Differentiation Of Leukemia Cell Lines Research Articles

Overexpression of RBM4 promotes acute myeloid leukemia cell differentiation by regulating alternative splicing of TFEB

Alternative splicing (AS) is an efficient and ubiquitous transcriptional regulatory mechanism that expands the coding capacity of the genome and is associated with the occurrence and progression of cancer. The differentiation-promoting regimen is a potential therapeutic approach in cancer treatment. In this study, we screened NPMc-positive and NPMc-negative AML samples from the Cancer Genome Atlas (TCGA), focusing on the splicing factor RBM4 and its splicing mechanism on the target gene TFEB, which are most relevant to the prognosis of AML. We also investigated the impact of the TFEB-dominant spliceosome on autophagy and differentiation of THP-1 and K562 cells. The results showed that RBM4 recognized the CU-rich sequence in intron 8 of TFEB, increasing the production of the TFEB-L spliceosome, which promoted autophagy. Overexpression of RBM4 increased autophagy and promoted cell differentiation. The combination of TFEB-L with the therapeutic drug rapamycin further promoted the differentiation of leukemia cell lines and primary leukemia cells in AML patients. This study suggested that overexpression of RBM4 could promote cell differentiation by promoting the production of the TFEB-dominant spliceosome, demonstrating the potential of the TFEB-dominant spliceosome combined with chemotherapy drugs to promote leukemia cell differentiation and improve patient prognosis.

Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small molecule inhibitors with a well-defined mechanism of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary AML samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

Olfactomedin 4 Inhibits Erythroid Differentiation of Leukemic Cell Lines Induced By TGF-β: A Model of Preferential Commitment of Del(13q) Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

[Background] Olfactomedin 4 (OLFM4), a member of the olfactomedin-related protein family, is constitutively expressed in bone marrow (BM) cells and many gastrointestinal organs and is involved in a variety of cellular functions, including proliferation, differentiation, apoptosis, and cell adhesion in tumor cells. Previous studies have suggested that OLFM4 supports the survival of leukemic stem cells derived from iPS cells of patients with chronic myeloid leukemia. Our recent study revealed a somatic mutation of OLFM4 in HLA allele-lacking granulocytes of a patients with acquired aplastic anemia (AA) in long-term remission (Blood advances, 2(9):1000-1012, 2018). The OLFM4 gene is located at chromosome 13q14.3, which is a commonly deleted region in AA patients with del(13q) who show a good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein(GPI-AP)-deficient cells (Haematologica, 97(12):1845-9, 2012). There may be common mechanisms underlying the preferential commitment between GPI-AP- and del(13q) hematopoietic stem and progenitor cells (HSPCs), like insensitivity to inhibitory cytokines such as TGF-β, in immune-mediated BM failure. To confirm this hypothesis, we studied the effect of OLFM4 knockout on the erythroid differentiation of erythroid leukemia cell lines induced by TGF-β. [Methods] We established OLFM4 knockout (KO) K562 and TF-1 cells using a CRISPR-Cas9 system. OLFM4-knockdown (KD) cells were also prepared using siRNA to validate the results of OLFM4-KO cells. The OLFM4 mRNA and protein levels were determined using quantitative polymerase chain reaction, flow cytometry (FCM), Western blotting, immunocytochemistry, and immunofluorescence methods. The erythroid differentiation was assessed by measuring the expression of glycophorin A (GPA) with FCM, GATA-1 protein expression using Western blotting, and iron staining of the cells. [Results] The OLFM4 KO cells showed slower proliferation than wild-type (WT) cells. Both OLFM4-KO cells and OLFM4-KD cells showed a higher GPA expression than WT cells (median fluorescence intensity [MFI] of K562: 2924 and 2143 vs. 1469 and TF-1: 950 and 870 vs. 694, respectively). OLFM4-KO cells showed erythroid morphology, an elevated expression of GATA-1, and positivity for iron granules, suggesting that OLFM4 KO promoted the erythroid differentiation of K562 and TF-1 cells in RPMI1640 containing 10% fetal bovine serum (Figure 1). When WT cells were cultured in a serum-free culture medium (Steampro34) with or without TGF-β (6 ng/ml) for 8 days, the GPA expression was induced in both TF-1 (MFI: 4358 vs. 883), and K562 cell lines (33440 vs. 25655). The OLFM4 protein levels in these cell lines were significantly decreased by the TGF-β treatment in a dose-dependent manner, suggesting that TGF-β directly downregulated the OLFM4 expression in WT cells; the relative expression of OLFM4 was 1, 0.64, and 0.12 while that of TF-1 was 1, 0.12, and 0.05 at 0, 2, and 6 ng/ml of TGF-β, respectively (Figure 2). [Conclusion] OLFM4 prevents K562 and TF-1 cells from differentiating into erythroid cells in response to TGF-β. The erythroid differentiation of these leukemic cells may be mediated by the downregulation of OLFM4 induced by TGF-β. Haploinsufficiency of OLFM4 due to either a loss of function mutation or del(13q) may be related to the mechanisms underlying the preferential commitment of the mutant HSPCs to erythroid cells in patients with immune-mediated BM failure where TGF-β is abundantly present. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Novartis Pharma K.K: Honoraria; Kyowa Kirin: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; SynBio Pharmaceuticals: Consultancy.

