Lsd1 as a therapeutic target in Gfi1-activated medulloblastoma

Drugs that modify the epigenome are powerful tools for treating cancer, but these drugs often have pleiotropic effects, and identifying patients who will benefit from them remains a major clinical challenge. Here we show that medulloblastomas driven by the transcription factor Gfi1 are exquisitely dependent on the enzyme lysine demethylase 1 (Kdm1a/Lsd1). We demonstrate that Lsd1 physically associates with Gfi1, and that these proteins cooperate to inhibit genes involved in neuronal commitment and differentiation. We also show that Lsd1 is essential for Gfi1-mediated transformation: Gfi1 proteins that cannot recruit Lsd1 are unable to drive tumorigenesis, and genetic ablation of Lsd1 markedly impairs tumor growth in vivo. Finally, pharmacological inhibitors of Lsd1 potently inhibit growth of Gfi1-driven tumors. These studies provide important insight into the mechanisms by which Gfi1 contributes to tumorigenesis, and identify Lsd1 inhibitors as promising therapeutic agents for Gfi1-driven medulloblastoma.

A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

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Software and code
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Data collection
No software was used to collect data.

Data analysis
Affymetrix data was pre-processed using the RMA algorithm. Differential gene expression was determined using the "limma" package in R, and the BH method was used for multiple testing correction. Pathway analysis of the differentially expressed genes was performed using the ClueGO plug-in for Cytoscape.
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April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability  All studies must disclose on these points even when the disclosure is negative.

Sample size
Based on previous experience with studies of this type, power analysis indicates 8 mice per group will have 90% power to detect an average survival difference of 14 days with a significance of p = 0.05. Thus, most experiments included at least 8 mice/group and were repeated at least 3 times. In some cases where limited amounts of cells were available for transplant into mice, fewer than than 8 mice were used and the sample size is indicated as such.
Data exclusions For survival studies evaluating tumorigenesis, no data were excluded from analysis. For drug treatment studies on subcutaneous flank tumors, a few mice were excluded because they died prematurely before the experiment endpoint (ie. death due to circumstances unrelated to tumor burden). Mice excluded from analysis were not included in reported sample sizes.

Replication
Experiments were done at least three times. Attempts at replication were successful.
Randomization For in vivo treatment experiments, tumor-bearing mice were subjected to bioluminescent imaging and caliper measurements. Animals with comparable tumor sizes were randomized into treatment groups; this prevented outcomes from being influenced by initial differences in tumor burden.

Blinding
Investigators were not blinded to group allocation.
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Antibodies
Antibodies used Details about all antibodies used in this study are provided in the Materials and Methods section of the manuscript.

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Cell line source(s) NIH-3T3 and HEK 293T/17 were obtained from ATCC.

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Cell lines have been authenticated by ATCC.

Mycoplasma contamination
Authors declare that cell lines used were not tested for mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) N/A

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Laboratory animals
Animal strains used for the study are described in Methods section. Male and female NSG mice are used as hosts for transplantation at 6-7 weeks of age. 5-7 day old pups from C57/BL6, CAG-CreER, and CAG-CreER Lsd1 flox mice were used to generate neural stem cells, which were then transduced with oncogenes to generate primary mouse medulloblastoma.

Wild animals
This study did not involve wild animals.

Field-collected samples
This study did not involve field-collected samples.

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