Stable expression of small interfering RNA sensitizes TEL-PDGFβR to inhibition with imatinib or rapamycin
Jing Chen, Nathan R. Wall, Kerry Kocher, Nicole Duclos, Doriano Fabbro, Donna Neuberg, James D. Griffin, Yang Shi, D. Gary Gilliland
Jing Chen, Nathan R. Wall, Kerry Kocher, Nicole Duclos, Doriano Fabbro, Donna Neuberg, James D. Griffin, Yang Shi, D. Gary Gilliland
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Article Hematology

Stable expression of small interfering RNA sensitizes TEL-PDGFβR to inhibition with imatinib or rapamycin

Abstract

Small molecule inhibitors, such as imatinib, are effective therapies for tyrosine kinase fusions BCR-ABL–TEL-PDGFβR–mediated human leukemias, but resistance may develop. The unique fusion junctions of these molecules are attractive candidates for molecularly targeted therapeutic intervention using RNA interference (RNAi), which is mediated by small interfering RNA (siRNA). We developed a retroviral system for stable expression of siRNA directed to the unique fusion junction sequence of TEL-PDGFβR in transformed hematopoietic cells. Stable expression of the siRNA resulted in approximately 90% inhibition of TEL-PDGFβR expression and its downstream effectors, including PI3K and mammalian target of rapamycin (mTOR). Expression of TEL-PDGFβR–specific siRNA (TPsiRNA) significantly attenuated the proliferation of TEL-PDGFβR–transformed Ba/F3 cells or disease latency and penetrance in mice induced by intravenous injection of these Ba/F3 cells. Although a 90% reduction in TEL-PDGFβR expression was insufficient to induce cell death, stable siRNA expression sensitized transformed cells to the PDGFβR inhibitor imatinib or to the mTOR inhibitor rapamycin. TPsiRNA also inhibited an imatinib-resistant TEL-PDGFβR mutant, and the inhibition was enhanced by siRNA in combination with PKC412, another PDGFβR inhibitor. Although siRNA delivery in vivo is a challenging problem, stable expression of siRNA, which targets oncogenic fusion genes, may potentiate the effects of conventional therapy for hematologic malignancies.

Authors

Jing Chen, Nathan R. Wall, Kerry Kocher, Nicole Duclos, Doriano Fabbro, Donna Neuberg, James D. Griffin, Yang Shi, D. Gary Gilliland

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Retroviral delivery of siRNA directed against TEL-PDGFβR (T/P) breakpoin...
Retroviral delivery of siRNA directed against TEL-PDGFβR (T/P) breakpoint into hematopoietic cells. (A) The upper panel shows a schematic representation of TEL-PDGFβR fusion tyrosine kinase. The fusion junction that the siRNA was designed to target is indicated (upper and lower case letters correspond to TEL and PDGFβR sequence, respectively). The lower panel shows the predicted short hairpin transcript with a 3′_U5 pol III stop signal. (B) Schematic representation of pQCXIP_ H1_TPsiRNA retroviral vector. The cassette of siRNA hairpin structure driven by pol III promoter H1 was subcloned into the 3′ ØLTR of the self-inactivating retroviral vector pQCXIP. Ø indicates deletion. Pcmv, CMV promoter; IRES, internal ribosomal entry site; Puror, puromycin-resistance; H1, pol III RNAse P RNA H1 promoter. (C) Expression of TEL-PDGFβR in stably transformed Ba/F3 cells. pQCXIP_H1_TPsiRNA was introduced into TEL-PDGFβR stable Ba/F3 cells following retroviral transduction. TEL-PDGFβR was detected with a rabbit polyclonal antibody recognizing PDGFβR C-terminal tail. Ba/F3 cells transduced with an empty retroviral vector, as well as stable Ba/F3 cells expressing TEL-PDGFβR transduced with pQXCIP retroviral vector encoding a nonspecific siRNA construct, were included as controls. WB, Western blot. (D) RT-PCR analysis with total RNA from different stable Ba/F3 cells was performed with a pair of primers amplifying approximately 350 bp sequence overlapping the breakpoint of TEL-PDGFβR. The mRNA level of TEL-PDGFβR was normalized to the endogenous GAPDH mRNA level as indicated. (E) Northern blot analysis with total RNA from different stable Ba/F3 cells was performed and probed with 32P-labeled antisense 19-nt oligonucleotide for TPsiRNA. The control 5S-rRNA band was detected with ethidium bromide staining. Ba/F3 cells transduced with empty retroviral vector were included as a control.