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. 2012 Jun 12;109(24):9545-50.
doi: 10.1073/pnas.1121119109. Epub 2012 May 23.

Functional genomics identifies therapeutic targets for MYC-driven cancer

Masafumi Toyoshima  1 , Heather L Howie, Maki Imakura, Ryan M Walsh, James E Annis, Aaron N Chang, Jason Frazier, B Nelson Chau, Andrey Loboda, Peter S Linsley, Michele A Cleary, Julie R Park, Carla Grandori
Affiliations

Functional genomics identifies therapeutic targets for MYC-driven cancer

Masafumi Toyoshima et al. Proc Natl Acad Sci U S A. .

"V体育安卓版" Abstract

MYC oncogene family members are broadly implicated in human cancers, yet are considered "undruggable" as they encode transcription factors. MYC also carries out essential functions in proliferative tissues, suggesting that its inhibition could cause severe side effects. We elected to identify synthetic lethal interactions with c-MYC overexpression (MYC-SL) in a collection of ~3,300 druggable genes, using high-throughput siRNA screening. Of 49 genes selected for follow-up, 48 were confirmed by independent retesting and approximately one-third selectively induced accumulation of DNA damage, consistent with enrichment in DNA-repair genes by functional annotation. In addition, genes involved in histone acetylation and transcriptional elongation, such as TRRAP and BRD4, were identified, indicating that the screen revealed known MYC-associated pathways. For in vivo validation we selected CSNK1e, a kinase whose expression correlated with MYCN amplification in neuroblastoma (an established MYC-driven cancer) VSports手机版. Using RNAi and available small-molecule inhibitors, we confirmed that inhibition of CSNK1e halted growth of MYCN-amplified neuroblastoma xenografts. CSNK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms, whereas our data provide a previously unknown link with oncogenic MYC. Furthermore, expression of CSNK1e correlated with c-MYC and its transcriptional signature in other human cancers, indicating potential broad therapeutic implications of targeting CSNK1e function. In summary, through a functional genomics approach, pathways essential in the context of oncogenic MYC but not to normal cells were identified, thus revealing a rich therapeutic space linked to a previously "undruggable" oncogene. .

