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Endocrine

Endocrine/Head & Neck Parallel Session

36: Phosphoproteomic Mass Spectrometry Reveals A Novel Therapeutic Target in Well-Differentiated Gastroenteropancreatic Neuroendocrine Tumors

Thursday, March 21, 2024
10:57am – 11:07am ET
Location: C102
Disclosure(s):
Tracey Pu, MD: No financial relationships to disclose
Amber Leila Sarvestani, MD: No financial relationships to disclose
Introduction:

Though surgical debulking is an accepted therapeutic strategy for gastroenteropancreatic neuroendocrine tumors (GEP-NETs), most patients will develop liver recurrence limiting overall survival, highlighting need for additional therapy. Given limited utility of mutational analysis to identify new drug targets, we sought to employ novel techniques to unveil tumor signaling pathways and kinases of interest.


Methods:

Phospho- and total proteomic analyses were performed on snap-frozen small bowel NET (SBNET) and pancreatic NET (PNET) liver metastases and adjacent normal liver. Identified phosphorylation sites were back-mapped onto upstream kinases using the Kinase Library, a computational motif dataset of the entire human serine/threonine kinome, and used to nominate kinases driving these tumors based on statistically significant enriched substrates in the mass-spec dataset.

Small molecule inhibitors were selected based on candidate kinase targets. Patient-derived organoids (PDOs) were generated for pharmacologic testing. PDO drug response was measured by CellTiter-Glo and fluorescent ­­­­imaging after 96-hours of drug treatment.


Results:

Liver metastases and adjacent liver parenchyma were obtained from nine SBNET patients (Grade I n=6, Grade II n=3) and four PNET patients (Grade I n=1, Grade II n=3). In total, we identified ~19,000 peptides and 2,176 independent phosphopeptides. The number and distribution of total peptides/proteins detected by mass spectrometry were similar between metastatic NETs and hepatic parenchyma. However, mean abundance of phosphopeptides in NET samples increased by over 2-fold as compared to normal hepatic parenchyma (Figure B). After computational analysis of tumor-enriched phospho-sites, several kinase signatures emerged including regulators of MAPK signaling, protein secretion, and activation of casein kinase (CK) isoforms.  We selected CX-4945 (silmitasertib), a casein-kinase 2 inhibitor, for PDO validation.

PDOs were derived from 13 unique PNET metastases from three patients and 8 unique SBNET metastases from three patients. Establishment rate was 84.6% (11/13) for PNET and 75% (6/8) for SBNET. IHC demonstrated GEP-NET marker expression in PDOs. Silmitasertib exhibited an IC­50 0.18-0.75 µM in SBNETs and IC50 0.79-4.75 µM in PNETS.


Conclusions:

Phosphoproteomic mass spectrometry reveals novel signal dysregulation in well-differentiated GEP-NET liver metastases. Our data suggests CK-2 inhibition has potential therapeutic efficacy for Grade I and Grade II SBNETs and PNETs.

Learning Objectives:

  • Understand applications of phosphoproteomic mass spectrometry in identifying signal dysregulation in cancer and discover pathways of interest.
  • Describe using patient-derived tumor organoids.as a translational tool to measure patient drug response to novel treatments.
  • Analyze identified phosphorylation sites using a computational serine/theronine kinome dataset to nominate novel kinase targets for potential pharmacologic treatment in cancer.