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Combination treatment has proven effective for patients with acute promyelocytic leukemia, exemplifying the importance of therapy targeting multiple components of oncogenic regulation for a successful outcome. However, recent studies have shown that the mutational complexity of acute myeloid leukemia (AML) precludes the translation of molecular targeting into clinical success. Here, as a complement to genetic profiling, we used unbiased, combinatorial in vitro drug screening to identify pathways that drive AML and to develop personalized combinatorial treatments. First, we screened 513 natural compounds on primary AML cells and identified a novel diterpene (H4) that preferentially induced differentiation of FLT3 wild-type AML, while FLT3-ITD/mutations conferred resistance. The samples responding to H4, displayed increased expression of myeloid markers, a clear decrease in the nuclear-cytoplasmic ratio and the potential of re-activation of the monocytic transcriptional program reducing leukemia propagation in vivo. By combinatorial screening using H4 and molecules with defined targets, we demonstrated that H4 induces differentiation by the activation of the protein kinase C (PKC) signaling pathway, and in line with this, activates PKC phosphorylation and translocation of PKC to the cell membrane. Furthermore, the combinatorial screening identified a bromo- and extra-terminal domain (BET) inhibitor that could further improve H4-dependent leukemic differentiation in FLT3 wild-type monocytic AML. These findings illustrate the value of an unbiased, multiplex screening platform for developing combinatorial therapeutic approaches for AML.

Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the differentiation of primary AML cells, thus allowing for mechanistic studies in vitro. Additionally, the co-culture on OP9-M2 stromal is superior in preserving surface marker expression of primary (adult and pediatric) AML cells in comparison to stroma-free culture. Thus, by combining the co-culture with multicolor, high-throughput FACS, we can evaluate the effect of hundreds of small molecules on multi-parametric processes including: cell survival, stemness (leukemic stem cells), and myeloid differentiation on the primary AML cells at a single-cell level. This method streamlines the identification of potential therapeutic agents, but also facilitates combinatorial screening aiming, for instance, at dissecting the regulatory pathways in a patient-specific manner. Graphic abstract: [Image: see text] Schematic representation of the ex vivo small molecule screening of primary human acute myeloid leukemia. Irradiated, sub-confluent OP9-M2 stromal cells are plated in half-area 96 wells plates 4–16 h prior to adding primary AML cells. Compounds are added 36–48 h later and effects on cell number, leukemic stem cell population, and myeloid differentiation are quantifed by FACS after 4 days of treatment.

Additional file 3. Supplementary Figures: Fig. S1, S2, S3, S4, S6.

Additional file 1: Table S1. Clinical Information regarding GBM cell lines. Table S2. Cell markers used for GBM CD105+ cell characterization. Table S3. Differentiation assay of GBM CD105+ cells. Table S4. Panel metrics summary of GBM CD105+ cell exome sequencing. Table S5. Variant calling metrics of GBM CD105+ cell exome sequencing.

Supplemental material, Supplemental_Material_for_PhenotypicscreeningassayidentifiesmodulatorsofDBA_by_Siva_et_al for A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia by Kavitha Siva, Fredrik Ek, Jun Chen, Abdul Ghani Alattar, Kristmundur Sigmundsson, Roger Olsson, Marcin Wlodarski, Thomas Lundbäck and Johan Flygare in SLAS Discovery

Purpose - Previous studies suggest that regulatory changes result in an increased audit effort. Given a competitive market setting and audit price negotiation between the auditor and the auditee, it is unclear if an increased audit effort results in higher audit prices. The purpose of this study is to provide the reader with a deeper understanding concerning pricing of audit services. Moreover, we examine if the EU audit reform package, comprising of Directive 2014/56/EU and Regulation (EU) No 537/2014, has had an impact on audit prices. Method - This study was conducted through semi-structured interviews with public authorized accountants from Big 4-firms in order to gain a deeper insight in audit pricing and the EU audit reform. Results - The results in this study show that audit effort has increased as a result of the EU audit reform. In addition, this study provides some support that the increase in audit effort has resulted in higher audit prices. Conclusion - Our main conclusion in this study is that the EU audit reform has resulted in higher audit prices, despite indications that an increased audit effort does not necessarily result in higher audit prices. The main reason behind this uncertainty is due to factors such as the competitive market setting and price negotiation. Syfte - Tidigare studier indikerar att regelverksförändringar resulterar i en ökad resursåtgång för revisorer. Givet att det råder konkurrens på revisionsmarknaden och att prisförhandlingar sker mellan revisorer och kunder, innebär det oklarheter om en ökad resursåtgång leder till en högre prissättning av revisionstjänster. Syftet med denna studie är att bidra med en djupare förståelse kring prissättning av revisionstjänster. Vidare undersöker viom EU:s revisionspaket, bestående av Direktiv 2014/56/EU och Förordning (EU) Nr 537/2014, har haft en inverkan på prissättningen. Metod - Denna studie utfördes genom semistrukturerade intervjuer med auktoriserade revisorer tillhörande Big 4-byråer, för att få en djupare insikt i prissättning av revisionstjänster samt EU:s revisionspaket. Resultat - Resultatet i denna studie visar att resursåtgången för revisorer har ökat som ett resultat av EU:s revisionspaket. Vidare ger denna studie ett visst stöd att ökningen i resursåtgång har lett till en ökad prissättning av revisionstjänster. Slutsats - Vår huvudsakliga slutsats i denna studie är att EU:s revisionspaket har resulterat i en högre prissättning av revisionstjänster, trots att en ökad resursåtgång inte entydigt leder till ökade revisionspriser. Anledningen till denna tvetydighet beror på osäkerhetsfaktorer såsom den konkurrensmässiga marknadssituationen och prisförhandling.

Supplementary data. (DOCX 1430 kb)

Supplementary material and methods. (PDF 198 kb)

Additional methods including gene expression studies and western blotting. Figure S1. The expression of isoform a1 of V-ATPase on a) mRNA is partly anti-correlated to SOX11, as observed here in two different cell line models with altered expression of SOX11. However, no correlation of V-ATPase to SOX11 is visible on b) protein level. The mRNA expression of V-ATPase was assessed by HuGene ST 1.0 arrays. Each data point represents a unique sample. GAPDH expression was used as a protein-loading control for western blot analysis. (DOCX 111 kb)

Library of 75 small molecule inhibitors targeting proteins of the Wnt signaling pathway. Table S2. Small molecule inhibitors with an anti-proliferative effect observed in three different MCL cell lines at a minimum of two different concentrations per cell line. Reduction in proliferation is presented as mean percentage compared to the used vehicle control, DMSO. Table S3. Small molecule inhibitors with an anti-viability effect on Z138 cells. Reduction in viability is presented as mean percentage compared to the used vehicle control, DMSO. (DOCX 28 kb)