RNAi screen for rapid therapeutic target identification in leukemia patients

Abstract

Targeted therapy has vastly improved outcomes in certain types of cancer. Extension of this paradigm across a broad spectrum of malignancies will require an efficient method to determine the molecular vulnerabilities of cancerous cells. Improvements in sequencing technology will soon enable high-throughput sequencing of entire genomes of cancer patients; however, determining the relevance of identified sequence variants will require complementary functional analyses. Here, we report an RNAi-assisted protein target identification (RAPID) technology that individually assesses targeting of each member of the tyrosine kinase gene family. We demonstrate that RAPID screening of primary leukemia cells from 30 patients identifies targets that are critical to survival of the malignant cells from 10 of these individuals. We identify known, activating mutations in JAK2 and K-RAS, as well as patient-specific sensitivity to down-regulation of FLT1, CSF1R, PDGFR, ROR1, EPHA4/5, JAK1/3, LMTK3, LYN, FYN, PTK2B, and N-RAS. We also describe a previously undescribed, somatic, activating mutation in the thrombopoietin receptor that is sensitive to down-stream pharmacologic inhibition. Hence, the RAPID technique can quickly identify molecular vulnerabilities in malignant cells. Combination of this technique with whole-genome sequencing will represent an ideal tool for oncogenic target identification such that specific therapies can be matched with individual patients.

Fig. 1.

RAPID functional profiling of a patient with ASM with associated CMML. (A) White blood cells were incubated with 1 μM siRNA, individually targeting each member of the tyrosine kinase family. Cells were electroporated and replated into culture media, and cell viability was determined by addition of a tetrazolium salt (MTS assay) at day 4 postelectroporation. Values represent percentage mean (normalized to the median value on the plate) ± SEM (n = 3). (B) Cells from patient 07-079 were treated with a dose curve of AG490. After 3 days, cell viability was measured with an MTS assay (Left). Alternatively, colonies were counted after 10 days (Right). Values represent percentage mean (normalized to untreated controls) ± SEM (n = 3). *, P < 0.05.

Fig. 2.

MPL^1886InsGG is hypersensitive to thrombopoietin and transforms BA/F3 cells. (A) BA/F3 cells were plated in a dose gradient of thrombopoietin. After 3 days, cell proliferation was assessed by using an MTS assay. Values represent percentage mean (normalized to 10 ng/mL TPO wells) ± SEM (n = 6). *, P < 0.05. (B) BA/F3 cells were stimulated for 15 min with 0–10 ng/mL thrombopoietin. Whole-cell lysates were subjected to immunoblotting with antibodies specific for total (Middle) or phospho-STAT5 (Top), as well as β-actin (Bottom). (C) BA/F3 cells were plated in medium lacking WEHI-conditioned medium, and total viable cells were counted daily for 1 week. Values represent mean viable cells ± SEM (n = 3). *, P < 0.05.

Fig. 3.

Midostaurin is effective against JAK2-dependent cells. (A) HEL cells or BA/F3 cells were treated with a dose gradient of AG490 (Left) or midostaurin (Right). Cell proliferation was determined after 3 days. Values represent percentage mean (normalized to untreated control wells) ± SEM. (n = 3). (B) BA/F3 cells expressing MPL 1886InsGG were incubated for 1 h in 0–1,000 nM midostaurin. Cells were then stimulated for 15 min with thrombopoietin (10 ng/mL), and cell lysates were subjected to immunoblot analysis for phospho or total-JAK2, STAT5, STAT3, AKT, and ERK1/2, as well as β-actin.

Fig. 4.

Midostaurin exhibits patient-specific therapeutic efficacy in patient 07-079. (A) Cells from patient 07-079 were treated with a dose curve of midostaurin. Colonies were counted after 10 days. Values represent mean colonies per plate ± SEM (n = 3). *, P < 0.05. (B) Cells from patient 07-079, as well as 8 other CMML patients, were treated with a dose curve of midostaurin. After 3 days, cell viability was measured with an MTS assay. Values represent percentage mean (normalized to untreated controls) ± SEM (n = 3). (C) Patient 07-079 was treated with midostaurin (50-mg bid), briefly in combination with hydroxyurea, and white blood cell counts were monitored over 9 days. Normal white blood cell counts range from 4 to 12 × 10³ per mL.