The relative rarity of salivary gland adenoid cystic carcinoma (ACC) and its slow growing yet aggressive nature has complicated the development of molecular markers for patient stratification. To analyze molecular differences linked to the protracted disease course of ACC and metastases that form 5 or more years after diagnosis, detailed RNA-sequencing (RNA-seq) analysis was performed on 68 ACC tumor samples, starting with archived, formalin-fixed paraffin-embedded (FFPE) samples up to 25 years old, so that clinical outcomes were available. A statistical peak-finding approach was used to classify the tumors that expressed MYB or MYBL1, which had overlapping gene expression signatures, from a group that expressed neither oncogene and displayed a unique phenotype. Expression of MYB or MYBL1 was closely correlated to the expression of the SOX4 and EN1 genes, suggesting that they are direct targets of Myb proteins in ACC tumors. Unsupervised hierarchical clustering identified a subgroup of approximately 20% of patients with exceptionally poor overall survival (median less than 30 months) and a unique gene expression signature resembling embryonic stem cells. The results provide a strategy for stratifying ACC patients and identifying the high-risk, poor-outcome group that are candidates for personalized therapies.

Long term high altitude exposure is known to cause tissue hypoxia and exposure of fetuses as well as adults to rarefied environments increases morbidity and mortality. In particular, hypoxia can cause developmental abnormalities in the fetal pulmonary vasculature and contribute to pulmonary arterial hypertension in the adult. β2 adrenergic receptor (β‐AR) agonists are a major treatment for asthma, however far less is known about their role in pulmonary arterial function. Even still, previous evidence illustrates that β‐AR stimulation holds promise for the treatment of pulmonary arterial hypertension. Other evidence illustrates that β‐AR stimulation modulates the activity of Ca2+‐activated K+ channels (KCa), which are important mediators of pulmonary vasorelaxation. Dysregulation of KCa may contribute to chronic hypoxia induced pulmonary hypertension and β‐AR signaling may be involved in the process. In these studies we tested the hypothesis that β‐AR ‐ mediated pulmonary arterial vasodilation may be preserved following long term high altitude hypoxia due to dependency on KCa channels. This hypothesis was addressed in isolated pulmonary arteries from adult or fetal sheep that gestated at 700 meters (normoxic) or 3,801 meters for 110+ days (hypoxic). Myography was performed on pulmonary arterial rings to evaluate the vasorelaxation in response to the β‐AR agonist isoproterenol (ISO) and the methylxanthine phosphodiesterase inhibitor, IBMX. ISO‐mediated relaxation was preserved following hypoxia in fetal sheep and relaxation was greater in arteries from adult hypoxic animals relative to fetuses. Inhibition of BKCa with TEA blunted ISO‐mediated vasorelaxation in fetal normoxic and adult hypoxic arteries. IBMX caused a dose dependent relaxation in arteries from all groups and TEA did not influence this effect. Overall, these studies provide evidence that β‐AR stimulated pathways cause pulmonary arterial vasodilation, but with varying reliance on BKCa activation. Targeting β‐AR signaling pathways could therefore be therapeutically significant and functional alterations in the pathways may contribute to the development of disease.Support or Funding InformationThis material is based upon work supported by NIH HD‐069746, P01HD083132, and P20MD006988. MA and BP were Walter E Macpherson Medical Student Summer Research Fellows while AV was an Undergraduate Summer Research Fellow through the Center for Health Disparities and Molecular Mechanisms at Loma Linda University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adenoid cystic carcinoma (ACC) of the salivary gland is a rare head and neck malignancy with poor long-term prognosis. Hallmark chromosomal translocations result in MYB to NFIB (t(6;9)) and MYBL1 to NFIB (t(8;9)) gene fusions. Gene rearrangement and aberrant expression of the MYB and MYBL1 genes, along with the encoded transcription factors, are the oncogenic driver in the majority of ACC tumors. Previous RNA-sequencing (RNA-seq) data collected from FFPE fixed ACC tumors illustrated MYB and MYBL1 elicit similar gene expression profiles in ACC, suggesting these transcription factors drive ACC oncogenes via similar mechanisms. Using these RNA-seq data we have identified EN1 and SOX4 as important Myb regulated genes and demonstrated activation of their promoters in reporter gene assays. We hope to utilize these reporters in high-throughput screens to identify new drug candidates for ACC. We have utilized RNA-seq data to identify an additional mechanism leading to MYB overexpression that appears unique to ACC tumors. In these tumors transcription of the MYB gene is aberrantly activated through use of an alternative promoter located within the first intron of the gene. Transcripts originating from this promoter have been confirmed in an ACC tumor via 5'RACE and the resulting Myb protein has a small N-terminal truncation eliminating the epitope recognized by a common Myb antibody. The alternative promoter is active and regulated in a cell type specific manner in reporter gene assays. Previous studies have shown chromosomal translocations juxtapose NFIB enhancers to activate MYB gene transcription. ChIP and 3C assays in fresh frozen ACC tumors will be utilized to test the hypothesis that NFIB enhancers are specifically recruited to the alternative MYB promoter, thereby activating gene transcription. Thus, multiple mechanisms lead to the aberrant over-expression and truncation of MYB and drives ACC oncogenesis. Citation Format: Candace Frerich, Kathryn Brayer, Brandon Painter, Scott Ness. Multiple mechanisms of Myb gene activation revealed through transcriptome analysis of salivary gland adenoid cystic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1507.

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