ATRT-09. IDENTIFICATION OF POTENTIAL GENETIC DRIVERS OF METHOTREXATE (MTX) RESISTANCE IN ATYPICAL TERATOID RHABDOID TUMOURS (ATRT) THROUGH A GENOME-WIDE RNAI SCREEN

Abstract ATRT of the CNS constitute a group of rare and aggressive early childhood tumors with poor prognosis. While there are differing chemotherapeutic regimens for ATRT, high-dose MTX is a crucial component of many therapeutic protocols. Currently, the biological mechanisms contributing to the generation of MTX resistance in ATRT are unknown. To identify genes involved in MTX resistance in ATRT, an unbiased genome-wide RNAi screen on ATRT cell lines was conducted using 24,000 distinct shRNAs covering 8,000 genes. ATRT cells were transfected with a retrovirus containing pRS-shRNA vectors and treated with puromycin for selection. The resulting cells were treated with MTX to identify resistant clones and resistant colonies were then isolated and amplified individually. Presence of shRNA inserts in each colony was determined by PCR using pRS forward and reverse primers. PCR products within each of the three resistant colonies were sequenced, leading to the identification of three distinct genes, TGIF1, HIF3A and PGAM2, as potential indicators of resistance. Western blotting verified depletion of these proteins in their respective colonies. Proliferation assays were then conducted on cells from each resistant colony alongside control cells to confirm that the identified drivers conferred resistance. Sensitivity to MTX was significantly lower in TGIF1-depleted (IC50=212±8.48nM, n=3), HIF3A-depleted (IC50=52±4.68nM, n=3) and PGAM2-depleted (IC50=41±4.13nM, n=3) cells compared to control cells (IC50=19±2.87nM, n=3), (p<0.001). In addition, more than 60% of TGIF1, HIF3A, and PGAM2-depleted cells survived the maximum MTX treatment (100nM), while less than 20% of control cells survived this treatment. Our study using an unbiased genome-wide RNAi screen approach has shown that depletion of TGIF1, HIF3A and PGAM2 are potential molecular markers of MTX resistance in ATRT. Screening for their occurrence may help to identify patients at high risk of MTX resistance and may also serve as targets for future novel therapeutics development.

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Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

Klinische Pädiatrie ◽

10.1055/s-0034-1374830 ◽

2014 ◽

Vol 226 (03) ◽

Author(s):

F Ponthan ◽

D Pal ◽

J Vormoor ◽

O Heidenreich

Keyword(s):

Rnai Screen ◽

Novel Genes ◽

Genome Wide ◽

A Genome

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Faculty Opinions recommendation of A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.10182956.14730113 ◽

2011 ◽

Author(s):

Miriam Merad ◽

Brian Brown

Keyword(s):

Antigen Presentation ◽

Mhc Class Ii ◽

Class Ii ◽

Rnai Screen ◽

Genome Wide ◽

A Genome ◽

Class Ii Antigen ◽

Mhc Class Ii Antigen

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Faculty Opinions recommendation of A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1031465.363758 ◽

2006 ◽

Author(s):

Toshinori Nakayama

Keyword(s):

Rnai Screen ◽

Genome Wide ◽

A Genome

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Faculty Opinions recommendation of A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718026493.793483761 ◽

2013 ◽

Author(s):

Megerditch Kiledjian

Keyword(s):

Rnai Screen ◽

Mrna Decapping ◽

Genome Wide ◽

A Genome

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Genome-Wide Analysis of Glucocorticoid-Responsive Transcripts in the Hypothalamic Paraventricular Region of Male Rats

Endocrinology ◽

10.1210/en.2018-00535 ◽

2018 ◽

Vol 160 (1) ◽

pp. 38-54 ◽

Cited By ~ 2

Author(s):

Keiichi Itoi ◽

Ikuko Motoike ◽

Ying Liu ◽

Sam Clokie ◽

Yasumasa Iwasaki ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Receptor Gene ◽

Male Rats ◽

Regulatory Mechanisms ◽

High Dose ◽

Sequencing Analysis ◽

Reverse Transcription Pcr ◽

Genome Wide ◽

A Genome

Abstract Glucocorticoids (GCs) are essential for stress adaptation, acting centrally and in the periphery. Corticotropin-releasing factor (CRF), a major regulator of adrenal GC synthesis, is produced in the paraventricular nucleus of the hypothalamus (PVH), which contains multiple neuroendocrine and preautonomic neurons. GCs may be involved in diverse regulatory mechanisms in the PVH, but the target genes of GCs are largely unexplored except for the CRF gene (Crh), a well-known target for GC negative feedback. Using a genome-wide RNA-sequencing analysis, we identified transcripts that changed in response to either high-dose corticosterone (Cort) exposure for 12 days (12-day high Cort), corticoid deprivation for 7 days (7-day ADX), or acute Cort administration. Among others, canonical GC target genes were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in vasopressin and apelin receptor gene expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, six immediate early genes were upregulated 1 hour after Cort injection, which was confirmed by quantitative reverse transcription PCR and semiquantitative in situ hybridization. This study may provide a useful database for studying the regulatory mechanisms of GC-dependent gene expression and repression in the PVH.

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