Correction to Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase InhibitorsStefano SainasStefano SainasMore by Stefano Sainashttp://orcid.org/0000-0001-5010-8536, Agnese C. PippioneAgnese C. PippioneMore by Agnese C. Pippionehttp://orcid.org/0000-0003-3778-8420, Elisa LupinoElisa LupinoMore by Elisa Lupinohttp://orcid.org/0000-0001-6631-8845, Marta GiorgisMarta GiorgisMore by Marta Giorgishttp://orcid.org/0000-0002-3282-1220, Paola CircostaPaola CircostaMore by Paola Circostahttp://orcid.org/0000-0001-8251-6905, Valentina GaidanoValentina GaidanoMore by Valentina Gaidanohttp://orcid.org/0000-0003-0701-7975, Parveen GoyalParveen GoyalMore by Parveen Goyalhttp://orcid.org/0000-0002-7808-8298, Davide BonanniDavide BonanniMore by Davide Bonannihttp://orcid.org/0000-0002-8832-0492, Barbara RolandoBarbara RolandoMore by Barbara Rolandohttp://orcid.org/0000-0001-6138-1503, Alessandro CignettiAlessandro CignettiMore by Alessandro Cignettihttp://orcid.org/0000-0003-2109-1194, Alex DucimeAlex DucimeMore by Alex Ducimehttp://orcid.org/0000-0003-3177-6111, Mikael AnderssonMikael AnderssonMore by Mikael Anderssonhttp://orcid.org/0000-0002-1188-8127, Michael JärvåMichael JärvåMore by Michael Järvåhttp://orcid.org/0000-0002-8519-5622, Rosmarie FriemannRosmarie FriemannMore by Rosmarie Friemannhttp://orcid.org/0000-0002-5633-2894, Marco PiccininiMarco PiccininiMore by Marco Piccininihttp://orcid.org/0000-0003-2287-0704, Cristina RamondettiCristina RamondettiMore by Cristina Ramondettihttp://orcid.org/0000-0002-0501-5696, Barbara BuccinnàBarbara BuccinnàMore by Barbara Buccinnàhttp://orcid.org/0000-0002-5843-7112, Salam Al-KaradaghiSalam Al-KaradaghiMore by Salam Al-Karadaghihttp://orcid.org/0000-0001-8608-0635, Donatella BoschiDonatella BoschiMore by Donatella Boschihttp://orcid.org/0000-0003-4929-4460, Giuseppe SaglioGiuseppe SaglioMore by Giuseppe Sagliohttp://orcid.org/0000-0002-1046-3514, and Marco L. Lolli*Marco L. LolliMore by Marco L. Lollihttp://orcid.org/0000-0002-3030-3163Cite this: J. Med. Chem. 2019, 62, 3, 1696Publication Date (Web):January 29, 2019Publication History Published online29 January 2019Published inissue 14 February 2019https://doi.org/10.1021/acs.jmedchem.9b00057Copyright © 2019 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views1007Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (570 KB) Get e-Alerts Get e-Alerts

Effects of all-trans retinoic acid (ATRA) in addition to chemotherapy for adults with acute myeloid leukaemia (AML) (non-acute promyelocytic leukaemia (non-APL)).

Acute myeloid leukaemia (AML) is the most common acute leukaemia affecting adults. Most patients diagnosed with AML are at advanced age and present with co-morbidities, so that intensive therapy such as stem cell transplantation (SCT) is impossible to provide or is accompanied by high risks for serious adverse events and treatment-related mortality. Especially for these patients, it is necessary to find out whether all-trans retinoic acid (ATRA), an intermediate of vitamin A inducing terminal differentiation of leukaemic cell lines, added to chemotherapy confers increased benefit or harm when compared with the same chemotherapy alone. This review aims to determine benefits and harms of ATRA in addition to chemotherapy compared to chemotherapy alone for adults with AML (not those with acute promyelocytic leukaemia (non-APL)). We searched the Central Register of Controlled Trials (CENTRAL), MEDLINE, study registries and relevant conference proceedings up to July 2018 for randomised controlled trials (RCTs). We also contacted experts for unpublished data. We included RCTs comparing chemotherapy alone with chemotherapy plus ATRA in patients with all stages of AML. We excluded trials if less than 80% of participants were adults or participants with AML, and if no subgroup data were available. Patients with myelodysplastic syndrome (MDS) were included, if they had a refractory anaemia and more than 20% of blasts. Two review authors independently extracted data and assessed the quality of trials. We contacted study authors to obtain missing information. We used hazard ratios (HR) for overall survival (OS) and disease-free survival (DFS; instead of the pre-planned event-free survival, as this outcome was not reported), and we calculated risk ratios (RR) for the other outcomes quality of life, on-study mortality and adverse events. We presented all measures with 95% confidence intervals (CIs). We assessed the certainty of evidence using GRADE methods. Our search resulted in 2192 potentially relevant references, of which we included eight trials with 28 publications assessing 3998 patients. Overall, we judged the potential risk of bias of the eight included trials as moderate. Two of eight trials were published as abstracts only. All the included trials used different chemotherapy schedules and one trial only evaluated the effect of the hypomethylating agent decitabine, a drug know to affect epigenetics, in combination with ATRA.The addition of ATRA to chemotherapy resulted in probably little or no difference in OS compared to chemotherapy only (2985 participants; HR 0.94 (95% confidence interval (CI) 0.87 to 1.02); moderate-certainty evidence). Based on a mortality rate at 24 months of 70% with chemotherapy alone, the mortality rate with chemotherapy plus ATRA was 68% (95% CI 65% to 71%).For DFS, complete response rate (CRR) and on-study mortality there was probably little or no difference between treatment groups (DFS: 1258 participants, HR 0.99, 95% CI 0.87 to 1.12; CRR: 3081 participants, RR 1.02, 95% CI 0.96 to 1.09; on-study mortality: 2839 participants, RR 1.02, 95% CI 0.81 to 1.30, all moderate-certainty evidence).Three trials with 1428 participants reported the adverse events 'infection' and 'cardiac toxicity': There was probably no, or little difference in terms of infection rate between participants receiving ATRA or not (RR 1.05, 95% CI 0.96 to 1.15; moderate-certainty evidence). We are uncertain whether ATRA decreases cardiac toxicity (RR 0.46, 95% CI 0.24 to 0.90; P = 0.02, very low certainty-evidence, however, cardiac toxicity was low).Rates and severity of diarrhoea and nausea/vomiting were assessed in two trials with 337 patients and we are uncertain whether there is a difference between treatment arms (diarrhoea: RR 2.19, 95% CI 1.07 to 4.47; nausea/vomiting: RR 1.46, 95% CI 0.75 to 2.85; both very low-certainty evidence).Quality of life was not reported by any of the included trials. We found no evidence for a difference between participants receiving ATRA in addition to chemotherapy or chemotherapy only for the outcome OS. Regarding DFS, CRR and on-study mortality, there is probably no evidence for a difference between treatment groups. Currently, it seems the risk of adverse events are comparable to chemotherapy only.As quality of life has not been evaluated in any of the included trials, further research is needed to clarify the effect of ATRA on quality of life.

Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5- a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors.

Human dihydroorotate dehydrogenase ( hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo[1,5- a]pyridine, that has been designed starting from brequinar, one of the most potent hDHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound 4, which shows brequinar-like hDHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of hDHODH inhibitors.

Histone Acetyltransferase p300/CREB-binding Protein-associated Factor (PCAF) Is Required for All-trans-retinoic Acid-induced Granulocytic Differentiation in Leukemia Cells

Differentiation therapy with all-trans-retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL); however, the molecular mechanism by which ATRA induces granulocytic differentiation remains unclear. We previously reported that the inhibition of the NAD-dependent histone deacetylase (HDAC) SIRT2 induces granulocytic differentiation in leukemia cells, suggesting the involvement of protein acetylation in ATRA-induced leukemia cell differentiation. Herein, we show that p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase (HAT), is a prerequisite for ATRA-induced granulocytic differentiation in leukemia cells. We found that PCAF expression was markedly increased in leukemia cell lines (NB4 and HL-60) and primary APL cells during ATRA-induced granulocytic differentiation. Consistent with these results, the expression of PCAF was markedly up-regulated in the bone marrow cells of APL patients who received ATRA-containing chemotherapy. The knockdown of PCAF inhibited ATRA-induced granulocytic differentiation in leukemia cell lines and primary APL cells. Conversely, the overexpression of PCAF induced the expression of the granulocytic differentiation marker CD11b at the mRNA level. Acetylome analysis identified the acetylated proteins after ATRA treatment, and we found that histone H3, a known PCAF acetylation substrate, was preferentially acetylated by the ATRA treatment. Furthermore, we have demonstrated that PCAF is required for the acetylation of histone H3 on the promoter of ATRA target genes, such as CCL2 and FGR, and for the expression of these genes in ATRA-treated leukemia cells. These results strongly support our hypothesis that PCAF is induced and activated by ATRA, and the subsequent acetylation of PCAF substrates promotes granulocytic differentiation in leukemia cells. Targeting PCAF and its downstream acetylation targets could serve as a novel therapeutic strategy to overcome all subtypes of AML.

Activation of G0S2 is coordinated by recruitment of PML/RARα and C/EBPɛ to its promoter during ATRA-induced APL differentiation

All-trans retinoic acid (ATRA) binds the promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion protein and is an effective oncogene-targeted therapy for acute promyelocytic leukemia (APL). However, the molecular basis of PML/RARα-mediated transcriptional control during ATRA-induced differentiation is unclear. Previous studies have shown that the PML/RARα fusion protein behaves as a type II nuclear receptor, binding to DNA regardless of ligand status. Here, we performed a series of chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) experiments, demonstrating that there is an additional mode of action of PML/RARα, wherein PML/RARα does not bind DNA in the absence of ATRA but binds DNA and activates adjacent genes in the presence of ATRA. This mode of action is similar to that of a type I nuclear receptor and is highlighted by activation of G0/G1 switch gene 2 (G0S2) during ATRA-induced neutrophil differentiation of leukemia cell lines (NB4 and PR9) and primary human APL cells. C/EBPε occupancy of the G0S2 promoter was elevated in parallel with recruitment of PML/RARα in ATRA-treated NB4, PR9, and primary APL cells. Furthermore, we verified that the p30 isoform of C/EBPε is crucial for activation of G0S2 and that PML/RARα interacts physically and cooperates functionally with C/EBPε to up-regulate G0S2 Our data not only demonstrate a new mode of action of PML/RARα but also suggest a novel model in which PML/RARα synergizes with C/EBPε to reactivate the C/EBPε target G0S2, thereby contributing to ATRA-mediated APL differentiation and potentially, clinical remission.

CATS (FAM64A) abnormal expression reduces clonogenicity of hematopoietic cells.

