Multiomics of azacitidine-treated AML cells reveals variable and convergent targets that remodel the cell-surface proteome

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are diseases of abnormal hematopoietic differentiation with aberrant epigenetic alterations. Azacitidine (AZA) is a DNA methyltransferase inhibitor widely used to treat MDS and AML, yet the impact of AZA on the cell-surface proteome has not been defined. To identify potential therapeutic targets for use in combination with AZA in AML patients, we investigated the effects of AZA treatment on four AML cell lines representing different stages of differentiation. The effect of AZA treatment on these cell lines was characterized at three levels: the DNA methylome, the transcriptome, and the cell-surface proteome. Untreated AML cell lines showed substantial overlap at all three omics levels; however, while AZA treatment globally reduced DNA methylation in all cell lines, changes in the transcriptome and surface proteome were subtle and differed among the cell lines. Transcriptome analysis identified five commonly up-regulated coding genes upon AZA treatment in all four cell lines, TRPM4 being the only gene encoding a surface protein, and surface proteome analysis found no commonly regulated proteins. Gene set enrichment analysis of differentially regulated RNA and surface proteins showed a decrease in metabolic pathways and an increase in immune defense response pathways. As such, AZA treatment led to diverse effects at the individual gene and protein levels but converged to common responses at the pathway level. Given the heterogeneous responses in the four cell lines, we discuss potential therapeutic strategies for AML in combination with AZA

Combination of azacitidine and enasidenib enhances leukemic cell differentiation and cooperatively hypomethylates DNA

•Enasidenib is an inhibitor of mutant IDH2 that releases the block in cellular differentiation imposed by aberrant production of 2-hydroxyglutarate.•Mechanisms of action of azacitidine and enasidenib involve regulation of the epigenome.•The enhanced anti-leukemic activity of combining azacitidine and enasidenib leads to greater reductions in DNA methylation. Azacitidine and enasidenib are two therapies available for treatment of acute myelogenous leukemia (AML), and the mechanisms of action of these drugs involve alteration of aberrant DNA methylation. We hypothesized that a combination of these agents could have interactive effects on DNA methylation and enhance differentiation in mIDH2 cells. Combination treatment enhanced cellular differentiation in TF-1 cells overexpressing IDJ2R140Q through increased hemoglobinization and increased hemoglobin γ RNA expression compared with the effects of single agents. Furthermore, in primary AML samples (IDH2R140Q or R172K), combination treatment reduced CD34+ cells and increased CD15+ cells to a greater extent than attained with single agents. To explore the mechanism of enhanced differentiation with combination treatment, the TF-1 epigenome was analyzed by profiling 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) DNA methylation changes. Enasidenib treatment alone increased 5hmC, consistent with reactivation of ten-eleven-translocation (TET) enzyme activity. Compared with treatment with azacitidine alone, combination treatment reduced 5mC levels at greater numbers of sites and these loci were significantly enriched in regions with increased 5hMC (25.8% vs. 7.4%). Results are consistent with a model in which enasidenib-mediated reactivation of ten-eleven-translocation enzymes cooperates with azacitidine-mediated inhibition of DNA methyltransferase enzymes, leading to greater reductions in DNA methylation and enhanced erythroid differentiation

The TALE homeodomain protein Pbx2 is not essential for development and long-term survival

Pbx2 is one of four mammalian genes that encode closely related TALE homeodomain proteins, which serve as DNA binding partners for a subset of Hox transcription factors. The expression and contributions of Pbx2 to mammalian development remain undefined, in contrast to the essential roles recently established for family members Pbx1 and Pbx3. Here we report that Pbx2 is widely expressed during embryonic development, particularly in neural and epithelial tissues during late gestation. Despite wide Pbx2 expression, mice homozygous mutant for Pbx2 are born at the expected Mendelian frequencies and exhibit no detectable abnormalities in development and organogenesis or reduction of long-term survival. The lack of an apparent phenotype in Pbx2 -/- mice likely reflects functional redundancy, since the Pbx2 protein is present at considerably lower levels than comparable isoforms of Pbx1 and/or Pbx3 in embryonic tissues. In postnatal bone marrow and thymus, however, Pbx2 is the predominant high-molecular-weight (NW)-isoform Pbx protein detectable by immunoblotting. Nevertheless, the absence of Pbx2 has no measurable effect on steady-state hematopoiesis or immune function in adult mice, suggesting possible compensation by low-MW-isoform Pbx proteins present in these tissues. We conclude that the roles of Pbx2 in murine embryonic development, organogenesis, hematopoiesis, immune responses, and long-term survival are not essential

