For a while now, researchers have speculated that RNA interference might be able to be boost the efficacy of small molecule drugs, such as cancer treatments. A paper in the most recent issue of the Journal of Clinical Investigation adds to the growing body of evidence supporting this theory, in demonstrating how stable expression of siRNAs in hematopoietic cells appears to increase the efficacy of Novartis’ Gleevec.
The paper was authored by Gary Gilliland of Brigham and Women’s Hospital, along with colleagues from Harvard Medical School, the Dana-Farber Cancer Institute, and Novartis Pharma. The authors noted that although small molecule inhibitors of tyrosine kinases, such as Gleevec, are effective in treating BCR-ABL and TEL-PDGFß receptor-mediated leukemias, drug resistance may develop.
“Therefore, the development of alternative molecularly targeted therapies that might enhance specificity and clinical efficacy, as well as overcome the problem of resistance, is of great clinical interest,” they wrote.
“Chromosomal translocations are recurrent findings in leukemias and some solid tumors, and they frequently result in abnormal expression of chimeric fusion oncoproteins,” the authors stated in their paper. “The unique fusion junctions of these molecules are attractive candidates for molecularly targeted therapeutic intervention using RNA interference.”
Gilliland and colleagues focused their experiments on the TEL-PDGFß receptor, which plays a role in chronic myelomonocytic leukemia. They developed a retroviral system that expresses siRNAs directed towards the unique fusion junction sequence of tyrosine kinase in transformed hematopoietic cells.
“We cloned the human pol III-dependent RNAse P RNA H1 promoter from genomic DNA and subcloned the H1 promoter followed by the siRNA hairpin cassette into the 3’ LTR of pQCXIP, a self-inactivating retroviral vector, to minimize potential promoter interference from the viral 5’ LRT,” the authors wrote.
“With this design, we would achieve a double copy of the siRNA expression cassette, one in each LTR,” Jing Chen, a Brigham and Women’s Hospital researcher and author of the JCI paper, told RNAi News. “This [would] enhance the expression level of the siRNA and therefore increase the effectiveness of the siRNA.”
According to the JCI paper, the researchers found that they could not only stably express siRNAs directed towards the TEL-PDGFß receptor fusion function sequence in hematopoietic cells, but that they could achieve a roughly 90 percent reduction in TEL-PDGFß receptor expression.
Testing the siRNAs in mice in order to evaluate therapeutic potential in vivo, they also found that the RNAi treatment was able to significantly prolong survival. “Ba/F3 cells stably expressing TEL-PDGFßR alone caused tumor development and death with a median latency of 24 days after injection,” the paper’s authors wrote. “In contrast, expression of inhibitory siRNA in cells stably transformed by TEL-PDGFßR resulted in a statistically significant prolongation in survival, with a median latency of 41 days.”
Despite the increased survival, by “using siRNA[s] as a single agent, it seems like it’s not sufficient to completely abrogate the TEL-PDGFß receptor-induced tumorigenesis,” Chen noted. “We successfully decreased [the] transforming activity of the fusion tyrosine kinase, but the residual, low-level expression of this protein can transform the hematopoietic cells so those cells can still form tumors.
“That’s why we later tested the idea [that there may be] synergistic effects between the siRNA and small molecule inhibitors such as Gleevec,” he added.
Testing the combination of the siRNAs and Gleevec in cell culture, Chen said that the research team “found out that the expression of siRNAs sensitizes the TEL-PDGFß receptor to the inhibition of Gleevec.”
Additionally, “Western blot results showed that stable expression of [the siRNAs] sensitized TEL-PDGFßR to inhibition by [Gleevec] in autophosphorylation activity of TEL-PDGFßR,” as well as activity of the tyrosine kinase’s downstream signaling pathway, the paper states.
The researchers also discovered that a Gleevec-resistant TEL-PDGFßR mutant could be inhibited with the siRNAs.
Commenting on the findings of the study, Chen was quick to note that while “the stable expression of siRNA can potentiate conventional cancer treatments, including small molecule drug treatment,” delivery issues remain a significant hurdle for the technology.
“If we can solve the problem of delivery of siRNA in vivo, this has the potential to be developed as a gene therapy to go along with small molecules,” he said.
— DM