Truncated p95HER2 receptor and novel anti-HER2 therapies
We previously showed that the expression of truncated forms of the HER2 receptor, p95HER2, correlate with a lack of response to the anti-HER2 antibody trastuzumab. Lapatinib, a HER2 tyrosine kinase inhibitor, has proven to be effi cacious in p95HER2-expressing cells in our preclinical models. We have now interrogated clinical samples from patients treated with lapatinib and found that patients with p95HER2-overexpressing tumors were as sensitive to this agent as p95HER2 negative tumors. This fi nding represents an additional step towards a rational subclassification of HER2-positive disease and a better selection of therapy for these patients. We have also explored new combinations of anti-HER2 therapies. Indeed, the combination of trastuzumab and lapatinib has already shown enhanced effi cacy in the neoadjuvant setting for patients with metastatic breast cancer. We had previously identifi ed the potential mechanism for the enhanced clinical activity of this combination, namely an enhanced immunemediated trastuzumab dependent cytotoxicity as a result of the lapatinib induced HER2 accumulation at the plasma membrane.
Additional mechanisms of resistance to targeted therapy against the HER2/PI3K pathway
As with the majority of anticancer agents, acquired resistance to anti-HER2 agents becomes an unavoidable phenomenon. Therefore, identifying potential “escape” mechanisms could lead to improved therapeutic strategies. In the light of these considerations, and in order to identify resistance mechanisms, we used a molecular barcode-screen approach to identify mediators of lapatinib resistance.
We showed that deregulation of the PI3K pathway, either through PTEN down-modulation, or through overexpression of the two most frequent breast cancer mutations in PI3KCA (E545K and H1047R), confers resistance to trastuzumab and lapatinib. We then confi rmed these observations by showing that PI3K-mediated trastuzumab and lapatinib resistance can be abrogated with the PI3K/mTOR inhibitor NVPBEZ235.
We understood that the enhanced effi cacy of combining anti-HER2 compounds with PI3K/ mTOR inhibitors is due to prevention of compensatory pathway activation. Such combinations are presently under investigation in phase Ib clinical trials. Using a diff erent approach, we also generated cells with acquired resistance to either trastuzumab or lapatinib. These cells represent a powerful tool in gaining a better understanding of the molecular mechanisms responsible for the resistant phenotype and, hence, the testing of new compounds that may overcome it. We explored the genetic aberrations in trastuzumab-resistant cell lines, by both microarray (Luminex) and genome-wide single nucleotide polymorphism (SNPs) analysis (Aff ymetrix 500K SNP array). These analyses have shown that cyclin E gene is amplifi ed and its protein product upregulated in our resistant cell lines. We then confirmed these fi ndings by FISH and IHC in a cohort of 50 HER2 positive patients where cyclin E amplifi cation/overexpression was found in approximately 30% of the cases.
We reasoned that deregulation of cyclin E expression may play a causative role in the acquisition of trastuzumab resistance, and we have tested this hypothesis both preclinically and in trastuzumab treated breast cancer patients. In cell lines with cyclin E overexpression we found that CDK2 inhibitors were very eff ective in inducing cell death and slowing down tumor growth.
In the clinical setting we confirmed that presence of cyclin E amplifi cation/overexpression negatively correlates with clinical benefi t rate and progression-free survival of patients treated with trastuzumab-based therapy. We plan to confi rm these results in a larger cohort of patients and, if these data are in line with the previous fi ndings, design ad hoc clinical trial with CDK inhibitors together with anti-HER2 agents. Finally, with the help of a cDNA library we have identifi ed novel kinases that mediate resistance to PI3K inhibition. We are further elucidating the precise mechanism by which these kinases confer resistance to PI3K-targeted therapy, with the aim of improving the clinical effi cacy of these agents upon overexpression of the given kinases.