scholarly journals Targeting the MTF2-MDM2 Axis Sensitizes Refractory Acute Myeloid Leukemia to Chemotherapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5232-5232
Author(s):  
Hani Jrade ◽  
Harinad B Maganti ◽  
Christopher Cafariello ◽  
Christopher J Porter ◽  
Julien Yockell-Lelièvre ◽  
...  

Abstract Next generation sequencing of acute myeloid leukemia (AML) patient samples has enabled more granular risk stratification of patients; however, refractory AML patients can be found across all risk groups, suggesting that non-genetic lesions regulate chemoresponsiveness. Consistent with this hypothesis is the finding that many of the mutated AML driver genes are encode epigenetic modifiers. Thus, unraveling the epigenetic dysregulation in AML is critical to better understand disease initiation and progression, as well as develop targeted therapies. Metal Response Element Binding Transcription Factor 2/Polycomblike 2 (MTF2/PCL2) plays a fundamental role in recruiting the Polycomb repressive complex 2 (PRC2) to chromatin and we show that it is commonly silenced in primary AML patient cells at diagnosis. Furthermore, the loss of MTF2 in hematopoietic stem and progenitor cells (HSPCs) leads to an altered epigenetic state that underlies refractory AML. By implementing unbiased systems analyses, we identified the E3 ubiquitin ligase MDM2 that inhibits p53 as a direct target of MTF2-PRC2. MTF2 deficiency leads to over-expression of MDM2 and inhibition of p53-mediated cell cycle regulation and apoptosis, leading to chemoresistance and refractory AML. Targeting this dysregulated signaling pathway by MTF2 overexpression or MDM2 inhibitors sensitized refractory patient leukemic cells to induction chemotherapeutics and prevented relapse in AML patient-derived xenograft (PDX) mice. Therefore, we have uncovered a direct epigenetic mechanism by which MTF2 functions as a tumor suppressor required for AML chemotherapeutic sensitivity and identified a potential therapeutic strategy to treat refractory AML. Disclosures Sabloff: Celgene: Membership on an entity's Board of Directors or advisory committees.

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2906-2912 ◽  
Author(s):  
D Haase ◽  
M Feuring-Buske ◽  
S Konemann ◽  
C Fonatsch ◽  
C Troff ◽  
...  

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3765-3765
Author(s):  
Cheuk-Him Man ◽  
David T. Scadden ◽  
Francois Mercier ◽  
Nian Liu ◽  
Wentao Dong ◽  
...  

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.


Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3678-3684 ◽  
Author(s):  
E.L. Sievers ◽  
F.R. Appelbaum ◽  
R.T. Spielberger ◽  
S.J. Forman ◽  
D. Flowers ◽  
...  

Abstract Leukemic blast cells express the CD33 antigen in most patients with acute myeloid leukemia (AML), but this antigen is not expressed by hematopoietic stem cells. We conducted a study to determine whether normal hematopoiesis could be restored in patients with AML by selective ablation of cells expressing the CD33 antigen. In a dose escalation study, 40 patients with relapsed or refractory CD33+ AML were treated with an immunoconjugate (CMA-676) consisting of humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicheamicin. The capacity of leukemic cells to efflux 3,3’-diethyloxacarbocyanine iodide (DiOC2) was used to estimate pretreatment functional drug resistance. Leukemia was eliminated from the blood and marrow of 8 (20%) of the 40 patients; blood counts returned to normal in three (8%) patients. A high rate of clinical response was observed in leukemias characterized by low dye efflux in vitro. Infusions of CMA-676 were generally well tolerated, and a postinfusion syndrome of fever and chills was the most common toxic effect. Two patients who were treated at the highest dose level (9 mg/m2) were neutropenic >5 weeks after the last dose of CMA-676. These results show that an immunoconjugate targeted to CD33 can selectively ablate malignant hematopoiesis in some patients with AML.


2020 ◽  
Vol 4 (8) ◽  
pp. 1722-1736 ◽  
Author(s):  
Ayaka Yamaoka ◽  
Mikiko Suzuki ◽  
Saori Katayama ◽  
Daiki Orihara ◽  
James Douglas Engel ◽  
...  

