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Abstract 1476 AIM: Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Materials & Methods: Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay. Results: AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p Conclusion: These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3978 Poster Board III-914 A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific kinases as cancer targets has been a slow process. Inhibition of cancer-causing tyrosine kinases offers a promising avenue of therapy, however this strategy of targeted therapy will require a detailed understanding of the oncogenic targets in each cancer patient. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. Combination of the RAPID screen with gene-specific therapeutic approaches promises to yield a powerful synthesis of methodologies by which cancer patients can be specifically treated on the basis of functionally diagnosed gene targets. Methods To detect gene targets necessary for viability of malignant cells, we screened primary cells from 150 patients with hematologic malignancies by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we measured cell viability and tabulated sensitivity to silencing of specific genes. Samples were also screened for sensitivity to small-molecule kinase inhibitors. The mechanism of oncogenesis was investigated for each positive result. Results In total, we have identified 40 patient-specific gene targets in primary leukemia samples. We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. Agreement between siRNA-sensitive gene targets and small-molecule inhibitor sensitivity profiles has been high. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with abnormalities including gene overexpression and patient-specific mis-splicing events. Conclusions We demonstrate that RNAi functional screening can determine sensitivity to individual genes in cells obtained directly from cancer patients. Thus, this technique offers the potential to match targeted therapies with patients in a personalized manner. Application of these technologies will enable efficient discovery of the genetic etiology of cancer as well as a means for gene-specific therapeutic intervention. Disclosures: Deininger: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Calistoga: Research Funding; Genzyme: Research Funding. Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding.

A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific tyrosine kinases as cancer targets has been a slow process. In the near future, whole-genome sequencing will enable vast amounts of sequence data to be collected, however clinical application of this information will require a detailed understanding of the functional consequences of each sequence change. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. These data have led to identification of novel oncogenic anomalies in cancer patients. Combination of the RAPID screen with whole-genome sequencing promises to yield a powerful synthesis of methodologies by which both functional targets and genetic lesions can be rapidly determined. Methods: To detect targets necessary for viability of malignant cells, we screened primary cells from 75 patients with AML, ALL, CMML, and other MPD as well as white blood cells from healthy individuals by electroporating siRNAs individually targeting each member of the tyrosine kinase family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. Where possible, results were confirmed by treating samples with small-molecule inhibitors with activity against the genes identified by the assay. In addition, the mechanism of oncogenesis was investigated for each positive result. Results: We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. In total, of 75 patients screened, this assay has yielded 25 cases that exhibit sensitivity to one or more tyrosine kinases. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with genetic abnormalities including evidence of chromosomal rearrangements as well as gene overexpression and mis-spicing events. Conclusions: We demonstrate that RNAi functional screening can determine sensitivity to individual tyrosine kinases in primary samples. Thus, this technique offers the potential to match specific therapies for targeted intervention with individual patients based on a functional assay. Additionally, in many cases, combination of the RAPID screen with whole-genome sequencing will enable efficient discovery of the genetic etiology of cancer.