Supplementary Materials and Methods

Supervised survival analysis. Analysis of CIS gene expression and AML patient survival was performed according to Beck et al, Sci. Transl. Med. 2011 (1) with the following modifications: TCGA AML samples were divided into high- or low-risk groups based on 2-year survival. We then built a CIS gene-based prognostic model using support vector machine (SVM) modeling (2-3). Model performance was assessed by leave-one out cross-validation (LOOCV). For each iteration of LOOCV, one sample was left out as the test sample and the rest of the samples were used to construct a SVM-based risk predictive model. This prediction model was then applied to the test sample and a risk score of the test samples was derived based on their CIS gene expression. After LOOCV, the samples were divided into equal-sized risk groups based on the median of the predicted risk scores (4). To assess the statistical significance of the survival fraction between the low-risk versus high-risk groups, we computed a log-rank P value using the survdiff function in R.

RT-PCR analysis of CIS genes. All quantitative real-time PCR analysis was performed using the 7900 HT Fast Real Time PCR system (Applied Biosystems). RNA from mutagenized Ba/F3 pools was reverse transcribed (cDNA First Strand Synthesis Kit, Invitrogen) and subjected to quantitative RT-PCR detection of transcripts using SYBR Green (Invitrogen) with primers that cross an exon-exon junction. All PCR assays were performed in triplicate and expression was determined relative to Actin. Primer sequences are included in Supplementary Table 4.

Antibodies. Western blotting was performed as described in Materials and Methods. The primary antibodies used were α-Phospho-Stat5 (CST, #9314), α-Stat5 (CST, #9363), α-USP32 (Bethyl, A302-287A), α-FARS2 (ProteinTech, 16436-1-AP), α-Pten (CST, #9552), and α-Ikaros (Abcam ab26083).

Immunohistochemistry. Paraffin embedded sections were de-paraffinized and rehydrated in a series of xylene and ethanol solutions, and then heated in a pressure cooker in Antigen Unmasking Solution (Vector labs, H-3300) at high heat for 5 minutes. After cooling for >3 hours, sections were blocked with BLOXALL solution (Vector labs, SP-6000) and Mouse Ig Blocking Reagent (Vector labs, BMK-2202). Sections were incubated with primary antibodies overnight at 4°C; then incubated with biotinylated antibodies against mouse or rabbit IgG for 30 minutes at room temperature; and then incubated with VECTASTAIN Elite ABC Reagent, R.T.U. (Vector labs, PK-7100) for 30 minutes at room temperature. Slides were developed using ImmPACT DAB Peroxidase (HRP) Substrate (Vector labs, SK-4105) and counterstained using Hematoxylin QS (Vector labs, H-3404). The primary antibodies used were CD117 (Dako, A4502), CD79a (Thermo, MA5-13212), CD11b (BioLegend, 101201), CD19 (BioLegend, 115537), and B220 (eBioscience, 14-0452-81).

CRISPR/Cas9-mediated knockout. CIS genes were knocked out in Ba/F3 or SU-DHL-1 cells using the lentiviral CRISPR/Cas9 system as previously described (5). The sequences of the sgRNAs used were: UPS32 sgRNA-1, 5’-TGTAGAGCTAAAACGACTGA-3’; USP32 sgRNA-2, 5’- CATGTAATATGAGAGTCCAC-3’; USP32 sgRNA-3, 5’-ATTATTGAAGTGCAGCCCTT-3’; EGFP sgRNA-1, 5’-GGGCGAGGAGCTGTTCACCG-3’.

Copy number determination of SB integrations using qPCR. Quantitation of SB insertions was performed by SYBR green analysis of genomic DNA isolated from Ba/F3 cells overexpressing candidate genes relative to a standard curve with a known number of SB transposon copies. To generate a standard curve, known copy numbers of the SB-T2/Onc plasmid were spiked into genomic DNA isolated from wild-type Ba/F3 cells. Primers were designed to match the left IR/DR of the Sleeping Beauty transposon. 5 ng of genomic DNA isolated from Ba/F3 cells expressing candidate CIS genes was used as template in 10 ul reactions containing 5 ul Power SYBR Green Master Mix (Applied Biosystems). Quantitative real time PCR was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems). Primers used were forward: 5’-TCTGACCCACTGGAATTGTG-3’ and reverse: 5’ TGTGCATGACACAAGTCATTTT-3’. All SYBR green PCR assays were performed in triplicate.

Expression of candidate genes in Ba/F3 cells using retroviral and lentiviral vectors. Select candidate genes were expressed using either a retroviral vector (pMIG) or a lentiviral vector (pLX302) and introduced into Ba/F3 cells following protocols as described previously (6,7). Briefly, candidate genes were cloned into pMIG or pLX302 vectors. 293T/17 cells were transfected with pMIG containing candidate genes and pCL-eco plasmid at a ratio of 2:1, or pLX302 containing candidate genes, psPAX2 and pMD2 plasmids at a ratio of 4:2:1. 48 hours following transfection, the viral supernatants were collected, filtered, and applied to Ba/F3 cells in the presence of 8µg/ml hexadimethrene bromide (Sigma). IL-3 was withdrawn 3 days after retroviral infection, or 7 days after puromycin selection for lentiviral infection.

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