Key Points• Large-scale loss-of-function RNAi screens in patientderived AML cells are feasible and able to pinpoint therapeutic targets.• ROCK1 inhibition exerts antileukemic effects in primary human AML cells in vitro and in vivo.Acute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral lossof-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient-derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling. (Blood. 2015;125(24):3760-3768)

Materials and methods Reagents Nilotinib was a gift from Novartis (Switzerland). It was dissolved in dimethylsulfoxide (DMSO) and 10 mM stock solution was prepared and stored at Ϫ 20 ° C. The final concentration of DMSO did not exceed more than 0.1% in

Although whole-genome sequencing has uncovered a large number of mutations that drive tumorigenesis, functional ratification for most mutations remains sparse. Here, we present an approach to test functional relevance of tumor mutations employing CRISPR/Cas9. Combining comprehensive sgRNA design and an efficient reporter assay to nominate efficient and selective sgRNAs, we establish a pipeline to dissect roles of cancer mutations with potential applicability to personalized medicine and future therapeutic use.

Ependymomas (EPN) are tumors of the central nervous system (CNS) that can arise in the supratentorial brain (ST-EPN), hindbrain or posterior fossa (PF-EPN) or anywhere in the spinal cord (SP-EPN), both in children and adults. Molecular profiling studies have identified distinct groups and subtypes in each of these anatomical compartments. In this review, we give an overview on recent findings and new insights what is driving PFA ependymomas, which is the most common group. PFA ependymomas are characterized by a young median age at diagnosis, an overall balanced genome and a bad clinical outcome (56% 10-year overall survival). Sequencing studies revealed no fusion genes or other highly recurrently mutated genes, suggesting that the disease is epigenetically driven. Indeed, recent findings have shown that the characteristic global loss of the repressive histone 3 lysine 27 trimethylation (H3K27me3) mark in PFA ependymoma is caused by aberrant expression of the enhancer of zeste homolog inhibitory protein (EZHIP) or in rare cases by H3K27M mutations, which both inhibit EZH2 thereby preventing the polycomb repressive complex 2 (PRC2) from spreading H3K27me3. We present the current status of the ongoing work on EZHIP and its essential role in the epigenetic disturbance of PFA biology. Comparisons to the oncohistone H3K27M and its role in diffuse midline glioma (DMG) are drawn, highlighting similarities but also differences between the tumor entities and underlying mechanisms. A strong focus is to point out missing information and to present directions of further research that may result in new and improved therapies for PFA ependymoma patients.