The CATS (FAM64A) protein interacts with CALM (PICALM) and the leukemic fusion protein CALM/AF10. CATS is highly expressed in leukemia, lymphoma and tumor cell lines and its protein levels strongly correlates with cellular proliferation in both malignant and normal cells. In order to obtain further insight into CATS function we performed an extensive analysis of CATS expression during differentiation of leukemia cell lines. While CATS expression decreased during erythroid, megakaryocytic and monocytic differentiation, a markedly increase was observed in the ATRA induced granulocytic differentiation. Lentivirus mediated silencing of CATS in U937 cell line resulted in somewhat reduced proliferation, altered cell cycle progression and lower migratory ability in vitro; however was not sufficient to inhibit tumor growth in xenotransplant model. Of note, CATS knockdown resulted in reduced clonogenicity of CATS-silenced cells and reduced expression of the self-renewal gene, GLI-1. Moreover, retroviral mediated overexpression of the murine Cats in primary bone marrow cells lead to decreased colony formation. Although our in vitro data suggests that CATS play a role in cellular processes important for tumorigenesis, such as cell cycle control and clonogenicity, these effects were not observed in vivo.

Diffron C, a Novel Oligonucleotide Based Gene-Silencing Agent, Induces Terminal Differentiation in Primary Murine Cells

Abstract Myelodysplastic syndrome (MDS) and acute myelogenous leukaemia (AML) are closely related diseases of haematopoietic stem cells (HSCs), characterized by ineffective terminal differentiation, effacement of normal blood cell production and accumulation of neoplastic cells, known as blasts, in the bone marrow, peripheral blood, and other tissues. In the past ten years it has become apparent that cancers are not composed of uniform populations of cancer cells. Instead, the great majority of the cells making up a cancer have very limited ability to undergo cell division, or have completely lost the ability to divide - only a minority of cells, known as cancer stem cells, retain the ability to divide in an unlimited way. To identify potential therapeutic targets, we studied the molecular signature of leukemia stem cells identifying the orphan nuclear receptor NR2F6 as a potential therapeutic target for differentiation therapy for MDS and AML. We've previously shown that NR2F6 is over expressed in patients with myelodysplastic syndromes and acute myeloid leukemia. In vitro, overexpression of NR2F6 inhibits differentiation of cell lines and primary bone marrow cells. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model lead to a condition that resembled myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. While overexpression of NR2F6 in 100% transduced bone marrow grafts that were sorted by flow cytometry prior to transplantation lead to a rapidly fatal erythroleukemia that was characterized by a block in terminal differentiation, pancytopenia and profound anemia. Furthermore, silencing of NR2F6 expression in human and mouse-leukemia cell lines causes terminal differentiation and death by apoptosis. This forms the basis of the therapeutic concept that we wished to develop further, by identifying gene-silencing methods that could be advanced to preclinical development for the treatment of myelodysplastic syndrome and acute leukemia. The discovery of the role of NR2F6 in leukemogenesis and the induction of differentiation of leukemia cell lines following silencing of NR2F6 expression suggests that this is a logical therapeutic target for differentiation therapy using gene silencing technology. Hence, we wished to show that silencing of NR2F6 using DiffronC, an oligonucleotide based gene-silencing technology, in primary mouse hematopoietic cells promoted terminal differentiation. Herein, we show that while DiffronC did not significantly reduce the number of colony forming units, it did significantly increase colony size. This was especially striking in cultures grown under erythroid conditions, where we observed a 3.21 increase in the number of cells per colony. We then showed that DiffronC reduced the clonal longevity of bone marrow cells: In replating experiments we observed a significant decrease in secondary colonies in cells treated with NR2F6 shRNA. Furthermore, DiffronC resulted in a drastic reduction in KSL stem and progenitor cells after six days of ex vivo culture, and caused a dramatic decline in lineage negative cells, and a concurrent increase in cells expressing the myeloid markers CD11b and Gr-1, suggesting that they had differentiated into cells of the neutrophil lineage. This was confirmed by examination of the cytomorphology of the bone marrow cultures. Taken together, these results establish that NR2F6 is a negative regulator of terminal differentiation of the hematopoietic lineage and lays the foundation for use of gene silencing technology using DiffronC as differentiation therapy for diseases with blocked differentiation such as leukemia and myelodysplastic syndromes. Disclosures Ichim: Entest BioMedical Inc: Employment, Equity Ownership, Research Funding; Regen BioPharma Inc: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Regen BioPharma Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wells:Alexion: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Effects of all-trans retinoic acid (ATRA) in addition to chemotherapy for adults with acute myeloid leukaemia (AML) (non-acute promyelocytic leukaemia (APL))

