Stable and efficient transfection of siRNA for mutated KRAS silencing using novel hybrid nanoparticles.

siRNA is currently the most widely studied form of RNAi, and it has promising therapeutic potential not just in cancer but also in other diseases such as autoimmune and infectious diseases. However, efficient delivery of siRNA to target cells is being limited by lack of an effective delivery system that ensures efficient transfection into cells while protecting the encapsulated siRNA from nuclease. We hypothesized that a hybrid nanoparticle system composed of human IgG and poloxamer-188, a stealth polymer, will efficiently deliver mutated KRAS siRNA to A549 cells, leading to an efficient knockdown of mutated siRNA while protecting the siRNA from serum nuclease. We also hypothesized that the nanoparticles will not elicit an immunostimulatory effect in murine macrophages and also avoid clearance by macrophages. These nanoparticles were found to efficiently deliver siRNA to the cytoplasm and nuclease of A549 cells in a controlled and sustained manner while avoiding recycling by endosomes. An effective knockdown of mutated KRAS was achieved, which subsequently led to an increased sensitivity to erlotinib. These nanoparticles successfully avoided uptake by murine macrophages and reduced immune responses normally associated with siRNA/nanoparticle therapy. These results demonstrate that the novel hybrid nanoparticles could potentially serve as a platform for efficient delivery of siRNA to cells for stable gene knockdown.

Reexpression of epigenetically silenced AML tumor suppressor genes by SUV39H1 inhibition.

Reexpression of hypermethylated tumor suppressor genes using DNA methyltransferase (DNMT) and histone deacetylase inhibitors occurs by a mechanism whereby promoter demethylation is the dominant event. In support of this model, we found in acute myeloid leukemia cells with hypermethylated p15INK4B and E-cadherin promoters that the DNMT inhibitor, 5-aza-2'-deoxycytidine, induced p15INK4B and E-cadherin expression, and decreased levels of DNA methylation, histone H3 lysine 9 (H3K9) methylation and SUV39H1 associated with p15INK4B and E-cadherin promoters. On the basis of these observations, we examined whether promoter demethylation was dominant to H3K9 demethylation in p15INK4B and E-cadherin reexpression. We observed that SUV39H1 short hairpin RNA and chaetocin, a SUV39H1 inhibitor, induced p15INK4B and E-cadherin expression and H3K9 demethylation without promoter demethylation. Reexpression of hypermethylated p15INK4B and E-cadherin required histone H3K9 demethylation that was achieved directly by inhibiting SUV39H1 expression or activity, or indirectly by decreasing the amount of SUV39H1 associated with the p15INK4B and E-cadherin promoters using 5-aza-2'-deoxycytidine. The results from this study highlight the potential of H3K9 methyltransferases as therapeutic targets for reactivating expression of hypermethylated genes.

Bcr-Abl induces autocrine IGF-1 signaling.

Bcr-Abl oncogene is responsible for the initial phase of chronic myelogenous leukemia (CML), which is effectively treated by the Bcr-Abl inhibitor imatinib. Over time patients become resistant to treatment and progress to blast crisis, an event that is driven by additional genetic and epigenetic aberrations. Recently, we showed that Riz1 expression decreases in blast crisis and that re-expression of Riz1 inhibits IGF-1 expression. IGF-1 signaling is required in many stages of hematopoiesis and inappropriate activation of autocrine IGF-1 signaling may facilitate transformation to blast crisis. We observed that in 8 out of 11 matched CML patient biopsies the IGF-1 expression is elevated in blast crisis. We examined mechanisms used by CML blast crisis cell lines to activate IGF-1 expression. We found that Bcr-Abl activates autocrine IGF-1 signaling using Hck and Stat5b. Inhibition of these signaling components using small molecule drugs or shRNA decreases proliferation and enhances apoptosis. Together, our study suggests that aberrant IGF-1 signaling is an important event in blast crisis transformation and it provides a mechanism to explain the activity of IGF-1R and Hck inhibitors in blocking CML blast crisis phenotypes.