 Hi, my name is Alex Toker and I'm a professor of pathology and chief of the division of signal transduction at the Bethesda Deaconess Medical Center and Harvard Medical School in Boston. Hi, my name is Maria Mancini. I was a fellow in the Toker lab and the first author of the publication. The Toker laboratory has for about 20 years been studying tumor progression with a particular emphasis on breast cancer and with a particular focus on a signal transduction pathway that is very frequently deregulated in most human tumors, both solid tumors and hematological malignancies. And specifically, the PSB kind is an AKT signaling pathway has garnered much attention in the context of tumor progression because of wide-ranging mutations that deregulate the pathway and elicit transformation and tumor progression. There is therefore significant interest, especially in the pharmaceutical field, to develop small molecule inhibitors for therapeutic intervention for patients that have mutations in this pathway. Many of the mutations that exist in this pathway have been well characterized. However, one specific mutation that was identified about 10 years ago in the AKT serine serine protein kinase known as E17K is less well understood. And so a few years ago, my laboratory undertook a study to evaluate the specific function of the E17K AKT1 mutation, which occurs with a frequency of approximately 6 to 10% across all molecular subtypes of breast cancer. This mutation has been studied extensively with respect to its oncogenic capacity using in vitro models. However, in vivo mouse models of this recurring somatic mutation had not been generated or studied in any detail. And so we took a study to generate a transgenic mouse that harbors AKT1 E17K mutation in the mouse mammary epithelium and to evaluate the consequences for mammary tumor genesis. So our main purpose, as Dr. Toko mentioned, in generating this AKT1 E17K transgenic mouse was to really ascertain whether this mutation would be on its own sufficient for transforming the mammary epithelial cells in vivo. And one of the things we had considered when we were generating this animal is we wanted also to be useful in the field for other cellular subtypes and other applications. And so what we did was we made it not only regulated by tetracycline so the transgene can be turned off and on, but we also made it so that it could be combined with different tissue-specific promoters so that you could study the role of the transgene and other tissue or cellular subtypes. We first examined the role of the transgene in the mammary epithelial compartment of a virgin female mice to see if in that steady state it would be sufficient for transformation of the mammary epithelium. And after a year's time we did not see any evidence of this transformation. What we did notice was the mammary gland became hyperplastic. And so that really sort of cascaded into essentially a bunch of follow-up questions where we wanted to understand what was really going on mechanistically in this tissue. So the next question that we wanted to understand was what was the role of this in a situation of multiple rounds of pregnancy. We understand that this mutation is associated with estrogen receptor-positive breast cancers and so we wanted to understand the role of this transgene in a situation where there would be a lot of hormone signaling in the mouse. And what we determined was that even after multiple rounds of pregnancy this transgene still was not going to be transformative in the mammary gland so we did not observe any tumors in animals that had undergone multiple rounds of pregnancy. Furthermore, when we added estrogen as a supplement to these animals it still was not sufficient for transformation. Based on previous work where AKT expression, sustained expression in vivo models had been placed onto the background of a HER2 amplification the literature had showed that the over-expression of AKT1 had accelerated tumor genesis and so we wanted to see if it would recapitulate that phenotype with this more physiologic mutation. And so to that end we combined our AKT1E17K transgenic mouse over-expressed specifically in the mammary gland. We combined that on the background of an over-expressing HER2 mouse model also in the mammary gland and what we saw was initially surprising and that was that these mice actually did not develop any mammary tumors at all. And so in the end what we, after doing further analysis what we ascertained is that the AKT1E17K actually participates in negative regulation of receptor tyrosine kinase signaling which in turn actually down regulates the signaling through the amplified HER2. The broader implications of this study, in addition to having generated the very first transgenic mouse model of a somatic mutation of AKT are that feedback regulation of the AKT pathway regulates receptor tyrosine kinase levels and therefore dampens the signal through in this case HER2 to decelerate tumor genesis which was, as Dr. Mazzini pointed out, a rather surprising finding. Another implication of this study is why do patients actually harbor this mutation? If this mutation as the transgenic mouse model indicated do not progress to frank carcinoma, why are there patients upwards of 10% of the breast cancer population actually harbor this mutation? And one of the most logical reasons for this might be that this mutation will not exist in isolation in the breast cancer population. It is highly likely that this mutation arises in the context of other hits or other somatic mutations such as p53 loss and other driver mutations that initiate tumor genesis and by which AKT1E17K contributes to whether this is indeed the situation remains to be demonstrated but this mouse model should provide a very useful tool to test these hypotheses. It will also provide a useful tool for testing therapeutic possibilities in this pathway. As I mentioned earlier, there are numerous small molecule inhibitors that are currently under clinical evaluation for therapeutic benefit for patients that harbor mutations in the ps3 kinase and AKT pathway. And we hope that this mouse model will provide a useful tool for both monotherapy and combination therapies that target one or more nodes in this pathway.