BACKGROUND Acute myeloid leukaemia (AML) is the most common acute leukaemia affecting adults. Most patients diagnosed with AML are at advanced age and present with co-morbidities, so that intensive therapy such as stem cell transplantation (SCT) is impossible to provide or is accompanied by high risks for serious adverse events and treatment-related mortality. Especially for these patients, it is necessary to find out whether all-trans retinoic acid (ATRA), an intermediate of vitamin A inducing terminal differentiation of leukaemic cell lines, added to chemotherapy confers increased benefit or harm when compared with the same chemotherapy alone. OBJECTIVES This review aims to determine benefits and harms of ATRA in addition to chemotherapy compared to chemotherapy alone for adults with AML (not those with acute promyelocytic leukaemia (non-APL)). SEARCH METHODS We searched the Central Register of Controlled Trials (CENTRAL), MEDLINE, study registries and relevant conference proceedings up to July 2018 for randomised controlled trials (RCTs). We also contacted experts for unpublished data. SELECTION CRITERIA We included RCTs comparing chemotherapy alone with chemotherapy plus ATRA in patients with all stages of AML. We excluded trials if less than 80% of participants were adults or participants with AML, and if no subgroup data were available. Patients with myelodysplastic syndrome (MDS) were included, if they had a refractory anaemia and more than 20% of blasts. DATA COLLECTION AND ANALYSIS Two review authors independently extracted data and assessed the quality of trials. We contacted study authors to obtain missing information. We used hazard ratios (HR) for overall survival (OS) and disease-free survival (DFS; instead of the pre-planned event-free survival, as this outcome was not reported), and we calculated risk ratios (RR) for the other outcomes quality of life, on-study mortality and adverse events. We presented all measures with 95% confidence intervals (CIs). We assessed the certainty of evidence using GRADE methods. MAIN RESULTS Our search resulted in 2192 potentially relevant references, of which we included eight trials with 28 publications assessing 3998 patients. Overall, we judged the potential risk of bias of the eight included trials as moderate. Two of eight trials were published as abstracts only. All the included trials used different chemotherapy schedules and one trial only evaluated the effect of the hypomethylating agent decitabine, a drug know to affect epigenetics, in combination with ATRA.The addition of ATRA to chemotherapy resulted in probably little or no difference in OS compared to chemotherapy only (2985 participants; HR 0.94 (95% confidence interval (CI) 0.87 to 1.02); moderate-certainty evidence). Based on a mortality rate at 24 months of 70% with chemotherapy alone, the mortality rate with chemotherapy plus ATRA was 68% (95% CI 65% to 71%).For DFS, complete response rate (CRR) and on-study mortality there was probably little or no difference between treatment groups (DFS: 1258 participants, HR 0.99, 95% CI 0.87 to 1.12; CRR: 3081 participants, RR 1.02, 95% CI 0.96 to 1.09; on-study mortality: 2839 participants, RR 1.02, 95% CI 0.81 to 1.30, all moderate-certainty evidence).Three trials with 1428 participants reported the adverse events 'infection' and 'cardiac toxicity': There was probably no, or little difference in terms of infection rate between participants receiving ATRA or not (RR 1.05, 95% CI 0.96 to 1.15; moderate-certainty evidence). We are uncertain whether ATRA decreases cardiac toxicity (RR 0.46, 95% CI 0.24 to 0.90; P = 0.02, very low certainty-evidence, however, cardiac toxicity was low).Rates and severity of diarrhoea and nausea/vomiting were assessed in two trials with 337 patients and we are uncertain whether there is a difference between treatment arms (diarrhoea: RR 2.19, 95% CI 1.07 to 4.47; nausea/vomiting: RR 1.46, 95% CI 0.75 to 2.85; both very low-certainty evidence).Quality of life was not reported by any of the included trials. AUTHORS' CONCLUSIONS We found no evidence for a difference between participants receiving ATRA in addition to chemotherapy or chemotherapy only for the outcome OS. Regarding DFS, CRR and on-study mortality, there is probably no evidence for a difference between treatment groups. Currently, it seems the risk of adverse events are comparable to chemotherapy only.As quality of life has not been evaluated in any of the included trials, further research is needed to clarify the effect of ATRA on quality of life.

MiR-638 Regulates Differentiation and Proliferation in Leukemic Cells by Targeting Cyclin-dependent Kinase 2

MicroRNAs have been extensively studied as regulators of hematopoiesis and leukemogenesis. We identified miR-638 as a novel regulator in myeloid differentiation and proliferation of leukemic cells. We found that miR-638 was developmentally up-regulated in cells of myeloid but not lymphoid lineage. Furthermore, significant miR-638 down-regulation was observed in primary acute myeloid leukemia (AML) blasts, whereas miR-638 expression was dramatically up-regulated in primary AML blasts and leukemic cell lines undergoing forced myeloid differentiation. These observations suggest that miR-638 might play a role in myeloid differentiation, and its dysregulation may contribute to leukemogenesis. Indeed, ectopic expression of miR-638 promoted phorbol 12-myristate 13-acetate- or all-trans-retinoic acid-induced differentiation of leukemic cell lines and primary AML blasts, whereas miR-638 inhibition caused an opposite phenotype. Consistently, miR-638 overexpression induced G1 cell cycle arrest and reduced colony formation in soft agar. Cyclin-dependent kinase 2 (CDK2) was found to be a target gene of miR-638. CDK2 inhibition phenotypically mimicked the overexpression of miR-638. Moreover, forced expression of CDK2 restored the proliferation and the colony-forming ability inhibited by miR-638. Our data suggest that miR-638 regulates proliferation and myeloid differentiation by targeting CDK2 and may serve as a novel target for leukemia therapy or marker for AML diagnosis and prognosis.

The orphan nuclear receptor Ear-2 (Nr2f6) is a novel negative regulator of T cell development

We describe a novel role for the orphan nuclear receptor Ear-2 in regulating T cell development. Retrovirus-mediated overexpression of Ear-2 (EAR-2++) in a bone marrow (BM) transplantation assay resulted in limited T cell development and a greater than tenfold decrease in thymus size and cellularity relative to controls. Ear-2-transduced murine BM hematopoietic stem cells (HSCs) in OP9-DL1 cultures showed a proliferation deficit during days 1-5 after induction of differentiation, which corresponded to increased expression of the cell cycle regulators p21 (cdkn1a) and p27 (cdkn1b), as well as increased expression of Hes1, Notch3, Egr1, and Scl (Tal1) and decreased expression of Gli1, Gfi-1, HoxA9, PU.1, Nrarp, and Tcf1. In addition, there was a block in differentiation at the DN4 to double-positive (DP) transition accompanied by an increase in apoptosis, similar to the deficit seen in the RORγt null mouse. Gene expression profiling revealed that, like the RORγt-deficient mouse, EAR-2++ DP cells had decreased expression of BclXL and increased expression of the proapoptosis gene Bad. In addition, EAR-2++ DP cells had decreased expression of Bcl11b, PU.1, and HoxA9, and increased expression of Id2. Based on these findings, we conclude that EAR-2++ cells were able to migrate to, but not fully repopulate, the thymus because of a cell-intrinsic defect in the proliferation of DN1 cells followed by a block in differentiation from the DN4 to DP stage of T cell development. We conclude that Ear-2 is a novel negative regulator of T-cell development and that downregulation of Ear-2 is indispensable for the proliferation of DN1 cells and the survival of DN4-DP cells.

