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    Research Grants Awarded

    A Novel Preemptive Strategic Combinational Molecular Therapeutics Targeting Cancer Bioenergetics And Growth Regulatory Signaling Network In Breast Cancer

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    Mutations in genes in key cellular pathways drive tumor development, prognosis and predict response to therapy making it important to develop accurate methods to detect mutations to provide useful biomarkers. PIK3CA, which encodes the p110alpha catalytic domain of phosphatidylinositol 3 kinase (PI3K), is the most frequently mutationally activated gene in breast cancer being aberrant in approximately 25% of breast cancers. We and others have shown that changes in signaling pathways can predict sensitivity or resistance to targeted therapies and that mutations in key genes drive these changes. For example, in breast cancer patients, we have demonstrated that PIK3CA activation or PTEN loss confer resistance to Herceptin, whereas HER2 amplification indicates sensitivity. In lung cancer patients, RAS mutations confer resistance to Iressa, Tarceva and EGFR targeted antibodies while EGFR mutations cause sensitivity. Indeed, the package insert for some anti-EGFR targeted therapies in Europe requires that RAS status be determined prior to therapy. In preclinical models, PIK3CA activation or PTEN loss confer resistance to MEK inhibitors while RAF mutations confer sensitivity and RAS/RAF mutations confer resistance to SRC and AKT inhibitors while PIK3CA mutations confer sensitivity (unpublished data). This makes it imperative that the mutational status of a tumor be accurately determined so that patients can be directed to chemotherapy agents to which their tumor is sensitive and avoid agents to which they are resistant. We have developed assays using a mass spectrometry approach that can detect single nucleotide polymorphism (SNPs) as representations for mutations for hotspot regions of PIK3CA, KRAS and AKT1. The mass spectrometry approach is much more sensitive than standard Sanger sequencing being able to detect mutations present in as little as 0.1% of the DNA present. For Sanger sequencing, the mutation must be present in at least 20% of the DNA present. If the mutation is heterozygous, it must be present in at least 40 percent of the cells present. Thus contamination of the tumor with stroma or inflammatory cells or if a mutation arises late in tumor development and is present in only a subset of tumor cells ie subclonal, then mutations will be missed by conventional sequencing approaches to the potential detriment of the patient. Selection of any subclonal resistant clones could then lead to the formation of a more aggressive tumor since treatment would only destroy the sensitive tumor cell population. As an example, mutations in BCR/ABL causing resistance to imatinib mesylate and EGFR mutations implicated in response to EGFR inhibitors have been demonstrated to be present in rare subclones within patient tumors. Thus, the development and validation of an approach to reliably detect mutations is critical to patient management. Hypothesis: Our hypothesis is that sub-clones within some breast cancers gain PIK3CA activating mutations late in tumor development. As a corollary, these mutations will be selected by current and emerging targeted therapies. Subclonal PIK3CA mutations could potentially alter response to chemotherapy and to emerging targeted therapeutics against the PI3K pathway. Thus is it imperative to be able to accurately predict PIK3CA mutational status. We will test the hypothesis and corollary through the following specific aims: Specific Aim 1: Determine whether PIK3CA subclonal mutations are present in breast cancers. We will use two strategies to test whether subclonal PIK3CA activating mutations are present. The first method will be to determine whether microdissection-based enrichment of tumor cells will increase the concordance between Sanger and mass-spectrometry based sequencing with 454 sequencing added as an additional sensitive approach for confirmation. The second approach will be based on a multiplex FISH single cell-based assay in patient tumors. If subclonal PIK3CA mutations are present in breast cancers, this will have an impact on the design of biomarker-driven trials of PI3K pathway targeted therapies as well as increase the number of breast cancer patients likely to benefit from these therapies. It could also influence the selection of patient therapy as for example, our demonstration that PIK3CA mutations alter the response to Herceptin. Specific Aim 2: Detect subclonal PIK3CA mutations in patient samples before and after treatment. We will use the validated approaches from Aim1 and an existing and robust set of patient samples obtained before and after therapy to determine whether chemotherapy increases the fraction of cells with PIK3CA mutations. Based on the studies above, we would predict that the fraction of cells with PIK3CA mutations will increase in patients with chemoresistant tumors. If this is the case, it will further warrant analysis of subclonal PIK3CA mutations as a predictor of outcome, selection of therapy and further to identify an additional population of patients likely to respond to PI3K targeted therapy. Specific Aim 3: Determine whether PIK3CA somatic mutations are selected during therapy. To test whether chemotherapy agents will select for a subpopulation of mutant cells, we will inject mice with cell lines which do not contain a PIK3CA mutation, or varying amounts of GFP/Luc+ labeled isogenic cells stably transformed with the PIK3CA-H1047R activating mutation. We will then monitor selection of labeled cells during therapy to determine whether subclonal mutations are selected or alter response to therapy.

