Unknown,Transcriptomics,Genomics,Proteomics

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In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

ABSTRACT: Primary leukemia stem cells (LSCs) reside in an in vivo microenvironment that supports the growth and survival of malignant cells. We used an in vivo short hairpin RNA (shRNA) screening approach to identify novel genes that are essential for primary murine MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is selectively essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation in long term transplantation assays. We explored the signaling pathways downstream of Itgb3 using an additional in vivo shRNA screen and identified Syk as a critical mediator of Itgb3 activity in leukemia. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using the Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML, and demonstrate the utility of in vivo RNA interference screens. We examined the effect of Itgb3 knockdown by gene expression profiling in primary leukemia cells.

ORGANISM(S): Mus musculus

SUBMITTER: Fatima Al-Shahrour

PROVIDER: E-GEOD-46307 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To design an effective antibody therapy to improve clinical outcomes in leukemia, the identification of novel cell surface antigens is needed. Herein, we demonstrate a role for transmembrane tumor necrosis factor-αin leukemia. To characterize tmTNF-α expression in acute leukemia, normal hematopoietic cells, and non-hematopoietic tissues, we used a monoclonal antibody, termed C1, which specifically recognizes the tmTNF-α domain. We found that tmTNF-α was preferentially expressed by acute leukemia and leukemia stem cells. More abundant expression correlated with poor risk stratification, extramedullary infiltration, and adverse clinical parameters. Moreover, knockdown of tmTNF-α+ expression rendered leukemia cells more sensitive to chemotherapy in vitro and delay regeneration of leukemia in NOD–SCID mice. Targeting tmTNF-α by C1 resulted in leukemia cell killing via antibody-dependent cell-mediated and complement -dependent cytotoxicity in vitro, and inhibited leukemia cell growth in vivo while simultaneously sparing normal hematopoietic cells. Notably, C1 administration impaired the regeneration of leukemia in secondary serial transplantationin to NOD-SCID mice. To further understand the different expression patterns in leukemia cells and potential mechanisms underlying the effects caused by decreased expression of tmTNF-α, total RNA was extracted with TRIzol from SKM-1 stably transfected with control or tmTNF-α shRNA, tmTNF-α+ or - cells sorted from a primary AML sample, and human CD45+ leukemia cells from micetreated with C1 or IgG1 after first transplantation of 7052, and were then subjected to the microarray analysis. We extracted total RNA from SKM-1 stably transfected with control or tmTNF-α shRNA, tmTNF-α+ or - cells sorted from a primary AML sample, and human CD45+ leukemia cells from mice treated with C1 or IgG1 after first transplantation of 7052 (leukemia cell line initially derived from patients with primary acute leukemia who remained stably transplantable in NOD–SCID mice). These were then subjected to the GeneChip probe arrays (Affymetrix GeneChip® Human Genome U133 Plus 2.0). Using SKM-1 stably transfected with control (NC), tmTNF-α+ cells sorted from the primary AML sample and CD45+ leukemia cells from mice treated with IgG1 as reference samples respectively, we compared the different expression patterns in leukemia cells caused by decreased expression of TNF-α (SKM-1 89-1/NC-1, 89-2/NC-2; 70-C1-2/70-IgG-1, 70-C1-3/70-IgG-2; AMLTNFα- 1/+ 1; AMLTNFα- 2/+ 2). Two replicates were included for each pair of samples.

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In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

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