Unraveling the importance of the post-transcriptional RNA regulon in leukemic stem cells by in vivo RNAi

Abstract

Accumulating evidence suggests that Acute Myeloid Leukemia (AML) is propagated by leukemic stem cells (LSCs), which drive the continuous replenishment of malignant clones through dysregulated self-renewal. Unique LSC characteristics have not yet been identified, severely hindering the development of effective therapeutics for AML patients. Besides transcriptional regulation, cellular fate decisions are subject to an additional layer of post-transcriptional control. Essential players at this ‘RNA-centric’ level of regulation include RNA-binding proteins (RBPs), which have recently been identified to play critical roles in regulating self-renewal and cancer pathogenesis. This interest however has not yet extended into malignant stem cells of the blood system, prompting us to hypothesize that the dysregulated self-renewal characteristic of LSCs is dependent on RBP-directed posttranscriptional networks. Using an in silico approach, we defined a set of candidate LSC-regulating RBPs whose function in these cells will be assayed by in vivo pooled shRNA dropout screening. To reveal RBPs regulating self-renewal specifically, we will pool bone marrow from all primary mice and passage these cells again through secondary and tertiary transplants. Our screen will rely on a custom lentiviral library of hairpins encoded within the GIPZ lentiviral backbone, embedding each shRNA within a miR-30 transcript and including a turboGFP marker as an indicator of transduction in our experiments. Significantly depleted shRNAs will be validated with independent hairpins, followed by functional assessment of candidate RBP knockdown by means of various in vivo and in vitro assays. As a validation of our experimental setup, we have generated an AML mouse model induced by the MLL-AF9 oncogene and show that leukemic bone marrow cells retain their proportionality within grafts after transduction with GIPZ-encoded shRNAs targeting non-mammalian transcripts. With this screening strategy we hope to shed light on the potential role of RBPs as critical regulators of LSC stemness, ultimately making inroads into identifying potential new AML therapeutic targets. Accumulating evidence suggests that Acute Myeloid Leukemia (AML) is propagated by leukemic stem cells (LSCs), which drive the continuous replenishment of malignant clones through dysregulated self-renewal. Unique LSC characteristics have not yet been identified, severely hindering the development of effective therapeutics for AML patients. Besides transcriptional regulation, cellular fate decisions are subject to an additional layer of post-transcriptional control. Essential players at this ‘RNA-centric’ level of regulation include RNA-binding proteins (RBPs), which have recently been identified to play critical roles in regulating self-renewal and cancer pathogenesis. This interest however has not yet extended into malignant stem cells of the blood system, prompting us to hypothesize that the dysregulated self-renewal characteristic of LSCs is dependent on RBP-directed posttranscriptional networks. Using an in silico approach, we defined a set of candidate LSC-regulating RBPs whose function in these cells will be assayed by in vivo pooled shRNA dropout screening. To reveal RBPs regulating self-renewal specifically, we will pool bone marrow from all primary mice and passage these cells again through secondary and tertiary transplants. Our screen will rely on a custom lentiviral library of hairpins encoded within the GIPZ lentiviral backbone, embedding each shRNA within a miR-30 transcript and including a turboGFP marker as an indicator of transduction in our experiments. Significantly depleted shRNAs will be validated with independent hairpins, followed by functional assessment of candidate RBP knockdown by means of various in vivo and in vitro assays. As a validation of our experimental setup, we have generated an AML mouse model induced by the MLL-AF9 oncogene and show that leukemic bone marrow cells retain their proportionality within grafts after transduction with GIPZ-encoded shRNAs targeting non-mammalian transcripts. With this screening strategy we hope to shed light on the potential role of RBPs as critical regulators of LSC stemness, ultimately making inroads into identifying potential new AML therapeutic targets.