    Lay Abstract:
    Breast cancer is the most commonly diagnosed cancer in women. The fact that is not also the most common cause of cancer deaths is due to early diagnosis and targeted treatments that can completely eradicate the tumor. Most of the cancer deaths due to breast cancer are caused because the tumor develops resistance to therapy which allows the cancer to progress and spread beyond the breast. At our current level of understanding, we cannot predict which individuals will not benefit from current therapies or those who will go on to develop chemoresistance and tumor recurrence. The accumulation of deleterious genomic changes, those leading to uncontrolled cell growth or increased cell invasion and metastases, drives the formation of breast tumors. Once a tumor is formed, the process to eliminate the accumulation of more mutations is often disrupted. Breast tumors are known to have a high degree of heterogeneity due to this genomic instability, allowing for a large repertoire from which chemoresistant cells can be selected. Which specific mutations are required for tumor formation or chemotherapy resistance is not known; however, we do have a list of potential candidates. These were obtained by the work of many researchers looking at the cellular processes that go awry in tumor formation and by brute-force analysis (gene sequencing) of many breast cancer tumor samples to get a consensus of which genes are frequently mutated. The most frequently mutated gene in breast cancer is PIK3CA, a gene that encodes part of the key regulatory complex PI3K which controls the biological pathways for cell growth, cell division, cell differentiation, cell mobility and metastases. Importantly, activation of PI3K contributes to resistance to chemotherapy, herceptin (traztuzumab) and hormonal therapy in breast cancer. All of these processes are implicated in the progression of tumor development and patient outcomes. Our preliminary data indicates that mutation in PIK3CA may occur late in tumor development and may thus only be present initially in only a few cells in the patient?s tumor (subclonal). Current techniques cannot detect mutations in tumors if they occur in less than 20% of the cells in a tumor. Thus, we predict that PIK3CA is mutated in more tumors than has been previously reported, but initially at such a low level that it is not detected by standard sequencing methods. However, if these clones are present and if they are confer resistance to chemotherapy agents, the drugs will preferentially kill non-PIK3CA mutant clones, giving PIK3CA mutant clones a selective advantage. Chemotherapy agents block the signal that allows the cell to grow, or overgrow in the case of tumors. An additional reason that we want to accurately know whether PIK3CA mutated cells are present even at low subclonal levels is that new drugs that specifically target PI3K and the proteins located downstream of it are beginning to enter clinical trials. If a tumor has a mutation in PIK3CA, the patient is likely to benefit from these drugs, particularly when they are combined with other targeted drugs such as traztuzumab or hormone manipulation, as well as other standard chemotherapy agents. The PI3K-targeted therapy will sensitize tumor cells to the effects of other targeted therapies or chemotherapy, killing more of the tumor cells so that they cannot re-form the tumor. Since these drugs are now in clinical trials, identifying patients that can benefit from these drugs could have an immediate impact on patient management. Hypothesis: Our hypothesis is that sub-clones within some breast cancers gain PIK3CA activating mutations late in tumor development. As a corollary, these mutations will be selected by current and emerging targeted therapies. Thus subclonal PIK3CA mutations could potentially alter response to chemotherapy and to emerging targeted therapeutics against the PI3K pathway. Thus is it imperative to be able to accurately predict PIK3CA mutational status. We will test the hypothesis and corollary through the following specific aims: Specific Aim 1: Determine whether PIK3CA subclonal mutations are present in breast cancers. Specific Aim 2: Detect subclonal PIK3CA mutations in patient samples before and after treatment. Specific Aim 3: Determine whether PIK3CA somatic mutations are selected during therapy.