Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi.
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.
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. bhousden@genetics.med.harvard.edu perrimon@receptor.med.harvard.edu.
Benjamin E
BE
Housden
Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
Alexander J
AJ
Valvezan
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Colleen
C
Kelley
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Richelle
R
Sopko
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Yanhui
Y
Hu
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Charles
C
Roesel
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Shuailiang
S
Lin
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Michael
M
Buckner
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Rong
R
Tao
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Bahar
B
Yilmazel
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Stephanie E
SE
Mohr
Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
Brendan D
BD
Manning
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Howard Hughes Medical Institute, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. bhousden@genetics.med.harvard.edu perrimon@receptor.med.harvard.edu.
Norbert
N
Perrimon
D000818:Animals; D064113:CRISPR-Cas Systems; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D055785:Gene Knockdown Techniques / Q000379:methods*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014402:Tuberous Sclerosis / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D034741:RNA, Small Interfering; C515347:TSC1 protein, Drosophila; C117726:gig protein, Drosophila