By Doug Macron
Researchers from Cold Spring Harbor Laboratory last week reported on the use of an RNAi screen to identify a novel therapeutic target for acute myeloid leukemia.
While the paper, which appeared in the online edition of Nature, points to the therapeutic potential of a small-molecule inhibitor of the protein bromodomain-containing 4, the team also notes that their data “highlight the utility of RNA interference screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.”
AML represents “a paradigm for understanding how patterns of cooperating genetic and epigenetic alterations lead to tumorigenesis,” the authors wrote. “Although this complexity poses a challenge for the development of targeted therapies, diverse gene mutations in AML generally converge functionally to deregulate similar core cellular processes.”
Key to AML is the “corruption” of cell-fate programs, which generates leukemia stem cells that self-renew and advance the disease.
“Although it is incompletely understood, this process has been linked to changes in regulatory chromatin modifications,” according to the paper. “For example, common AML oncogenes such as those encoding the AML1-ETO and MLL fusion proteins induce self-renewal programs at least in part through reprogramming of epigenetic pathways.”
Because epigenetic changes associated with cancers may be reversible, chromatin regulators are currently considered as possible oncology drug targets, the authors wrote.
To examine the epigenetic pathways required for AML maintenance, the researchers constructed a custom library of shRNAs targeting 243 known chromatin regulators, with three to six hairpins per gene. Specifically, the library was built in TRMPV-Neo, a vector optimized for negative-selection RNAi screening, and was transduced as one pool into an established Tet-on competent AML mouse model.
Tet-on RNAi involves a two-component conditional expression system: a reverse tetracycline transactivator and a tetracycline-responsive element promoter that drives shRNA expression, according to a recent overview of the approach published by the CSHL team and collaborators at the University of Zurich.
“In this system, shRNA expression is induced by doxycycline, enabling synchronous and reversible gene knockdown in established cell populations,” according to the paper.
Doxycycline was administered to the mouse to induce shRNA expression, and changes in library representation after two weeks of culture were monitored using deep sequencing of the shRNA guide stranded amplified from genomic DNA.
Of the roughly 1,000 shRNAs under evaluation, 177 were “strongly depleted,” the team noted. “These included all eight positive-control shRNAs targeting essential genes, as well as several shRNAs targeting two known MLL-AF9 co-factors.
Genes for which at least two independent shRNAs scored were subjected to extensive one-by-one validation, and in both the initial screen and validation stages, several shRNAs targeting Brd4 were among the most strongly depleted, identifying the gene as the “top scorer in the screen,” they added.
Five independent shRNAs against Brd4 showed a “close correspondence between knockdown efficiency and growth inhibition, indicating on-target effects,” the researchers wrote. “Suppression of Brd4 led to cell-cycle
arrest and apoptosis of leukemia cells,” while similar knockdown in immortalized murine embryonic fibroblasts resulted in only “modest” cell-cycle inhibition without cytotoxicity.
Moreover, Brd4 knockdown did not alter the growth of non-transformed G1E erythroblast cells, while shRNAs targeting the gene were capable of inducing cell-cycle arrest in two MLL-AF91 human AML lines, all of which suggests that Brd4 is a “critical requirement” in MLL-AF9-induced AML, according to the paper.
Further experimentation showed that suppressing Brd4, using either shRNAs or the small-molecule inhibitor JQ1, led to “robust anti-leukemic effects” in vitro and in vivo, along with terminal myeloid differentiation and elimination of leukemia stem cells.
“By taking a non-biased screening approach targeting epigenetic regulators, our study has identified Brd4 as a critical factor required for AML disease maintenance,” the investigators concluded. “Because Brd4 is not evidently mutated or over-expressed in AML, the exquisite sensitivity of leukemia cells to Brd4 inhibition would not have been revealed simply through genetic or transcriptional characterization of this disease.
“Our results unambiguously highlight the utility of RNAi screening for revealing candidate drug targets in cancer,” they added.
Contributing to the Nature paper were researchers from the Research Institute of Molecular Pathology in Austria; Stony Brook University; the Medical University of Vienna; Johns Hopkins University School of Medicine; the Dana-Farber Cancer Institute; the Cincinnati Children's Hospital Medical Center; and the University of California, San Francisco.
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