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. 2015 Sep 8;8(393):rs9.
doi: 10.1126/scisignal.aab3729.

Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi

Benjamin E Housden  1 , Alexander J Valvezan  2 , Colleen Kelley  3 , Richelle Sopko  3 , Yanhui Hu  3 , Charles Roesel  3 , Shuailiang Lin  3 , Michael Buckner  3 , Rong Tao  3 , Bahar Yilmazel  3 , Stephanie E Mohr  3 , Brendan D Manning  2 , Norbert Perrimon  4
Affiliations

Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi

Benjamin E Housden et al. Sci Signal. .

Abstract

The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.

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Conflict of interest statement

Competing interests: B.E.H., A.J.V., B.D.M., and N.P. have filed a provisional patent regarding the targeting of CCNT1, RNGTT, and CDK11 in tuberous sclerosis complex. The other authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. Optimization of the CRISPR system for Drosophila cell culture
(A) Graph showing relative mutation rates from 75 sgRNAs used to target a single sequence cloned into a luciferase reporter. Mutation rate is calculated as 1/firefly luciferase activity normalized to Renilla luciferase activity to control for differential transfection efficiency. Bars show mean relative mutation rates from three biological replicates using sgRNAs with 0 mismatches (blue bar), 1 mismatch (gray bars), 2 mismatches (green bars), or ≥ 3 mismatches (black bars) or in the absence of sgRNA (red bar). Dashes indicate nucleotides that are matched between sgRNA and the target sequence. Crosses indicate the position of mismatches. (B) Matrix showing the enrichment P values of each nucleotide in each position among high-efficiency sgRNAs. (C) Validation of efficiency scores generated using the matrix in (B) by correlating score (horizontal axis) with efficiency (vertical axis) from two independent publications (see fig. S3D for comparison with an additional data set).
Fig. 2
Fig. 2. Generation of mutant cell lines
(A) Survival rates of single S2R+ cells seeded into different medium formulations. “Clones” represents the number of seeded samples that produced viable populations of cells 3 weeks after seeding. Schneider’s medium was supplemented with FBS at the indicated concentrations and was preconditioned using S2R+ cells where indicated. (B) HRMA results for single S2R+ cells from a population 4 days after treatment with CRISPR targeting the yellow gene. The graph shows the difference in fluorescence between each sample and a mean control curve against temperature (scale from 76° to 84°C). (C) Graph showing relative firefly luciferase activity normalized to Renilla luciferase activity for either wild-type (black bars) or STAT92E (gray bars) mutant cells in the presence or absence of JAK/STAT pathway activation (upd ligand overexpression) and with or without activation in the presence of two different dsRNAs targeting STAT92E (RNAi-1 and RNAi-2). Bars show the mean from five biological replicates; error bars represent SEM. All differences between wild-type and STAT92E cells were significant (P < 0.05).
Fig. 3
Fig. 3. Characterization of TSC mutant cell lines
(A to C) Images of representative fields from wild-type (A), TSC1 mutant (B), or TSC2 mutant (C) cell lines. All images were taken at the same magnification and using the same settings. Scale bars, 50 μm. (D) Graph showing frequency of cell sizes for the cell lines indicated, divided into “low diameter” (gray bars) or “high diameter” (black bars) using a cutoff at which most wild-type cells fall into the low-diameter category. Bars represent the mean from three biological replicates; error bars indicate SEM. (E) Graph showing the relative rates of population growth for the indicated cell lines in either complete medium (10% FBS; blue bars), under partial serum starvation conditions (1% FBS; red bars), or under complete serum starvation conditions (0% FBS; green bars). Note that these values represent a combination of cell growth and proliferation. Bars show the mean of 24 samples per cell line and condition; error bars represent SEM. (F) Images of Western blots stained for phosphorylated S6K (p-S6K) or α-tubulin as indicated. Samples represent biological triplicates from S2R+, TSC1, and TSC2 cells. p-S6K amounts were normalized to α-tubulin because an antibody for Drosophila total S6K was not available. (G) Quantification of p-S6K for the indicated cell lines as shown in the Western blots in (F). Bars represent mean change in p-S6K normalized to α-tubulin for three biological replicates in each case. Error bars represent SEM; asterisks indicate significant differences from control (P =0.01), determined by t tests. (H) Graph indicating the fold enrichment of the indicated GO categories in phosphoproteomic data from TSC1 and TSC2 mutant cells compared to wild type. All samples are enriched with P values <0.05.
Fig. 4
Fig. 4. Identification of TSC-specific drug targets using synthetic screening
(A) Scatter plot showing the results of screens in Drosophila TSC1 and TSC2 mutant cell lines. dsRNAs that showed changes (see Materials and Methods) in wild-type cells are not shown in the graph. Points indicate the z-scores from three replicate screens in TSC1 cells (horizontal axis) and TSC2 cells (vertical axis). Dots represent candidates with no significant effect (black circles), TSC1-specific candidates (red circles), TSC2-specific candidates (blue circles), and candidates from TSC1 and TSC2 cells (purple crosses). The three genes showing synthetic reductions in population growth with both TSC1 and TSC2 are labeled. In addition, results for eIF3-S4 are plotted on the same graph for comparison (purple circle). (B) Graph showing relative viability (measured using CellTiter-Glo) for S2R+ cells treated with control (lacZ) dsRNA (blue bars) or two different dsRNAs targeting CG6182 (red and green bars) in combination with dsRNAs targeting CycT, Pitslre, mRNA-cap, or lacZ. Bars represent mean values from three biological replicates normalized to controldsRNAtreatments;error bars indicate SEM.(C) Summary plots showing onetimepoint from population growthassays in TSC2-deficient or wild-type MEFs treated with the indicated siRNAs (see fig. S7 for full time courses). Boxplots represent median (thick black lines), interquartile range (boxes), and min/max (error bars) from two biological replicates for the indicated genes in TSC2-deficient or wild-type background. The vertical axis represents change in ATP concentrations after 48 hours of culture relative to cells treated with control siRNA, measured using CellTiter-Glo assays. (D) Summary plots showing one time point from population growth assays in TSC2-deficient AML cells. Boxplots are as described in (C) and represent three biological replicates (see fig. S7 for full time courses). All differences between TSC-deficient and wild-type cells are significant (P < 0.05). (E) Graph showing the relative cell numbers after siRNA-mediated knockdown of the indicated genes in AML cells with (black bars) or without (gray bars) TSC2 addback. Bars represent the average of at least four biological replicates; error bars indicate SEM. Differences between TSC2 addback and empty vector conditions were significant for all three genes tested (P < 0.05).
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