Unique In Vitro and In Vivo Thrombopoietic Activities of Ingenol 3,20 Dibenzoate, A Ca++-Independent Protein Kinase C Isoform Agonist

Thrombopoiesis following severe bone marrow injury frequently is delayed, thereby resulting in life-threatening thrombocytopenia for which there are limited treatment options. The reasons for these delays in recovery are not well understood. Protein kinase C (PKC) agonists promote megakaryocyte differentiation in leukemia cell lines and primary cells. However, little is known about the megakaryopoietic effects of PKC agonists on primary CD34+ cells grown in culture or in vivo. Here we present evidence that the novel PKC isoform-selective agonist 3,20 ingenol dibenzoate (IDB) potently stimulates early megakaryopoiesis of human CD34+ cells. In contrast, broad spectrum PKC agonists failed to do so. In vivo, a single intraperitoneal injection of IDB selectively increased platelets in mice without affecting hemoglobin or white counts. Finally, IDB strongly mitigated radiation-induced thrombocytopenia, even when administered 24 hours after irradiation. Our data demonstrate that novel PKC isoform agonists such as IDB may represent a unique therapeutic strategy for accelerating the recovery of platelet counts following severe marrow injury.

Association of Deferasirox and Vitamin D Promotes Cell Differentiation and Improves Overall Survival in Acute Myeloid Leukemia (AML) Elderly Patients After Demethylating Agents Failure: A Retrospective Case Control Study

AbstractAbstract 3622 Background:AML is a group of heterogeneous malignant diseases characterized by uncontrolled cell growth and differentiation arrest. Following the success of differentiating therapies in APL, great hopes were placed in Vitamin D (VD) and its ability to promote differentiation of non-APL AML cells. However, results of clinical studies were disappointing and trials were interrupted due to the occurrence of life-threatening hypercalcemia. Our group has shown that iron chelators such as deferasirox (DFX) are able to promote monocytes differentiation in both normal hematopoietic progenitors and AML cells (Callens et al Jexp Med 2010). Moreover, iron deprivation synergized with VD to promote cell differentiation on leukemic cells. Most elderly patients diagnosed with AML suffer from secondary iron overload because of in some cases ineffective erythropoiesis and iterative red blood cell transfusions. Furthermore, in myelodysplastic syndromes, retrospective studies have suggested that iron chelators may increase life expectancy and decrease the risk of transformation into AML. In AML of the elderly, the use of demethylating agents such as 5-azacytidine or decitabine may induce hematological response and increase life expectancy. However, response is often of short duration. Since VD deficiency and iron overload prevalence is high in the elderly, the association of VD and DFX was given to a subgroup of patients following demethylating agents failure. Methods:A retrospective chart review of 17 elderly AML patients after demethylating agents failure was performed in three French centers. Patients treated by the combination of DFX/VD were matched to patients treated with best supportive care (BSC). Based on ferritin, and creatinin levels the dose of DFX was adapted in each case. DFX dose was up to 2000 mg a day and VD was used at 100,000 units orally weekly. The tolerance and the overall survival (OS) were analyzed. Pre-clinical studies were conducted in vitro on cell lines (HL60, U937, OCI-AML3, THP-1, MOLM 13) to evaluate cell differentiation induced by DFX and a new VDR agonist (Inecalcitol) by cell morphology and flow cytometry (expression of CD11b and CD14 markers). VDR activity and expression were evaluated by flow cytometry, immunoblotting, luciferase reporter assays and qPCR to detect VDR-targeted genes. Results:Median age of DFX/VD patients and BSC control group were 76 (range 63–84) and 71 (58–85) respectively. Most patients were diagnosed with AML with multilineage dysplasia (cases 70%, controls 76%). Prognosis groups were distributed homogeneously between the treated patients and controls. There were no significant differences in blast infiltration, leukocytosis, neutropenia, systemic iron and phosphocalcium parameters. All patients received 5-azacytidine (median of 8 courses for the cases and 7 for the controls).No renal insufficiency, hepatotoxicity or hypercalcemia were observed in DFX/VD patients. At 3 months, 4 treated patients (23.5%) had significant monocyte level increase an evidence of the enhanced monocyte differentiation efficacy. The treatment did not decrease the need of transfusion. Most interestingly median survival of treated patients was significantly increased (10.4 m vs 4 m, p=0.002).In vitro studies were conducted in parallel aiming to characterize new potential alternative therapeutic associations, which could improve patients' response. We show that the use of a new highly potent VDR agonist (Inecalcitol) potentiated the effect of DFX in promoting terminal monocyte differentiation of leukemic cell lines. It also increased VDR activity evaluated by VDR expression and phosphorylation and expression of VDR-targeted genes. In vivo studies in mice model of AML using combined DFX/inecalcitol therapy will be presented. Conclusions:The prognosis of elderly patients diagnosed with AML after demethylating agents remains poor. Here we show that the differentiating therapy by the association of Deferasirox and Vitamin D was able to improve overall survival with low toxicity. New generation of highly-potent VDR agonists (which are devoided of hypercalcemic properties) significantly enhanced VDR activation and terminal monocyte differentiation of AML cells and represent potential therapeutic alternatives in the near future. These encouraging results should be verified in a large randomized prospective multicenter study. Disclosures:No relevant conflicts of interest to declare.