Analysis of patient-level data from 3 cooperative group trials confirms a survival advantage for NPM1m patients achieving MRD-negative CR after intensive induction

Background: NPM1 mutations (NPM1m), predominantly tetra-nucleotide insertions in exon 12, are clonal driver events in acute myeloid leukemia (AML) and are found exclusively in leukemic cells, making them ideal for sensitive detection of measurable residual disease (MRD) using quantitative polymerase chain reaction (qPCR)- or next generation sequencing (NGS)-based methods. Over 50 peer-reviewed publications across multiple cooperative group trials have shown that NPM1m AML patients in complete remission (CR), CR with incomplete count recovery (CRi), or CR with partial hematologic recovery (CRh) have significantly better survival if they are MRD negative compared to MRD positive. Based on this body of evidence, the European LeukemiaNet recommends monitoring molecular MRD in NPM1m AML patients during treatment to help guide treatment decisions. Purpose: To provide further evidence for the value of NPM1 MRD as a surrogate endpoint for prospective, randomized trials in patients receiving novel agents in combination with intensive chemotherapy by evaluating the relationship between MRD negativity in patients with CR after 2 cycles of chemotherapy and event-free survival (EFS) and overall survival (OS) in a pooled analysis of patient-level data from 3 large cooperative group trials. MRD in all 3 studies was assessed using reverse transcriptase-mediated qPCR (RT-qPCR) of NPM1m transcripts normalized to ABL transcripts. Methods: Deidentified patient data for a total of 1,128 patients who achieved CR, CRi, or CRh, and who had MRD data after 2 cycles of chemotherapy, were provided by the UK National Cancer Research Institute AML Study Group (SG) for the AML17 (N = 539), the AMLSG for the AMLSG 09-09 (N = 497), and the Study Alliance Leukemia (SAL) for the AML2003 (N = 105) trials and standardized using Standard Data Tabulation Models. Data included demographic information, disease history, induction and consolidation treatment received, morphologic response, MRD data after 2 cycles of treatment, relapse, and survival. Cutoffs for MRD negativity were defined according to the definitions used in the individual studies (Ivey A, et al. N Engl J Med. 2016; Kapp-Schwoerer S, et al. Blood. 2020; Shayegi N, et al. Blood. 2013). In addition, sensitivity analyses were conducted using a range of cutoff values. CR was defined as <5% blasts in the bone marrow (BM) with count recovery, occurring within 42 days of the start of the last induction cycle; only these patients were included in the MRD analysis. EFS and OS were estimated using the Kaplan-Meier method and defined according to FDA criteria. Briefly, EFS was measured from day 1 of randomization to the date of treatment failure, hematologic relapse from CR or death from any cause; treatment failure was defined as not achieving CR after two cycles of chemotherapy. OS was measured from day 1 to the date of death from any cause. The absolute risk difference among MRD response status groups in EFS/OS at the 2-month, 1-year, 2-year, 3-year, and 5-year landmarks and their corresponding 95% confidence intervals (CI) were calculated using complementary log-log transformation. Hazard ratios (HR) were fitted using a Cox regression model with EFS/OS as the dependent variable and with MRD-response status and study as independent variables. Results: Among the 515 patients who achieved a CR within 42 days at the start of chemotherapy Cycle 2, MRD negativity confers a substantial survival advantage with EFS HR (95% CI) = 1.6 (1.1-2.3) when measured in BM and 1.8 (1.4-2.4) in peripheral blood (PB) using the study-defined cutoffs for positive and negative. This advantage is also observed using cutoff definitions of MRD negativity ranging from <10 to <1,000 NPM1m/104 ABL transcripts. A survival advantage for OS was also observed with HR (95% CI) = 1.5 (1.0-2.4) and 1.7 (1.2-2.3) in BM and PB, respectively. Analysis of the predictive value of MRD negativity in the 613 patients who achieved CRi/CRh within 42 days of the start of Cycle 2 is underway. Conclusions: Analysis of pooled patient-level data confirms the survival advantage of achieving MRD-negative CR as previously reported in 3 independent, cooperative group trials using different induction regimens. The prognostic value of MRD negativity in NPM1m AML is consistent across a broad range of cutoff definitions in both BM and PB providing further evidence for the value of NPM1 MRD as a surrogate endpoint for clinical trials