Abstract Chromosomal rearrangements between 3q21 and 3q26 elicit high-risk acute myeloid leukemia (AML), which is often associated with elevated platelet and megakaryocyte (Mk) numbers. The 3q rearrangements reposition a GATA2 enhancer near the EVI1 (or MECOM) locus, which results in both EVI1 overexpression and GATA2 haploinsufficiency. However, the mechanisms explaining how the misexpression of these 2 genes individually contribute to leukemogenesis are unknown. To clarify the characteristics of differentiation defects caused by EVI1 and GATA2 misexpression and to identify the cellular origin of leukemic cells, we generated a system to monitor both inv(3) allele-driven EVI1 and Gata2 expression in 3q-rearranged AML model mice. A cell population in which both EVI1 and Gata2 were highly induced appeared in the bone marrows before the onset of frank leukemia. This population had acquired serial colony-forming potential. Because hematopoietic stem/progenitor cells (HSPCs) and Mks were enriched in this peculiar population, we analyzed the independent EVI1 and GATA2 contributions to HSPC and Mk. We found that inv(3)-driven EVI1 promotes accumulation of Mk-biased and myeloid-biased progenitors, Mks, and platelets, and that Gata2 heterozygous deletion enhanced Mk-lineage skewing of EVI1-expressing progenitors. Notably, inv(3)-directed EVI1 expression and Gata2 haploinsufficient expression cooperatively provoke a leukemia characterized by abundant Mks and platelets. These hematological features of the mouse model phenocopy those observed in human 3q AML. On the basis of these results, we conclude that inv(3)-driven EVI1 expression in HSPCs and Mks collaborates with Gata2 haploinsufficiency to provoke Mk-lineage skewing and leukemogenesis with excessive platelets, thus mimicking an important feature of human AML.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2905-2905
Author(s):  
Marta Cabezon ◽  
Joan Bargay ◽  
Blanca Xicoy ◽  
Laura Palomo ◽  
Sílvia Marcé ◽  
...  

Abstract INTRODUCTION: Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms originated in hematopoietic stem cells, characterized by citopenias, dysplasia in one or more cell lines, ineffective hematopoiesis and an increased risk of progression to acute myeloid leukemia (AML). Treatment of MDS depends on subtype and prognostic category. DNA methyltranferase inhibitors are approved for high risk MDS. Over the past decade, the application of new high-throughput technologies to the study of MDS has led to the identification of several recurrently mutated genes. These include genes producing proteins involved in RNA splicing, DNA methylation, chromatin modification, transcription, DNA repair control, cohesin function, RAS pathway, and DNA replication. There is a significant overlap between the genes mutated commonly in MDS with those found in AML. Mutation status is not widely used to select treatment in MDS. The aim of this study is to define the mutational status of MDS and secondary AML (sAML) patients at diagnosis that have been treated with azacitidine (AZA) to see if it could help to discriminate which patients will respond from those who will not. MATERIAL AND METHODS: A prospective study was performed on 36 patients with MDS and sAML treated with AZA. Genomic DNA was obtained from bone marrow at diagnosis. SeqCap EZ and KAPA Library Preparation Kit (Roche) reagents have been used to enrich DNA of 83 genes implicated in myeloid neoplasm. The customized panel has been analyzed in MiSeq Illumina platform with 150bp paired-end reads. Samples were preliminary analyzed using Illumina MiSeq Reporter and Variant Studio softwares. Data from response to treatment and survival have been collected from all patients. RESULTS:The mean depth of the targeted resequencing per base was 685-fold. After filtering all the variations obtained for quality, biological consequence and discard the known SNPs, we have obtained 162 variations, including 145 single nucleotide variants (SNV) and 17 insertions/deletions. All patients harbored at least 1 alteration with a mean of 4.5 variants per sample. The average of alterations detected in each cytological category can be observed in Table 1.Table 1.Average abnormalities detected by cytological category.Nº patientsAverage of alterations detected for patient (range)sAML104,8 (1-8)RAEB-274,9 (2-8)RAEB-1123,7 (1-6)RCDM54,4 (3-7)RCDM-RS16RARs11The most frequent altered genes have been TP53, TET2 and DNMT3A. The numbers of variations detected for each gene are represented in Table 2.Complete results, including correlation with treatment response will be presented in the meeting.Table 2.Number of variations in each gene.GeneNº of variations foundNº of diferent variationsNº of patients with variationsFrequency of variationsTP5322191952,8%TET214101027,8%DNMT3A88822,2%CREBBP75719,4%SRSF271719,4%ASXL165616,7%U2AF162616,7%EP30053513,9%STAG255513,9%CUX144411,1%ETV643411,1%MLL (KMT2A)43411,1%RUNX14438,3%BCOR3338,3%CDH133338,3%CTNNA13238,3%EZH23338,3%GCAT3338,3%MLL2 (KMT2D)3338,3%NF13338,3%PDGFRB3338,3%SH2B33338,3%TGM23238,3%UMODL13338,3%CEBPA2125,6%CSF3R2225,6%GATA22125,6%PHLPP12225,6%RAD212225,6%SF3B12125,6%SUZ122225,6%TIMM502125,6%Others*1112,8%*ABL1, BCORL1, CALR, CDH3, IDH2, KRAS, LUC7L2, NPM1, NRAS, PHF6, SF3A1, SFPQ, SMC3, TERT, WT1, ZRSR2. CONCLUSIONS: Targeted deep-sequencing technique is a good tool to study mutational profile in MDS and sAML. SNV are the most frequent type of alteration found in our cohort. The patients with sAML and RAEB-2 present more variations than patients with RAEB-1. The rest of groups are less representing to be evaluated. The most affected genes match with those described in the literature, with some exceptions that need to be studied in more detail. We expect to predict in advance which patients are going to respond when we study the correlation of mutational analysis with treatment response. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Josep Carreras, Obra Social "La Caixa" and Celgene Spain. Diana Domínguez for her technical assistance Disclosures Valcarcel: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3684-3684
Author(s):  
Valentina Salvestrini ◽  
Stefania Orecchioni ◽  
Francesca Reggiani ◽  
Giovanna Talarico ◽  
Elisa Orioli ◽  
...  

