[show abstract][hide abstract] ABSTRACT: Aberrant regulation of the erythroblastosis oncogene B (ErbB) family of receptor tyrosine kinases (RTKs) and
their ligands is common in human cancers. ErbB3 is required in luminal mammary epithelial cells (MECs) for
growth and survival. Since breast cancer phenotypes may reflect biological traits of the MECs from which they
originate, we tested the hypothesis that ErbB3 drives luminal breast cancer growth. We found higher ERBB3
expression and more frequent ERBB3 gene copy gains in luminal A/B breast cancers compared with other
breast cancer subtypes. In cell culture, ErbB3 increased growth of luminal breast cancer cells. Targeted depletion of ErbB3 with an anti-ErbB3 antibody decreased 3D colony growth, increased apoptosis, and decreased
tumor growth in vivo. Treatment of clinical breast tumors with the antiendocrine drug fulvestrant resulted
in increased ErbB3 expression and PI3K/mTOR signaling. Depletion of ErbB3 in fulvestrant-treated tumor
cells reduced PI3K/mTOR signaling, thus decreasing tumor cell survival and tumor growth. Fulvestrant treatment increased phosphorylation of all ErbB family RTKs; however, phospho-RTK upregulation was not seen
in tumors treated with both fulvestrant and anti-ErbB3. These data indicate that upregulation of ErbB3 in
luminal breast cancer cells promotes growth, survival, and resistance to fulvestrant, thus suggesting ErbB3 as
a target for breast cancer treatment.
The Journal of clinical investigation 09/2013; · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40% to 50% of cases are positive. To uncover other potential targetable mutations, we conducted whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, and D816) wild-type melanoma. Surprisingly, we found a somatic BRAF(L597R) mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF(V600) mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition. A patient with BRAF(L597S) mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data show clinical significance to BRAF(L597) mutations in melanoma.
Cancer Discovery 07/2012; 2(9):791-797. · 10.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: ErbB3 harbors weak kinase activity, but strongly activates downstream phosphatidylinositol 3-kinase/Akt signaling through heterodimerization with and activation by other ErbB receptor tyrosine kinases. We report here that ErbB3 loss in the luminal mammary epithelium of mice impaired Akt and MAPK signaling and reduced luminal cell proliferation and survival. ERBB3 mRNA expression levels were highest in luminal mammary populations and lowest in basal cell/stem cell populations. ErbB3 loss in mammary epithelial cells shifted gene expression patterns toward a mammary basal cell/stem cell signature. ErbB3 depletion-induced gene expression changes were rescued upon activation of Akt and MAPK signaling. Interestingly, proliferation and expansion of the mammary basal epithelium (BE) occurred upon ErbB3 targeting in the luminal epithelium, but not upon its targeting in the BE. Multiple cytokines, including interleukin 6, were induced upon ErbB3 depletion in luminal epithelium cells, which increased growth of BE cells. Taken together, these results suggest that ErbB3 regulates the balance of differentiated breast epithelial cell types by regulating their growth and survival through autocrine- and paracrine-signaling mechanisms.
Proceedings of the National Academy of Sciences 12/2011; 109(1):221-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, they develop resistance, often due to acquisition of a second-site mutation (T790M). Current EGFR TKIs select for T790M in preclinical models of acquired resistance. We explored whether all EGFR TKIs similarly select for the T790M mutation using data from early clinical trials and established in vitro models of acquired resistance.
We analyzed the clinical characteristics of eight patients with metastatic EGFR-mutant lung adenocarcinoma who were treated first-line with XL647 and then progressed. XL647 is an ATP-competitive inhibitor of EGFR, HER2, KDR, and EPHB4. Additional molecular preclinical studies were performed to characterize resistance.
Four patients displayed confirmed partial responses (PRs), three patients had unconfirmed PRs, and one patient displayed stable disease. Only one of five patients' tumor samples available for analysis after disease progression harbored the T790M mutation. Eight patients subsequently received erlotinib, with (n = 3) or without (n = 5) chemotherapy. Three of five patients treated with single-agent erlotinib derived additional benefit, staying on drug up to 9 months. EGFR-mutant PC-9 cells with acquired resistance to XL647 did not harbor the T790M mutation, displayed a distinct mRNA profile from PC-9 cells with T790M-mediated resistance, and were moderately sensitive to erlotinib in growth inhibition assays. Crystal structure analyses of XL647/EGFR T790M did not reveal a different binding mode from that of erlotinib.
