RAPID siRNA Screen for Identification of Therapeutic Gene Targets in Patients with Hematologic Malignancies.
Authors: Tyner Jeffrey W , Loriaux Marc , Willis Stephanie G , Chang Bill H , Bicocca Vincent T , Oh Stephen T , Hollink Iris H.I.M. , Segers Stefanie , DenBoer Monique L. , van den Heuvel-Eibrink Marry M , Zwaan C. Michel , Gotlib Jason R , Deininger Michael , Druker Brian J. 
Published: October 2019

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.