Abstract ATP is the key energy molecule as well as an ubiquitous extracellular messenger. Depending on its dose and the engaged purinergic P2 receptor (P2R) subtype, ATP can trigger many different cell responses, including proliferation and cell death. Recent studies have shown that high ATP level exhibits direct cytotoxicity on many tumor cell types. Among the receptors engaged by ATP, P2X7 is the most consistently expressed by tumor cells and its overexpression is related to tumor growth and progression. The P2X7 is an ATP-gated ion channel that, upon sustained stimulation with millimolar ATP concentrations, drives the opening of a non-selective large conductance pore, triggering cell-death signal. We previously demonstrated that ATP is a potent stimulator of normal hematopoietic stem cell compartment while inhibiting acute myeloid leukemia (AML) cells. Based on this observation, we studied AML samples (n=20) collected from the bone marrow or the peripheral blood of leukemic patients at diagnosis before treatment (percentage of circulating blasts >90%). In addition, normal hematopoietic stem cells (HSC) were isolated from leukapheresis products of 5 healthy donors receiving G-CSF. Our data demonstrate that AML cells express high level of P2X7 and that its activation with high dose of ATP reduces blast cell viability while is not effective on normal CD34+ cells. The cytotoxic effect is due to the induction of apoptosis, associated with reduction of mithocondrial membrane potential and activation of caspase cascade. Interestingly, P2X7 is also expressed by leukemic stem/progenitor cells (LSC) and ATP treatment exerts a direct cytotoxicity on different subsets of stem/progenitor cell compartment i.e. CD34- CD38-, CD34+ CD38-, CD34+ CD38+ and CD34- CD38+. Of note, this cytotoxic effect was not observed on HSC subpopulations. Furthermore, we transplanted 1x106 human AML cells into NSG immunodeficient mice followed by intraperitoneal administration of ATP every other day for thirty days post-transplantation. Our results show a 40% inhibition of AML engraftment in ATP-treated mice vs controls. Different P2X7 splice variants have been identified among which only two are functional: P2X7A, which shows both pro-apoptotic and trophic activity and P2X7B, which retains only the growth promoting phenotype. In order to explain ATP different effects on LSCs and normal HSCs, we assumed a different P2X7 isoforms expression on normal and leukemic cells. Preliminary results showed a reduced expression of both P2X7A and P2X7B on normal CD34+ compared to leukemic cells. In particular normal CD34+ express very low level of P2X7A, which is responsible for pore formation after ATP stimulation. Moreover, since P2X7 pore formation facilitates the passage of hydrophilic chemotherapeutic agents, we hypothesized that ATP may potentiate the cytotoxic effect of antineoplastic drugs. Our results showed that ATP potentiates the cytotoxic effect of ARA-C, by significantly reducing cell proliferation and increasing apoptosis of leukemia cell lines. In conclusion, overall survival of adult AML remains poor due to the lack of novel and effective therapies. Novel compounds that have the potential to improve the treatment efficacy with low toxicity are highly warranted. Overall, our results may provide the biological rationale to use P2X7 as a target for novel therapeutical approaches against AML. Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3428-3428
Author(s):  
Van Hung Tran ◽  
Florence Persat ◽  
Sophie Gardes ◽  
Jeremy Monfray ◽  
Sophie Ducastelle-Leprêtre ◽  
...  