The findings of this exploratory study suggest that different EGFR TKIs may select for distinct mechanisms of resistance. These results raise the possibility that different EGFR TKIs could be sequentially used to improve outcomes in patients with EGFR-mutant lung cancer. Further work investigating this hypothesis is warranted.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 12/2011; 7(2):434-42. · 4.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gene fusions involving the catalytic domain of tyrosine kinases (TKs) are found in a variety of hematological and solid tumor malignancies. Clinically, TK fusions have emerged as prime targets for therapy with small molecule kinase inhibitors. Unfortunately, identification of TK fusions has been hampered by experimental limitations. Here, we developed version 2.0 of a genomically based systematic kinase fusion screen and used it to detect a novel imatinib-sensitive C6orf204-PDGFRB fusion in a patient with precursor T lymphoblastic lymphoma (T-ALL) and an associated myeloproliferative neoplasm with eosinophilia. These data validate the ability of this targeted capture-sequencing approach to detect TK fusion events in small amounts of DNA extracted directly from patient samples.
Genes Chromosomes and Cancer 09/2011; 51(1):54-65. · 3.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Non-small cell lung cancers (NSCLCs) that harbor mutations within the epidermal growth factor receptor (EGFR) gene are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Unfortunately, all patients treated with these drugs will acquire resistance, most commonly as a result of a secondary mutation within EGFR (T790M). Because both drugs were developed to target wild-type EGFR, we hypothesized that current dosing schedules were not optimized for mutant EGFR or to prevent resistance. To investigate this further, we developed isogenic TKI-sensitive and TKI-resistant pairs of cell lines that mimic the behavior of human tumors. We determined that the drug-sensitive and drug-resistant EGFR-mutant cells exhibited differential growth kinetics, with the drug-resistant cells showing slower growth. We incorporated these data into evolutionary mathematical cancer models with constraints derived from clinical data sets. This modeling predicted alternative therapeutic strategies that could prolong the clinical benefit of TKIs against EGFR-mutant NSCLCs by delaying the development of resistance.
Science translational medicine 07/2011; 3(90):90ra59. · 10.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: The identification of somatically acquired tumor mutations is increasingly important in the clinical management of cancer because the sensitivity of targeted drugs is related to the genetic makeup of individual tumors. Thus, mutational profiles of tumors can help prioritize anticancer therapy. We report herein the development and validation of two multiplexed assays designed to detect in DNA from FFPE tissue more than 40 recurrent mutations in nine genes relevant to existing and emerging targeted therapies in lung cancer. The platform involves two methods: a screen (SNaPshot) based on multiplex PCR, primer extension, and capillary electrophoresis that was designed to assess for 38 somatic mutations in eight genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN) and a PCR-based sizing assay that assesses for EGFR exon 19 deletions, EGFR exon 20 insertions, and HER2 exon 20 insertions. Both the SNaPshot and sizing assays can be performed rapidly, with minimal amounts of genetic material. Compared with direct sequencing, in which mutant DNA needs to compose 25% or more of the total DNA to easily detect a mutation, the SNaPshot and sizing assays can detect mutations in samples in which mutant DNA composes 1.56% to 12.5% and 1.56% to 6.25% of the total DNA, respectively. These robust, reliable, and relatively inexpensive assays should help accelerate adoption of a genotype-driven approach in the treatment of lung cancer.
The Journal of molecular diagnostics: JMD 01/2011; 13(1):74-84. · 3.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tyrosine kinase (TK) fusions are attractive drug targets in cancers. However, rapid identification of these lesions has been hampered by experimental limitations. Our in silico analysis of known cancer-derived TK fusions revealed that most breakpoints occur within a defined region upstream of a conserved GXGXXG kinase motif. We therefore designed a novel DNA-based targeted sequencing approach to screen systematically for fusions within the 90 human TKs; it should detect 92% of known TK fusions. We deliberately paired 'in-solution' DNA capture with 454 sequencing to minimize starting material requirements, take advantage of long sequence reads, and facilitate mapping of fusions. To validate this platform, we analyzed genomic DNA from thyroid cancer cells (TPC-1) and leukemia cells (KG-1) with fusions known only at the mRNA level. We readily identified for the first time the genomic fusion sequences of CCDC6-RET in TPC-1 cells and FGFR1OP2-FGFR1 in KG-1 cells. These data demonstrate the feasibility of this approach to identify TK fusions across multiple human cancers in a high-throughput, unbiased manner. This method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 µg of DNA, and circumvents the need for complex computational sequence analysis.
Nucleic Acids Research 11/2010; 38(20):6985-96. · 8.28 Impact Factor