In vivo pool-based shRNA screens to identify modulators of disease progression in hematopoietic malignancies

• Main Author: Meacham, Corbin Elizabeth
• Other Authors: Michael T. Hemann.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2012
• Subjects: Biology.
• Online Access: http://hdl.handle.net/1721.1/70104

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012. === This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. === Cataloged from student-submitted PDF version of thesis. === Includes bibliographical references. === shRNA screens have been very effective in identifying novel cancer genes in mammalian cells, but they have primarily been limited to in vitro applications in tumor cell lines. Whereas in vivo retroviral mutagenesis screens typically identify gain-of-function alterations in positive selection screens, shRNA screening approaches allow for the systematic interrogation of the impact of loss of function events across large gene sets. Using transplantable mouse models of E[mu]-myc lymphoma and Bcr-Abl driven B-cell acute lymphoblastic leukemia, we have adapted shRNA screening approaches to in vivo applications and have performed large-scale loss of function genetic screens in tumors. Initial work suggested that screens with complex shRNA libraries could be performed in the in vivo setting using a model of E[mu]-myc lymphoma. Here, the introduction of an shRNA library into lymphoma cells ex vivo, followed by transplantation, showed that as many as 1000 unique hairpins were represented in tumors in individual recipient animals. The set of genes targeted by shRNAs that negatively impacted lymphoma cell growth in vivo was highly enriched for regulators of cell motility, and suppression of these genes, which included Rac2, CrkL, and the poorly characterized actin monomer binding protein Twf, impaired lymphoma progression and tumor cell dissemination to sites of terminal metastatic disease. Additionally, suppression of Rac2 and Twf improved chemotherapeutic outcome, suggesting that lymphoma cell migration following therapy, from sites of minimal residual disease to sites of terminal disease, may be an important event in relapse. In a second screen using a BCR-Abl B-cell acute lymphoblastic leukemia model, we found that as many as 9,000 unique shRNAs could be identified in individual animals following tumor cell transplantation. Based on this result, we were able to perform an unbiased screen for modulators of leukemia cell growth in vivo using a genome-scale shRNA library composed of five pools of 10,000 shRNAs each. shRNAs that negatively impacted leukemia cell growth specifically in vivo were selected for validation, which included a set of hairpins targeting poorly characterized C2H2 zinc finger proteins. While neutral in the in vitro setting, a number of these shRNAs validated as single constructs in vivo. As an additional filter to select candidates for validation, we compared the set of genes targeted by candidate hairpins with genes that are transcriptionally upregulated in leukemia cells in vivo, and the leukemia genes Runx and Lmo2 met both of these criteria. Suppression of Runx and Lmo2 was selected against in tumors, suggesting that oncogenic pathways downstream of these genes may be used for leukemia cell growth or survival in vivo. We also compared the set of genes targeted by depleting hairpins with published DNA copy number alteration data from human ALL patients. In validation studies, we identified genes within regions of genomic alteration that impacted tumor cell proliferation in vivo. One of these genes was the plant homeodomain finger protein Phf6. While inactivating mutations in Phf6 are a common alteration in T cell malignancies, we found that suppression of this gene negatively impacted the growth of B cell malignancies in vivo, indicative of lineage-specific role for Phf6 in hematopoietic tumors. === by Corbin Elizabeth Meacham. === Ph.D.