
Amanda G Davis ◽  
Daniel T. Johnson ◽  
Dinghai Zheng ◽  
Ruijia Wang ◽  
Nathan D. Jayne ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Alternative Polyadenylation ◽  
Hematopoietic Stem ◽  
Global Trends ◽  
Protein Levels ◽  
Mtorc1 Signaling ◽  
Differentiation Block ◽  
Acute Myeloid

Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site (PAS) usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in acute myeloid leukemia (AML) patients by performing 3' Region Extraction And Deep Sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in PAS usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS dataset. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block of AML patients.

scholarly journals Selective targeting of NAMPT by KPT-9274 in acute myeloid leukemia

2019 ◽  
Vol 3 (3) ◽  
pp. 242-255 ◽  
Author(s):  
Shaneice R. Mitchell ◽  
Karilyn Larkin ◽  
Nicole R. Grieselhuber ◽  
Tzung-Huei Lai ◽  
Matthew Cannon ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Treatment Options ◽  
Tumor Development ◽  
Xenograft Model ◽  
Nicotinamide Phosphoribosyltransferase ◽  
P21 Activated Kinase ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Treatment options for acute myeloid leukemia (AML) remain extremely limited and associated with significant toxicity. Nicotinamide phosphoribosyltransferase (NAMPT) is involved in the generation of NAD+ and a potential therapeutic target in AML. We evaluated the effect of KPT-9274, a p21-activated kinase 4/NAMPT inhibitor that possesses a unique NAMPT-binding profile based on in silico modeling compared with earlier compounds pursued against this target. KPT-9274 elicited loss of mitochondrial respiration and glycolysis and induced apoptosis in AML subtypes independent of mutations and genomic abnormalities. These actions occurred mainly through the depletion of NAD+, whereas genetic knockdown of p21-activated kinase 4 did not induce cytotoxicity in AML cell lines or influence the cytotoxic effect of KPT-9274. KPT-9274 exposure reduced colony formation, increased blast differentiation, and diminished the frequency of leukemia-initiating cells from primary AML samples; KPT-9274 was minimally cytotoxic toward normal hematopoietic or immune cells. In addition, KPT-9274 improved overall survival in vivo in 2 different mouse models of AML and reduced tumor development in a patient-derived xenograft model of AML. Overall, KPT-9274 exhibited broad preclinical activity across a variety of AML subtypes and warrants further investigation as a potential therapeutic agent for AML.

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