Abstract Introduction Invasive aspergillosis (IA) remains an important cause of mortality in immunocompromised acute myeloid leukemia (AML) patients receiving induction chemotherapy and in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematological malignancies. Early diagnostic is critical and challenging given the efficacy and availability of several new anti-fungal therapies. In this study, we evaluated the performance of different factors in predicting the occurrence of IA, including the Aspergillus antigen galactomannan (GM) detection in sera. Methods We included all AML patients receiving induction chemotherapy and patients undergoing allo-HSCT for any hematological malignancy at our center between April 2006 and April 2014 with available data on Aspergillus antigen GM. Serologic detection of circulating GM fungal biomarker was considered during the 100 days following the first day of induction chemotherapy in AML patients or from the day of allo-HSCT. Usual follow-up included two GM tests per week, only patients with at least three serum GM results were considered. The GM tests have been performed routinely using the ELISA kit (Platelia Aspergillus antigen ELISA, Biorad), giving the results in index values. Demographic, GM index results and diagnostic data were collected. IA cases were classified as proven or probable according to the EORTC criteria. The value of the first antigen test, the delay to positivity, and the slope of the progression of the index value between the first two antigens concentrations were considered as predictors of IA. ROC curves for each predictor and their combination were performed and prognostic scores were established. Results A total of 775 patients were included : i) 292 AML patients, 153 (52%) males with a median age of 62 years (range: 17-79), 15% were classified as favorable, 8% as intermediate I, 18% as intermediate II and 59% as unfavorable according to cytogenetics and molecular markers; ii) 483 allo-HSCT patients, 293 (61%) were males, median age was 48 years (range: 18-70), among them 234 (48%) AML, 66 (14%) multiple myeloma, 46 (10%) Myelodysplastic syndromes, 38 (8%) Non-Hodgkin Lymphoma and the rest of patients had other hematological disorders; 233 (48 %) patients received reduced intensity conditioning and 250 (52%) myeloablative conditioning. The disease status at allo-HSCT was complete remission (CR) in 366 (76%) patients and the rest of patients were in less than CR. HSC source was peripheral blood in 42.2% (90 identical siblings, 150 10/10 matched unrelated, 54 9/10 mismatched unrelated), bone marrow in 42.6% (105 identical siblings, 162 10/10 matched unrelated, 45 9/10 mismatched unrelated) and cord blood in 15.2%. A total of 877 episodes with 16121 GM serum antigen results was considered (median: 18 GM tests per patient). During the follow-up, we identified 121 episodes with at least one positive GM test with a cumulative incidence at day 100 of 13.8%. We also diagnosed 48 IA (2 proven, 46 probable), with a cumulative incidence at day 100 of 5.5% in total, 7.2% in AML and 4.3% in allo-HSCT, respectively. We then classified the GM positive episodes in 82 false-positive (68%) and 39 true-positive episodes (32%) for IA, respectively. A majority of IA events occurred during the first 30 days of follow up, GM positivity showing a positive predictive value of 41% versus a negative predictive value of 99%. The three IA predicting factors had similar independent effects and their combinations were performed, allowing the establishment of an area under ROC of 0.79 (95% CI: 0.70-0.89). Cut off values of the first positive GM serum and slope were equal or higher than 1.04 and 0.04, respectively, and delay to positivity equal or less than 15 days. To simplify the practical use in clinical practice, the prognostic score defining the IA risk probability was defined as the number of predictors present (values from 0 to 3). This score was tested on positive follow-up giving values of 0, 1, 2 or higher for 45 (37%), 39 (32%) and 37 episodes (31%), respectively. A score superior or equal to 2 was indicative of IA in 62% of the cases (figure 1). Conclusion As IA has a significant impact on hematology patient's survival, this GM predictive score combining three predictors (value of the first antigen index, delay of positivity and slope of the index values) may help clinicians to conclude about starting an early preemptive IA treatment. Figure 1. Figure 1. Disclosures Nicolini: Ariad Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1356-1356
Author(s):  
Anup Kumar Singh ◽  
Xiaochun Yu