Vitamin D3 Modulates Definitive Hematopoiesis by Two Distinct Mechanisms in the Developing Zebrafish Embryo

Abstract 763Studies of the vitamin D3 signaling pathway have revealed a broad role for this hormone in tissue homeostasis and as a result there is great interest in exploring the therapeutic potential of vitamin D3 for the treatment of various diseases including cancer. Studies investigating the role of vitamin D3 in hematological malignancies have shown that in vitro vitamin D3 has anti-proliferative and pro-differentiation effects resulting in differentiation of leukemic cell lines towards the monocytic lineage. Surprisingly, the role of vitamin D3 in HSC homeostasis and leukemia progression in vivo is not well understood. To elucidate the mechanisms of action of vitamin D3 in vivo during HSC self-renewal and differentiation, we utilized the zebrafish (Danio rerio) as vertebrate model due to their evolutionary conserved blood system and their amenability for genetic and chemical manipulation. Vitamin D3 was identified in a chemical screen performed in our laboratory as a regulator of runx1 and c-myb expression in the aorta-gonad mesonephros (AGM), the first site of definitive hematopoiesis. Treatments of zebrafish embryos with active vitamin D3 (1,25OH D3) between 12–36 hours post fertilization (hpf) resulted in increased expression of HSC markers (runx1, c-myb, CD41) as determined by whole mount in situ hybridization (WISH). In contrast, treatment with the non-hydroxylated vitamin D3 precursor cholecalciferol (D3) resulted in decreased runx1 and c-myb expression. D3 treatment during HSC induction and expansion from 24–36 hpf did not affect runx1 and c-myb expression, suggesting that D3 is acting early during the establishment of the vascular niche. To quantify the difference in HSC progenitors observed by WISH, FACS analysis was performed on double positive Tg(Lmo2:dsRed), Tg(c-myb:gfp) embryos. 1,25OH D3 treated embryos had a 20% increase in the double positive cell population corresponding to HSCs, while treatment with D3 resulted in a 25% decrease in the number of HSCs. The differential effect of 1,25OH D3 and D3 suggest that these two compounds act via two distinct mechanisms. To determine if the HSC enhancement observed by 1,25OH D3 was acting through the canonical vitamin D3 receptor (VDR), loss of function experiments were performed by injecting morpholinos targeting the vitamin D receptors. Morpholino knockdown of VDRA and VDRB (zebrafish VDR orthologs) resulted in decreased expression of runx1 c-myb, and CD41 via WISH. The decrease in HSC positive cells was confirmed by fluorescence microscopy using the Tg(Runx1P2:gfp) and Tg(-6.0itga:gfp) reporter lines, further supporting our hypothesis. To address the decrease in HSCs by D3, we postulated that D3 was inhibiting hedgehog signaling, as vitamin D3 has been previously shown to act as a negative regulator of the hedgehog pathway. In support of our hypothesis, treatment with D3 resulted in decreased ptch2 mRNA expression a downstream target of hedgehog signaling. In addition, FACS analysis using the hedgehog reporter Tg(6xGli:mCherry) line showed a 15% reduction in the number of mCherry positive cells in D3 treated embryos compared to controls. Co-treatments of zebrafish embryos with the hedgehog antagonist cyclopamine and D3 resulted in additive loss of reporter activity (45%) and loss of runx1 positive cells in the AGM, revealing synergy between these two compounds. In addition, co-exposure with the hedgehog agonist SAG rescued the HSC defect in D3-treated embryos. Hedgehog signaling is known to regulate vein and artery specification through the activation of notch signaling. Consistent with our hypothesis that D3 reduces hedgehog signaling, an expansion of the venous marker flt4 and a reduction in the expression of the arterial marker ephrinb2a was observed by WISH on D3-treated embryos. Furthermore, treatment of the notch reporter line Tg(Notch:gfp) with D3 resulted in lowered notch activity. In summary, these studies reveal that the active 1,25OH D3 positively regulates HSC progenitors in vivo during the onset of definitive hematopoiesis. In contrast, the non-hydroxylated vitamin D3 precursor acts as negative regulator of hematopoiesis by inhibiting hedgehog signaling and affecting vascular niche formation. Based on our studies, the differential effect between D3 and 1,25OH D3 should be considered when investigating the therapeutic potential of vitamin D3 in the context of hematological malignancies and other cancers. Disclosures:Goessling:Fate Therapeutics: Consultancy. North:Fate Therapeutics: Consultancy.

MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia

AbstractAlthough microRNAs (miRNAs) are increasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up-regulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cell lines and CD34+ hematopoietic stem/progenitor cells. By gain-of-function and loss-of-function experiments, we demonstrated that both miRNAs promote the phorbol 12-myristate 13-acetate–induced monocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, preventing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent kinase 6 gene, and a target of miR-142-3p, TGF-β–activated kinase 1/MAP3K7 binding protein 2 gene, are involved in the regulation of both monocytic and granulocytic differentiation. A significant decrease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their targets and promoted myeloid differentiation. These findings confirm that miR-29a and miR-142-3p are key regulators of normal myeloid differentiation and their reduced expression is involved in acute myeloid leukemia development.

