Project Summary/Abstract Non-functional pancreatic neuroendocrine tumors (NF-PanNETs) are a heterogeneous group of neoplasms with increasing incidence and ill-defined pathobiology. While many NF-PanNETs are indolent and remain stable for years, a significant subset may behave aggressively and metastasize widely. Thus, the increasing and frequent detection of NF-PanNETs presents a treatment dilemma. Current prognostic parameters and systems are susceptible to interpretation errors, sampling issues, and do not accurately reflect the clinical behavior of these neoplasms. Hence, additional biomarkers are needed to improve the prognostic stratification of patients with NF-PanNETs. Previously, we and others have reported recurrent genomic alterations in ATRX and DAXX are associated with metastatic progression of NF-PanNETs. Mutations in these genes result in loss of their respective proteins and coincide with the alternative lengthening of telomeres (ALT), a telomerase-independent maintenance mechanism. We have also shown that loss of ATRX/DAXX and ALT correlate with the development of metastases, are prognostic biomarkers for shorter disease-free survival and are independent of other prognostic clinicopathologic parameters. Thus, ATRX/DAXX and ALT represent promising biomarkers. However, they have not been evaluated prospectively nor in preoperative specimens, where prognostic stratification is important for patient management. In addition, only 50% of metastatic NF-PanNETs harbor inactivation of ATRX/DAXX and the presence of ALT. To identify additional biomarkers, we recently profiled ATRX/DAXX wild type metastatic NF-PanNETs and reported recurrent alterations in SETD2/H3K36me3 and ARID1A, which similar to ATRX and DAXX are chromatin regulating genes. We therefore hypothesize that the metastatic progression of NF-PanNETs is characterized by changes in chromatin regulation and determining the key genomic and epigenomic hallmarks will not only improve the prognostic stratification of patients with NF-PanNETs, but advance our understanding of the pathogenesis of this increasingly prevalent disease. Aim 1 will be to develop and clinically validate preoperative prognostic biomarker assays for NF-PanNETs. Through both retrospective and prospective studies, we will determine the prognostic performance and significance of ATRX, DAXX, ALT, H3K36me3 and ARID1A. For Aim 2, we plan to define the chromatin patterns at different epigenetic states in the metastatic progression of NF-PanNETs. Considering ATRX, DAXX, SETD2 and ARID1A are chromatin regulators, we will evaluate the nanoscale chromatin structure and affected molecular pathways of various NF-PanNET states using PathSTORM and CUT&RUN/RNA-seq assays. Finally, Aim 3 will investigate the adaptive response of ATRX/DAXX inactivation and ALT initiation. We have discovered that the histone chaperone, HIRA, can reconstitute telomeric chromatin and function of ATRX/DAXX deficiency ALT cancer cells. Within this aim...