Abstract DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet. In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 11 ◽  
Author(s):  
Yuhang Li ◽  
Longcan Cheng ◽  
Chen Xu ◽  
Jianlin Chen ◽  
Jiangwei Hu ◽  
...  

Hypomethylating agents, decitabine (DAC) and azacitidine, can act as prophylactic and pre-emptive approaches after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a non-intensive bridging approach before allo-HSCT. However, they are rarely used as a part of conditioning regimens in patients with relapsed or refractory acute myeloid leukemia (AML). This retrospectively study included a total of 65 patients (median, 37; range, 13–63) with relapsed or refractory AML who were treated by allo-HSCT after myeloablative conditioning regimens without or with DAC (high-dose DAC schedule, 75 mg/m2 on day −9 and 50 mg/m2 on day −8; low-dose DAC schedule, 25 mg/m2/day on day −10 to −8). DAC exerted no impact on hematopoietic reconstitution. However, patients who were treated with the high-dose DAC schedule had significantly higher incidence of overall survival (OS, 50.0%) and leukemia-free survival (LFS, 35.0%), and lower incidence of relapse (41.1%) and grade II–IV acute graft versus host disease (aGVHD, 10.0%) at 3 years, when compared with those treated with standard conditioning regimens or with the low-dose DAC schedule. In conclusion, high-dose DAC combined with standard conditioning regimens before allo-HSCT is feasible and efficient and might improve outcomes of patients with relapsed or refractory AML, which provides a potential approach to treat these patients.


Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 1944-1950 ◽  
Author(s):  
W Terpstra ◽  
RE Ploemacher ◽  
A Prins ◽  
K van Lom ◽  
K Pouwels ◽  
...  

A subset of leukemic cells is assumed to maintain long-term growth of acute myeloid leukemia (AML) in vivo. Characterization of these AML progenitor cells may further define growth properties of human leukemia. In vitro incubations with 5-fluorouracil (5-FU) have been used for enrichment of normal primitive hematopoietic stem cells. By analogy to normal hematopoiesis, it was hypothesized that primitive leukemic stem cells might be kinetically more inactive than colony- forming cells (colony-forming units-AML [CFU-AML]). To examine this hypothesis, conditions were established for incubation with 5-FU that eliminated all CFU-AML. These conditions selected a 5-FU-resistant AML fraction that was evaluated for its capacity for long-term growth by transplantation into mice with severe combined immunodeficiency (SCID) and long-term culture in the quantitative cobblestone area-forming cell (CAFC) assay. Transplantation of the 5-FU-resistant fraction of four cases of AML into SCID mice resulted in growth of AML. Whereas no CFU- AML survived, 31% to 82% of primitive (week-6) CAFC were recovered from the 5-FU-treated cells. Hematopoietic cells proliferating in the CAFC assay were shown to be leukemic by cytologic, cytogenetic, or molecular analysis. The reduction of AML growth as determined by outgrowth of AML in SCID mice was in the same order of magnitude as the primitive (week- 6) CAFC reduction. This indicates that both assays measure closely related cell populations and that the CAFC assay can be used to study long-term growth of AML. These results show a hierarchy of AML cells that includes 5-FU-resistant progenitors. These cells are characterized as primitive (week-6) CAFC and as leukemia-initiating cells in SCID mice.


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