Examining Mechanisms of Sensitivity and Resistance to Phosphatidylinositol 3-kinase inhibitors in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a common and debilitating form of cancer with few effective treatment options. HNSCC tumors display a complex array of molecular changes, and sequencing studies have identified the phosphatidylinositol 3-kinase pathway (PI3K) as the most frequently mutated oncogenic and targetable pathway in this cancer type. PI3K signaling contributes to cell growth and survival and is most commonly dysregulated by alterations in the gene PIK3CA, which encodes the catalytic subunit and alpha isoform of PI3K. In spite of this, PI3K inhibition has shown underwhelming efficacy in HNSCC clinical trials to date. Thus, my thesis seeks to evaluate the hypothesis that resistance to PI3K targeting therapies is the result of compensatory signals, which are activated in the presence of PI3K inhibitors. To test this, I examined how aberrant PI3K signaling was influenced by co-expression of EGFR, co-alteration of NOTCH1, and co-dependence of multiple RTKs, including ALK and IGF-1R. EGFR is overexpressed in most HNSCCs and its signaling is a widely studied means by which HNSCC cells evade death in the presence of PI3K inhibition. Consistent with previous studies, I demonstrated activation of the Ras-MEK-ERK pathway, downstream of EGFR, following treatment with PI3K inhibitor monotherapy in multiple PIK3CA amplified UM-SCC cell lines. I also showed that co-inhibition of PI3K with MEK or EGFR was synergistic in a further subset of these cell lines. I then tested several PI3K and EGFR inhibitor combinations in additional in vitro models. My pharmacologic analysis revealed that combinations including irreversible EGFR inhibitors were more effective than those utilizing reversible EGFR inhibitors. In HNSCC, NOTCH1 acts as a tumor suppressor, and inactivating alteration in this gene is observed in nearly 20% of tumors. Emerging data suggests interplay between PI3K and NOTCH signaling in this cancer type. Our CRISPR/Cas9 partial knockout model of PIK3CA in UM-SCC-47, reveal the cooperativity between the PI3K and NOTCH pathways. We confirmed this relationship and its potential importance using a transgenic mouse model: following treatment with 4-nitroquinoline N-oxide, mice with overexpression of mutant Pik3ca and knockout of Notch1 reach endpoint faster than animals with alterations in just one of these genes. Finally, in order to characterize additional signaling pathways driving compensatory PI3K inhibitor resistance, we developed and optimized an unbiased, high-throughput screening approach. We used this assay to test ~1400 inhibitors as monotherapies and in combination with PI3K inhibitors HS-173 and BKM120 in ten HNSCC cell lines. Our initial screening data suggested that combinations of PI3K inhibitors and ALK/IGF-1R inhibitors were among the most effective drug pairs. Using viability, apoptosis and cell cycle assays to test single-agent and combined treatments, we validated the combinatory effects of FDA-approved agents PI3K inhibitor pictilisib and ALK inhibitor brigatinib in a subset of cell lines. These inhibitors were similarly effective in a xenograft model. Furthermore, we identified additional synergistic dual-therapies; many of these inhibited PI3K in combination with upstream receptor tyrosine kinases, while combining PI3K inhibition with inhibition of downstream pathway members did not display synergy. Collectively, these data deepen our understanding of the combined effects of PI3K activation and aberration of an additional signaling pathway in HNSCC. In doing so, they inform the use of targeted PI3K inhibitors, motivate further analyses of PI3K combination treatments and suggest dual-therapies that may result in improved prognoses for HNSCC patients.PHDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/151718/1/nmich_1.pdfDescription of nmich_1.pdf : Restricted to UM users only

Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms

The identification of drug resistance pathways and approaches to target these pathways remains a significant and important challenge in cancer biology. Here, we address this challenge in the context of ongoing efforts to advance phosphatidylinositol 3-kinase (PI3K) inhibitors for the treatment of PI3K-aberrant cancers. While PI3K inhibitors have had tremendous success in some diseases, such as breast cancer, early clinical trials in other malignancies, such as head and neck squamous cell carcinoma (HNSCC), have not had the same level of success. Since HNSCC and other cancers display relatively high PI3K pathway alteration rates (>45%), these underwhelming results suggest that additional or unexpected factors may contribute to the lower response rates. Here, we highlight some of the emerging functional genomic and sequencing approaches being used to identify predictive biomarkers of PI3K inhibitor response using both cancer cell lines and clinical trial specimens. Importantly, these approaches have uncovered both innate genetic and adaptive mechanisms driving PI3K inhibitor resistance. In this chapter, we describe recent technological advances that have revolutionized our understanding of PI3K inhibitor resistance pathways in HNSCC and highlight how these and other approaches lay the groundwork to make significant strides in our understanding of molecular pharmacology in the cancer field

