Integrative prognostic risk score in acute myeloid leukemia with normal karyotype

To integrate available clinical and molecular information for cytogenetically normal acute myeloid leukemia (CN-AML) patients into one risk score, 275 CN-AML patients from multicenter treatment trials AML SHG Hannover 0199 and 0295 and 131 patients from HOVON/SAKK protocols as external controls were evaluated for mutations/polymorphisms in NPM1, FLT3, CEBPA, MLL, NRAS, IDH1/2, and WT1. Transcript levels were quantified for BAALC, ERG, EVI1, ID1, MN1, PRAME, and WT1. Integrative prognostic risk score (IPRS) was modeled in 181 patients based on age, white blood cell count, mutation status of NPM1, FLT3-ITD, CEBPA, single nucleotide polymorphism rs16754, and expression levels of BAALC, ERG, MN1, and WT1 to represent low, intermediate, and high risk of death. Complete remission (P = .005), relapse-free survival (RFS, P < .001), and overall survival (OS, P < .001) were significantly different for the 3 risk groups. In 2 independent validation cohorts of 94 and 131 patients, the IPRS predicted different OS (P < .001) and RFS (P < .001). High-risk patients with related donors had longer OS (P = .016) and RFS (P = .026) compared with patients without related donors. In contrast, intermediate-risk group patients with related donors had shorter OS (P=.003) and RFS(P=.05). Donor availability had no impact on outcome of patients in the low-risk group. Thus, the IPRS may improve consolidation treatment stratification in CN-AML patients. Study registered at www.clinicaltrials.gov as #NCT00209833

Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization

<div><p>Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the ‘classical’ (M1) and ‘alternative’ (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.</p></div

Validation of OGT as a modulator of M2 polarization.

<p>(A) Flow cytometric analysis of the M2 (CD209) cell surface marker after inducing M2 polarization in OGT targeted or control cells. OGT was targeted with the small molecule OSMI-1 or CRISPR with two independent sgRNA constructs, and compared to the corresponding DMSO or scrambled controls. (B) The 25 OGT regulated genes identified from 84 probed cytokines and chemokines are shown. mRNA levels were quantified by qPCR on control and OGT targeted THP-1 M2 macrophages (n = 3). Data are presented as mean ± SD; unpaired Student’s t test: *p < 0.05; **p < 0.01, ***p < 0.001.</p

Contingency table showing gene-centric representation of top 10 ranked hits from the M1 / M2 screens.

<p>Contingency table showing gene-centric representation of top 10 ranked hits from the M1 / M2 screens.</p

Pooled shRNA screening identifies candidate modulators of macrophage polarization in THP-1 cells.

<p>(A) Screening workflow for the phenotypic pooled shRNA screens to identify genes influencing macrophage differentiation and M1 / M2 polarization. Below, schematic representation of the analysis set up comprising different comparisons in two analysis modes (phenotype suppressor or mediator). (B) Analysis of screening results using the five comparisons and two analysis modes described in panel A. Top 10 hits are highlighted. Gene level scores were derived from shRNA reagents by calculating both the Redundant siRNA Activity (RSA) p-values as well as upper / lower quartiles. These two quantities describe significance and effect size, respectively, of a gene's knockdown by all reagents targeting this gene. Each sample was scaled to a total 12 million reads.</p

THP-1 monocyte to macrophage differentiation and M1 / M2 polarization.

<p>(A) Schematic representation of treatments to differentiate THP-1 monocytes (Mo) into macrophages (M0) or polarized macrophages (M1, M2). (B) Morphological and cell surface marker expression (CD11c, CD11b) changes observed by microscopic and flow cytometric analysis of PMA (phorbol 12-myrisate 13-acetate) differentiated M0. FCS: forward-scatter; SSC: side-scatter. (C) Resting CD38- / CD209- M0 macrophages (top panel) are polarized in CD38+ / CD209- M1 fraction upon LPS / IFNγ treatment (middle panel) and in CD38- / CD209+ M2 fraction upon IL4 / IL13 treatment (bottom panel). (D) Cytokine and chemokine release profiling of THP-1 M1 and M2 macrophages detecting M1 (black) and M2 (grey) specific soluble mediators, respectively. Supernatants were collected from three independent experiments and then pooled for the analysis. *Exogenously added to the M2 polarization medium. (E) Correlation analysis of transcriptional fold change (FC) during Mo to M1 (top panel) or M2 (bottom panel) polarization comparing THP-1 and human primary cells. For macrophage polarization MCSF (human primary cells) or PMA (THP-1) differentiated macrophages were treated with IFNγ (M1) or IL4 (M2). Whole genome microarray measurements were used from three independent experiments and each dot corresponds to a probe on the array. Highlighted genes are previously reported human markers relevant for the M1 or M2 polarization [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183679#pone.0183679.ref032" target="_blank">32</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183679#pone.0183679.ref031" target="_blank">31</a>] (additional references listed in the main text). Highlighting was limited to genes which at least in one of the cellular models had at least one probe with 1 < or– 1 > FC. Dashed line is the y = x reference, solid line is fitted on the experimental data y = a + b * x (a = interception of y, b = slope).</p

Validation of selected M2 polarization modulators with small molecule inhibitors.

<p>(A) Representative images of indirect immunofluorescence staining for CD209 positive M2 macrophages. Cells were treated with OSMI-1, PFI-3 and LMK-235 targeting OGT, SMARCA2/4 and HDAC4/5, respectively, and compared to DMSO control. From the treatments a lower and the highest concentrations are shown. The representative images shown were processed with Fiji and CD209 signal was pseudo-colored yellow. (B) Dose response curves representing percent of CD209 positive cells at different concentrations of compound treatment. Values from 4 independent wells for each condition were averaged and plotted as data points. Error bars indicate the standard deviation. Dashed horizontal lines mark the average percent (11%) of positive cells in DMSO controls (n = 48 wells; standard deviation = 2.25). **The lowest or highest concentration with a significant effect (unpaired Student’s t-test, p < 0.01) compared to the DMSO control.</p