Phase I Study of CC-486 Alone and in Combination with Carboplatin or nab-Paclitaxel in Patients with Relapsed or Refractory Solid Tumors

IF 10.199 (2017-2018)International audiencePurpose: This large two-part, three-arm phase I study examined the safety and tolerability of CC-486 (an oral formulation of azacitidine, a hypomethylating agent) alone or in combination with the cytotoxic agents, carboplatin or nab-paclitaxel, in patients with advanced unresectable solid tumors.Patients and Methods: Part 1 (n = 57) was a dose escalation of CC-486 alone (arm C) or with carboplatin (arm A) or nab-paclitaxel (arm B). The primary endpoint was safety, MTD, and recommended part 2 dose (RP2D) of CC-486. In part 2 (n = 112), the primary endpoint was the safety and tolerability of CC-486 administered at the RP2D for each treatment arm, in tumor-specific expansion cohorts. Secondary endpoints included pharmacokinetics, pharmacodynamics, and antitumor activity of CC-486.Results: At pharmacologically active doses CC-486 in combination with carboplatin or nab-paclitaxel had a tolerable safety profile and no drug-drug interactions. The CC-486 RP2D was determined as 300 mg (every day, days 1-14/21) in combination with carboplatin (arm A) or as monotherapy (arm C); and 200 mg in the same dosing regimen in combination with nab-paclitaxel (arm B). Albeit limited by the small sample size, CC-486 monotherapy resulted in partial responses (three/eight) and stable disease (four/eight) in patients with nasopharyngeal cancer. Three of the stable disease responses lasted more than 150 days.Conclusions: CC-486 is well tolerated alone or in combination with carboplatin or nab-paclitaxel. Exploratory analyses suggest clinical activity of CC-486 monotherapy in nasopharyngeal cancer and provided the basis for an ongoing phase II clinical trial (ClinicalTrials.gov identifier: NCT02269943). Clin Cancer Res; 24(17); 4072-80. ©2018 AACR

The role of migrating leukocytes in IL-1β-induced up-regulation of kinin B(1) receptors in rats

1. The present study examines the role of migrating leukocytes in the ability of IL-1β to induce the functional up-regulation of B(1) receptors, as assessed by kinin B(1) agonist-induced oedema in the rat paw. 2. Pre-treatment with the PAF receptor antagonist WEB 2086 inhibited des-Arg(9)-BK-induced oedema in IL-1β-treated paws, while the LTB(4) receptor antagonist CP105696 had no effect. Des-Arg(9)-BK-induced paw oedema was also inhibited by pre-treatment with the selectin blocker fucoidin or by an anti-CD-18 monoclonal antibody. 3. I.d. injection of IL-1β produced a 5 – 10-fold increase of myeloperoxidase (MPO) activity in the rat paw. The increase in MPO activity was significantly inhibited by WEB 2086 (46±9%), fucoidin (68±5%) or the CD-18 antibody (84±3%). In contrast, i.d. injection of TNFα a dose known to upregulate the B(1) receptor functionally did not induce any significant increase in MPO activity. 4. Des-Arg(9)-BK alone had no effect in MPO activity but enhanced (by about 40%) the response induced by IL-1β, an effect prevented by the B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK. 5. The concentration of TNF-α was increased in the paws after i.d. injection of IL-1β. Pre-treatment with fucoidin, WEB 2086, anti-CD-18 or CP 105695, significantly reversed the local increases in TNF-α concentrations (80±2; 75±4, 73±3 and 40±2%), respectively. 6. Finally, IL-1β induced an increase of B(1) receptor mRNA levels in the rat paw, an effect which was prevented by fucoidin treatment. 7. Taken together, these results indicate that up-regulation of B(1) receptors in the rat paw following IL-1β seems to involve the local recruitment of neutrophils and subsequent local TNF-α production. The cross-talk between kinins, cytokines and leukocytes implicate B(1) receptors in chronic inflammatory diseases