Methane fluxes from a small boreal lake measured with the eddy covariance method

Fluxes of methane, CH4, were measured with the eddy covariance (EC) method at a small boreal lake in Sweden. The mean CH4 flux during the growing season of 2013 was 20.1 nmol m(-2) s(-1) and the median flux was 16 nmol m(-2) s(-1) (corresponding to 1.7 mmol m(-2) d(-1) and 1.4 mmol m(-2) d(-1)). Monthly mean values of CH4 flux measured with the EC method were compared with fluxes measured with floating chambers (FC) and were in average 62% higher over the whole study period. The difference was greatest in April partly because EC, but not FC, accounted for fluxes due to ice melt and a subsequent lake mixing event. A footprint analysis revealed that the EC footprint included primarily the shallow side of the lake with a major inlet. This inlet harbors emergent macrophytes that can mediate high CH4 fluxes. The difference between measured EC and FC fluxes can hence be explained by different footprint areas, where the EC system sees the part of the lake presumably releasing higher amounts of CH4. EC also provides more frequent measurements than FC and hence more likely captures ebullition events. This study shows that small lakes have CH4 fluxes that are highly variable in time and space. Based on our findings we suggest to measure CH4 fluxes from lakes as continuously as possible and to aim for covering as much of the lakes surface as possible, independently of the selected measuring technique.Funding Agencies|Swedish Research Council FORMAS, project Color of Water (CoW); Swedish Research Council FORMAS, project Landscape Greenhouse Gas Exchange (LAGGE)</p

Comprehensive review of genetic factors contributing to head and neck squamous cell carcinoma development in low‐risk, nontraditional patients

BackgroundThe past 2 decades have seen an increased incidence of head and neck squamous cell carcinoma (HNSCC) in a nontraditional, low‐risk patient population (ie, ≤45 years of age, no substance use history), owing to a combination of human papillomavirus (HPV) infection and individual genetic variation.MethodsArticles positing genetic variants as contributing factors in HNSCC incidence in low‐risk, nontraditional patients were identified using a PubMed search, reviewed in detail, and concisely summarized herein.ResultsRecent data suggest that common polymorphisms in DNA repair enzymes, cell‐cycle control proteins, apoptotic pathway members, and Fanconi anemia‐associated genes likely modulate susceptibility to HNSCC development in low‐risk, nontraditional patients.ConclusionAt present, there is a lack of robust, comprehensive data on genetic drivers of oncogenesis in low‐risk patients and a clear need for further research on genetic alterations underlying the rising incidence of HNSCC in low‐risk, nontraditional patients.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143606/1/hed25057_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143606/2/hed25057.pd

Head and Neck Squamous Cell Carcinoma Detection and Surveillance: Advances of Liquid Biomarkers

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150578/1/lary27725.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150578/2/lary27725_am.pd

Defining the Role of Nuclear Hormone Receptors in Response to Radiation Therapy: Targeted Therapies for Radiosensitization in Androgen Receptor-Positive and Estrogen Receptor-Positive Breast Cancers

Breast cancer is the most common form of invasive cancer diagnosed in women, and despite advances in therapeutic strategies, approximately 10% of women with breast tumors will experience locoregional recurrence. Therefore, we have identified the presence of nuclear hormone receptors, including the androgen and estrogen receptors, as potential therapeutic targets that may be aiding in the response to ionizing radiation. While the androgen receptor (AR) and the estrogen receptor (ER) are expressed both alone and together in breast cancer, the potential roles of AR or ER alone or in tandem have not been investigated. Previous work demonstrated that AR may be a mediator of radioresistance in AR-positive, ER-negative models of breast cancer, but little is known about the underlying mechanism of radiosensitization. First, we demonstrate that in AR+ triple negative breast cancer (TNBC) models, AR inhibition with the novel AR inhibitor and CYP17 lyase inhibitor, seviteronel, or AR knockdown results in radiosensitization through a delay in dsDNA break repair following treatment with radiation therapy (RT). While seviteronel is sufficient to radiosensitize AR+ TNBC cells, seviteronel appears to have a different mechanism of radiosensitization in comparison to the second-generation anti-androgen, enzalutamide. To further investigate the mechanism of radiosensitization with AR inhibition, we transcriptomic and proteomic data to nominate the MAPK/ERK signaling pathway as a mediator of radioresistance in AR+ TNBC models. Our data demonstrates an increase in p-ERK1/2 signaling in response to AR activation using synthetic androgens. This work suggests that AR may be activating the MAPK/ERK signaling cascade to promote DNA repair following RT, and inhibition of AR may be sufficient to block this phenotype. ER-positive (ER+) breast cancers account for 67-80% of all breast cancer diagnoses, and women with breast cancer receive multimodal therapies including surgery, radiation therapy, and endocrine therapies targeting estrogens and ER signaling. We have shown that combination treatment of endocrine therapies with RT results in an increase in radiosensitivity in AR-negative (AR-)/ER+ breast cancer models. This radiosensitization is due to a decrease in non-homologous end joining efficiency resulting in a delay in dsDNA break repair with endocrine therapy treatment. Co-expression of ER and AR has been observed in 70-90% of all ER+ breast cancers, and we sought to understand whether AR and ER had independent roles when co-expressed together in AR+/ER+ breast cancers. Abrogation of AR alone in AR+/ER+ breast cancer models, using pharmacologic or genetic approaches, had no effect on radiosensitivity, while targeting the ER had a limited effect on radiosensitization in some models. To understand whether there may be a compensation mechanism between AR and ER signaling, we assessed radiosensitization in AR+/ER+ models, seeing no added benefit from combined abrogation of AR and ER compared to monotherapy alone, suggesting that the role of AR in response to RT may be dependent on co-expression of other proteins, most notably, the estrogen receptor. Collectively, our data begin to uncover the nuances of nuclear hormone receptor signaling in response to ionizing radiation in breast cancer models, suggesting that AR and ER may play independent roles when expressed alone or in tandem. Together these findings will inform clinical trial design for patients with breast cancer, increasing the translational relevance of our work.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/174326/1/annamich_1.pd