Unique Thrombopoietic Activity of Novel PKC Agonist Ingenol 3,20 Dibenzoate.

Abstract 3622Poster Board III-558Despite recent advances in our understanding of megakaryocytic growth and platelet production, thrombocytopenia remains a difficult problem in the clinical management of patients with hematologic malignancies. Thrombopoietin (TPO) is the major cytokine involved in the normal production of platelets. However, the use of TPO has been relatively unsuccessful for the treatment of these patients and platelet transfusions remain the primary treatment for thrombocytopenia despite their significant cost and relatively short-lived responses. Thus, there remains an important clinical need for the development of novel approaches to generate platelets. Despite numerous reports on protein kinase C (PKC) agonists as promoters of megakaryocytic differentiation in leukemic cell lines and primary cells, little is known about their in vitro effects on primary CD34-selected progenitors or when administered in vivo. In the present study, we examine that effects of the novel PKC isoform agonist ingenol 3,20 dibenzoate (IDB) on megakaryocyte differentiation from CD34+ cells cultured in TPO and stem cell factor (SCF) or erythropoietin/SCF and its effects on platelet production in BALB/c mice. IDB potently stimulates early megakaryopoiesis and redirects the specificity of EPO to favor megakaryopoiesis over erythropoiesis. In contrast, broad spectrum PKC agonists such as phorbol myristate acetate, mezerein, and indolactam V fail to promote megakaryopoiesis. In vitro, IDB stimulates early expression of the promegakaryopoietic transcription factors egr1 and fli-1 and downregulates the proerythropoietic factors KLF1 and c-myb. Induction of the early megakaryocytic marker, CD9, was observed within the first 24 hrs of treatment with IDB and CD9 induction was blocked by the PKC inhibitor bisindolylmaleimide, which inhibits both novel and conventional PKC isoforms. In contrast, an inhibitor of conventional PKC isoforms, Gö6976, failed to block CD9 induction. In vivo, single intraperitoneal injections of IDB selectively increased platelet counts in BALB/c mice by 50% (plt= 630,000 vs. 985,000/μl; p<.005) at day 7 without affecting hemoglobin (Hgb) concentration or white counts (WBC). Mice treated with low dose radiation (2-4 Gy) had a transient drop in both platelet and WBC counts. Pretreatment with IDB 3 hrs prior to irradiation increased the platelet counts without improving WBC. More severe radiation exposure (6-8 Gy) causes pancytopenia. IDB treatment 3 hrs prior to 6 Gy irradiation significantly reduced the thrombocytopenia (plt=192,000 vs 594,000/μl; p<0.005) and anemia (hemoglobin=11.9 vs. 13.5gm/dl); p<0.005) without affecting the drop in WBC (WBC=1,200 vs. 1,300/μl; p=NS) at 14 days following irradiation. For mice treated with 8 Gy radiation, IDB pretreatment resulted in similar improvements in platelet counts (plt=111,000 vs. 443,000/μl; p<0.005) and hemoglobin (hgb=8.2 vs. 12.7 gm/dl; p<0.005) at 21 days following irradiation. The mitigation of thrombocytopenia is accompanied by marked increases in the megakaryocyte content in both the spleens and bone marrows of IDB-treated mice. Most importantly, IDB mitigated radiation-induced thrombocytopenia, even when administered 24 hrs after irradiation (plt=80,000 vs. 241,000/μl at 14 days following 6 Gy irradiation; p<0.01). Finally, IDB improved the survival of lethally irradiated mice. Our data suggest that the novel PKC isoform agonist IDB promotes the early differentiation of megakaryocytes from hematopoietic progenitors at the resulting in a significant improvement in platelet recovery following irradiation. IDB also improved Hgb levels following higher radiation doses. This may be due to improved hemostasis secondary to increased platelet numbers; however, an additional radioprotective effect on erythroid precursors cannot be excluded. These results strongly support our hypothesis that the novel PKC agonist IDB may be useful for the treatment of radiation and possibly drug-induced thrombocytopenia. Disclosures:No relevant conflicts of interest to declare.

Toll‐like Receptors (TLRs) are expressed by myeloid leukaemia cell lines, but fail to trigger differentiation in response to the respective TLR ligands

Toll‐like Receptors (TLRs) are expressed by myeloid leukaemia cell lines, but fail to trigger differentiation in response to the respective TLR ligands

NDRG1/2 expression is inhibited in primary acute myeloid leukemia

Expression of N-myc downregulated gene 1 (NDRG1) is associated with growth arrest and differentiation of tumor cells. In hematopoietic cells, NDRG1 was identified in a screen for differentiation-related genes in human myelomonocytic leukemic U937 cells. In the present study, we found significantly higher NDRG1 mRNA levels in granulocytes of healthy donors than in primary acute myeloid leukemia (AML) cells. Another NDRG family member, NDRG2, was significantly higher expressed in normal macrophages compared to primary AML cells. Moreover, NDRG1 mRNA levels increased in two acute promyelocytic leukemia (APL) patients as well as in NB4 and HT93 APL cells upon all- trans retinoic acid (ATRA) therapy. In line with these observations, silencing of NDRG1 diminished neutrophil differentiation of leukemic cell lines. In conclusion, we found an association of low NDRG1 levels with an immature cell phenotype and provide evidence that NDRG1 is functionally involved in neutrophil maturation.