Fuhrmann Fonden - Investigation and optimization of kras‐targeting SSO’s in cancer cells

Project: Private Foundations

Description

The RAS proteins (HRAS, NRAS and KRAS) are crucial for proliferation, growth and survival of cells. Their aberrant regulation in somatic cells usually leads to cancer (1). At least 1/3 of all cancers harbor activating RAS mutations and up to 97% change codons 12 or 13 (2,3). The HRAS c.35G>T mutation, p.G12V, was the first mutation reported to cause cancer (4,5). Our lab has recently established that this mutation is in fact located in a fundamental splicing regulatory element (SRE) (6). The KRAS oncogene is mutated in three of the cancers that cause most deaths, namely lung, colon and pancreatic cancer. Approximately 50% of colorectal tumors have oncogenic mutations in KRAS codon 12 or 13 (7). RAS proteins were early on recognized as highly promising direct therapeutic targets (8). A recent review provides an overview of suggested KRAS targeting strategies ranging from direct inhibition of the KRAS protein to RNA interference, and details how each of them has yet to produce an impressive treatment strategy. KRAS remains a difficult and yet attractive target for cancer therapy (9). We propose to use Splice Switching Oligonucleotides (SSOs) to cause exclusion of KRAS exon 2 by blocking binding of splicing regulatory factors to the important SRE previously identified (6). This inhibits the expression of the oncogenic protein and has proved to be a very promising approach used frequently in cell culture studies in our lab (6,10-16). Exon 2 is weakly defined in both HRAS and KRAS, hence it is vulnerable to SSO treatment, and its exclusion deactivates the transforming effects of RAS (6). Our research has demonstrated decreased cell growth (6,14-16, unpublished) and changes in the KRAS pathway in response to SSOs (14-16, unpublished). This use of SSOs in cancer treatment has been patented by the group (16). Chemically modified SSO-based treatment has the advantages of being sequence-, even mutation-, specific, long-term stable, not dependent on cellular machinery, and various SSOs can be combined and delivered together. This means that one exon can be targeted in multiple positions to increase skipping, but also that several genes can be simultaneously inhibited to potentially produce synthetic lethality. The lab has developed several efficient SSOs to induce KRAS exon 2 skipping, some of which are mutation specific. Modification of the SSOs with LNA and a tail region have proven to increase efficiency, and these versions will be used in this project (14-16, unpublished). A promising pilot study in xenografted mice has been conducted with a human colorectal cell line, but the sample size was too small to draw final conclusions. I will therefore use a different and better suited approach (intraperitoneal injection) and a sufficient number of mice to support a final conclusion in my thesis work. Unfortunately, we do not have the funding for this new experiment, because nude mice and SSOs are very expensive.
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