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of synthetic lethal genes with c-MYC overexpression by high-throughput siRNA screening. (A) Graphical schematic of the siRNA screen. Isogenic HFFs were plated, transfected with siRNAs targeting a total of 3,311 genes (1 gene per well), and assessed for viability using an Alamar Blue assay. (B) Graph of normalized viability of HFF-pB and HFF–c-MYC transfected with each siRNA pool (average of three replicates). MYC-SL “hits” (Z score ≥2) are highlighted in green. (C) mRNA levels following lentiviral-mediated shRNA knockdown in HFFs of three selected MYC-SL genes, CSNK1e, PES1, and CECR2. (D) Selective loss of viability of HFF-pBabe and HFF-MYC caused by knockdown of the genes shown in C. (E) Quantification of γ-H2AX staining following transduction of HFF-pBabe and HFF-MYC with siRNA pools corresponding to 40 MYC-SL genes. DDX18, a known MYC-target gene, and MYC were used as controls. y axis indicates the percent of cells stained with anti–γ-H2AX that scored for nuclear fluorescence levels above a threshold established from negative controls. (F) Representative images of γ-H2AX staining as described in E obtained with an INCell automated microscope (10× magnification). (G) Quantitative assessment of caspase-3 and -7 cleavage following transfection of the same siRNA pools as above (measured by the CaspaseGlo kit, Promega). Red line indicates background levels of caspase cleavage in HFF-c-MYC cells. Results were normalized for cell number by the Alamar Blue assay.
Fig. 2.
Fig. 2.
CSNK1e knockdown impairs growth of MYCN-amplified neuroblastoma cell lines. (A) Relative levels of CSNK1e mRNA following doxycycline or DMSO treatment of neuroblastoma cell-line SK-N-BE2, harboring lentivirus expressing shCSNK1e 1, 2, or sh control. Relative levels of each gene were calculated using the ΔΔCT method. GAPDH was used to normalize mRNA levels within each sample. (B) Viability assessment in two neuroblastoma lines, SK-N-BE2 and IMR32 (MYCN amplified) and SK-N-AS (MYCN not amplified) as measured by CellTiter Glo (Promega) following growth under DMSO or doxycycline-containing medium for 4 d. Values represent mean viability normalized to mock-treated cells. (C) Representative Western blot showing levels of CSNK1e protein in SK-N-BE2 cells shown in A. Actin is shown as a loading control. (D) Xenograft tumor growth of SK-N-BE2 cells transduced with doxycycline-inducible shRNA for CSNK1e or shControl in NOD/SCID mice. Doxycycline exposure was started when tumors reached a size of about 100 mm3. Knockdown of CSNK1e inhibited growth of established xenograft in three of four mice compared with no doxycycline-treated control (green arrows).
Fig. 3.
Fig. 3.
IC261 treatment blocks MYCN-amplified neuroblastoma tumor growth in vivo. (A) Representative images of MYCN-amplified neuroblastoma xenograft obtained with IMR32 cells in NOD/SCID mice before and after treatment with either DMSO or IC261. Tumors were engrafted and allowed to reach a size of about 100 mm3, then IC261 (21.5 mg/kg) or DMSO was injected s.c. daily for 8 d. (B) Quantitation of tumor size over the 8-d treatment regimen with either IC261 or DMSO control. Values represent mean tumor volume at each time point (n = 5 for each group, error bars indicate SD). (C) Immunohistochemical analysis of tumor sections from IC261 and DMSO treatment groups described in A and B. Representative images of H&E, TUNEL, and BrdU staining for each group are shown. BrdU was administered 2 h before collection. (D) Quantification of TUNEL+ and BrdU+ cells per field in DMSO- or IC261-treated xenograft tumors. Values represent mean number of positive cells per field, and error bars indicate SD of means.
Fig. 4.
Fig. 4.
CSNK1e expression correlates with poor prognosis and MYCN amplification in neuroblastoma. (A) Kaplan–Meier survival curves of neuroblastoma patients divided on the basis of CSNK1e expression; red indicates high expression and blue indicates low CSNK1e mRNA expression on the basis of microarray data accessible at the Oncogenomics neuroblastoma prognosis database (website: http://pob.abcc.ncifcrf.gov/cgi-bin/JK). (B) Graphical representation of expression levels of CSNK1e mRNA, derived from the website above. Each bar represents expression level in one sample. Solid yellow line indicates samples derived from MYCN-amplified neuroblastoma. (C) Representative Western blot of CSNK1e, MYCN, and Actin (loading control) protein levels in SK-N-AS (MYCN not amplified), SK-N-BE2 and IMR-32 (MYCN amplified) neuroblastoma cells. (D) Representative Western blot of CSNK1e, MYCN, and actin (loading control) protein levels in HFF pB and HFF c-Myc cells. (E) Representative Western blot of CSNK1e, MYCN, and actin (loading control) protein levels in Tet21N (MYCN Tet-Off) cells in the presence or absence of doxycycline. (F) Representative Western blot of relative c-MYC and MYCN levels in SKNAS, SKNBE2, IMR-32, HFF–c-MYC, and HFF-pBabe. Actin is shown as a loading control. (G) Real-time RT-PCR quantification of c-MYC and MYCN mRNA levels from the cells in G. Values were calculated using the ΔΔCT method, and represent the mean fold change compared with HFF-pBabe, using GAPDH for normalization. (H) Real time RT-PCR quantification of the relative levels of each casein kinase I isoform in neuroblastoma cell lines with or without MYCN amplification. Relative levels of each gene were calculated using the ΔΔCT method. GAPDH was used to normalize mRNA levels within each sample.
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