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In Vivo RNAi Screen Unveils PPAR? as a Regulator of Hematopoietic Stem Cell Homeostasis.

ABSTRACT: Hematopoietic stem cell (HSC) defects can cause repopulating impairment leading to hematologic diseases. To target HSC deficiency in a disease setting, we exploited the repopulating defect of Fanconi anemia (FA) HSCs to conduct an in vivo short hairpin RNA (shRNA) screen. We exposed Fancd2-/- HSCs to a lentiviral shRNA library targeting 947 genes. We found enrichment of shRNAs targeting genes involved in the PPAR? pathway that has not been linked to HSC homeostasis. PPAR? inhibition by shRNA or chemical compounds significantly improves the repopulating ability of Fancd2-/- HSCs. Conversely, activation of PPAR? in wild-type HSCs impaired hematopoietic repopulation. In mouse HSCs and patient-derived lymphoblasts, PPAR? activation is manifested in upregulating the p53 target p21. PPAR? and co-activators are upregulated in total bone marrow and stem/progenitor cells from FA patients. Collectively, this work illustrates the utility of RNAi technology coupled with HSC transplantation for the discovery of novel genes and pathways involved in stress hematopoiesis.

SUBMITTER: Sertorio M

PROVIDER: S-EPMC5425620 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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In Vivo RNAi Screen Unveils PPARγ as a Regulator of Hematopoietic Stem Cell Homeostasis.

Sertorio Mathieu M Du Wei W Amarachintha Surya S Wilson Andrew A Pang Qishen Q

Stem cell reports 20170413 5

Hematopoietic stem cell (HSC) defects can cause repopulating impairment leading to hematologic diseases. To target HSC deficiency in a disease setting, we exploited the repopulating defect of Fanconi anemia (FA) HSCs to conduct an in vivo short hairpin RNA (shRNA) screen. We exposed Fancd2<sup>-/-</sup> HSCs to a lentiviral shRNA library targeting 947 genes. We found enrichment of shRNAs targeting genes involved in the PPARγ pathway that has not been linked to HSC homeostasis. PPARγ inhibition b ...[more]

PMID: 28416286

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Project description:Glioblastoma multiforme is the most common and most aggressive type of primary brain tumor. The brain-infiltrative character of glioblastoma makes complete surgical removal of the tumor impossible and neither radiation nor current chemotherapy provide cure. Recent evidence shows that glioblastoma multiforme consists of heterogeneous cell populations which differ in tumor-forming potential. Enriched tumor-initiating capacity has been linked to poorly differentiated glioblastoma cells sharing features with neural stem cells. Thus, these cells are important targets for new therapeutic strategies. We aim to identify novel targets controlling maintenance and differentiation in glioblastoma-initiating cells through high throughput screening. To this end, we utilized libraries of small chemical compounds and small interference RNAs in combination with automated imaging and data analysis. Patient-derived glioblastoma cells were expanded and characterized using neural stem cell conditions. In culture, the cells showed low differentiation but expression of neural stem cell markers such as Nestin and Sox2. Upon intracranial injection into SCID mice these cells gave rise to tumors displaying the hallmarks of the human disease. Differentiation of glioblastoma-initiating cells (for example elicited through bone morphogenetic protein, BMP) was associated with strong morphological changes. Hence, cellular morphology, as well as markers specific for differentiation or death were used as screen readout. Lentiviral RNA interference-based screening yielded several gene knockdowns leading to ‘forced’ differentiation of glioblastoma-initiating cells. For example, knockdown of TRRAP (transformation/transcription domain associated protein) led to strongly increased differentiation and loss of proliferative and self-renewing capacity in these cells. TRRAP is an adapter protein implicated in oncogenic transformation through c-MYC transcription activation, also participating in chromatin remodeling and DNA repair. Glioblastoma-initiating cells with reduced TRRAP displayed increased apoptosis upon treatment with the genotoxic agent temozolomide. In vivo, Trapp knockdown cells were not able to give rise to glioblastoma upon transplantation into the brain of SCID mice. Together, these findings support a crucial role for TRRAP in maintenance and tumorigenicity of glioblastoma-initiating cells and might offer future therapeutic options. Two treatments compared to control: two different shRNA sequences for TRRAP were compared to a control shRNA sequence in their effects on global transcription in brain tumor initiating cells

Dataset Information

In Vivo RNAi Screen Unveils PPAR? as a Regulator of Hematopoietic Stem Cell Homeostasis.

Publications

In Vivo RNAi Screen Unveils PPARγ as a Regulator of Hematopoietic Stem Cell Homeostasis.

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