The Mouse Mammary Tumor Virus (MMTV) contains sequences in its proximal promoter region to which both glucocorticoid and progesterone receptors can bind. In transient transfection experiments both hormones are able to stimulate transcription from reporter plasmids containing either native or consensus hormone response elements (glucocorticoid response element/progesterone response element). Previous experiments have demonstrated that the MMTV long terminal repeat is reproducibly assembled into a phased array of nucleosomes when stably introduced into cells. Stimulation by glucocorticoids of endogenous templates led to a rapid but transient increase in transcription initiation and mRNA accumulation that can be correlated with increased sensitivity to restriction enzymes. In contrast, experiments using progesterone or a truncated glucocorticoid receptor failed to elicit a similar increase in mRNA levels as dexamethasone from stable chromatin templates. In an attempt to understand this differential response, we have compared the responsiveness of the MMTV promoter to glucocorticoids and progesterone when it is organized in either stable chromatin or in transiently acquired plasmids. Our results demonstrate that the native chromatin structure prevents activation of this locus by progesterone, but permits stimulation by glucocorticoids.

Sterol acquisition by soilborne plant pathogens of the genus Phytophthora is presumed to involve extracellular proteins belonging to class-I elicitins. However, little is known about the relationship between sterol availability and elicitin secretion. The objective of this study was to determine the expression of class-I elicitin genes in Phytophthora sojae when grown in a medium containing stigmasterol or cholesterol. P. sojae growth was stimulated by nanomolar concentrations of stigmasterol and cholesterol, which also resulted in the down-regulation of its elicitin genes over time when expression profiles were monitored using real time Reverse Transcription Polymerase Chain Reaction (RT-PCR). The down-regulation of elicitin genes in response to the two sterols also coincided with a reduction in the amount of elicitins detected in spent filtrates. Our study is the first to show the influence of sterols on elicitin gene expression in Phytophthora, which is important with respect to the ecology of elicitin secretion as sterol carrier proteins in the environment.

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Background: Recurrent/metastatic head and neck squamous cell carcinoma (RMHNSCC) is associated with poor quality of life and a poor prognosis with a median overall survival of 7 months. With limited systemic therapy options, there is an urgent need to identify predictive biomarkers for drug response. Patient derived xenografts (PDXs) have been demonstrated to preserve the histological features, heterogeneity, epigenetic and genetic profiles of the original tumor and appear to correlate well with objective tumor response in patients. This study aimed to test the feasibility of personalized PDXs for guiding systemic treatment in RMHNSCC. Methods: Eligible patients were consented and a fresh biopsy/surgical sample was obtained and implanted into mice (5-15 mice/patient) to establish TumorGraft® models. Engrafted tumors were excised and propagated into second generation models for drug testing with up to 4 drugs selected by the treating medical oncologist. Tumor dimensions were measured twice weekly and were reported as one of: progressive disease, stable disease (SD), partial response (PR), or complete response (CR) based on the percentage of tumor regression. Patients alive and suitable for chemotherapy were prescribed the regimen(s) observed to have the greatest response rate in their TumorGraft® models. Patients' responses to therapy were then observed. Results: Nine of 10 eligible patients had samples successfully engrafted with an average time to engraftment of 89.2 days (± SD 41.7 days). Drug testing was not performed on 5 patients as the patient either died or was not suitable for treatment. The remaining 4 patient TumorGraft® models underwent drug testing with the average time from engraftment completion to drug testing completion being 83.8 ± 59.9 days. Two of these patients then received xenograft-guided therapy. In one patient, paclitaxel demonstrated a partial response in the Tumorgraft®, however the patient's tumor did not respond and their clinical status rapidly deteriorated leading to death. In the second patient, cetuximab and paclitaxel demonstrated the best response in the TumorGraft® model. This patient had a sequential partial response to each drug including a 17 month response to cetuximab before progressing and transitioning to nivolumab. The patient remains alive with stable disease 3.5 years after diagnosis of recurrent disease. Conclusions: The main limitation of Tumorgraft® testing for this population is the time delay to obtain Tumorgraft® results. Despite this, Tumorgraft® testing is feasible for a subset of patients and appears to correlate with clinical benefit. Citation Format: Morgan D. Black, Allison Berger, Nicole Pinto, John Yoo, Kevin Fung, Danielle MacNeil, David A. Palma, Joseph S. Mymryk, Sara Kuruvilla, John W. Barrett, Suzanne Richter, Angela Davies, Eric W. Winquist, Anthony C. Nichols. Feasibility of real-time personalized patient-derived Tumorgraft® models for guiding systemic treatment in recurrent and/or metastatic head and neck cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1112.

Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with an incidence of ~600 000 new cases per year and a 50% mortality rate for advanced disease. The mutational landscape of HNSCC has been recently elucidated, introducing the possibility for targeted therapeutic approaches. PIK3CA—which encodes the α-catalytic subunit of PI3K (PI3Kα)—is the most frequently altered actionable target in HNSCC, however it is not presently clear which patients benefit most from PI3Kα-inhibition. BYL719 is a leading PI3Kα inhibitor in clinical development for HNSCC. We previously examined the responses of a large panel of genetically-characterized HNSCC cell lines to BYL719 and identified activating HRAS G12V mutations as strong predictors for BYL719 resistance. Here we examine if this mutation is able to individually modulate BYL719 response through overexpression and knockdown studies. Methods: To determine if HRAS G12V was able to modulate BYL719 sensitivity, we knocked down HRAS in HRAS G12V and HRAS wild-type (WT) cell lines using RNA interference, and then treated cells over 10-point dose ranges with BYL719. Sensitivity was determined by calculating IC50 (half-maximal inhibitory concentration) values at 72 hours using PrestoBlue®. Constructs expressing wildtype (WT) HRAS or HRAS G12V were transfected into BYL719-sensitive cells for overexpression studies and cells were selected using G418 Sulfate for 1 month. Proliferation and BYL719 sensitivity (IC50 values) were measured as described. Immunoblotting was used to examine activity of the PI3K-AKT axis with and without BYL719 (1μM) for 24 hours. Results & Conclusions: HRAS knockdown sensitized HRAS G12V lines to BYL719 (lower IC50), but did not affect the response of WT HRAS cells. WT HRAS and HRAS G12V overexpression significantly increased cellular proliferation and promoted BYL719 resistance. In HRAS G12V cell lines, we observed high levels of active phosphorylated AKT (S473/T308), even in the presence of BYL719. Collectively these findings highlight the predictive role of HRAS G12V for innate BYL719 resistance and contribute to our understanding of which patients may respond best on BYL719 therapy. Citation Format: Kara M. Ruicci, Ren Sun, Morgan Black, Nicole Pinto, John Yoo, Kevin Fung, Danielle Macneil, Lauire Aillies, Joseph S. Mymryk, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. HRAS G12V predicts for innate resistance to PI3Kα inhibition in head and neck squamous cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2017-3162