Upper GI
CME
Hyunjee V. Kwak, MD
Postdoctoral Research Fellow
University of Pennsylvania, United States
Disclosure(s): No financial relationships to disclose
Hyunjee V. Kwak, MD
Postdoctoral Research Fellow
University of Pennsylvania, United States
Disclosure(s): No financial relationships to disclose
Katherine J. Tardy, MD
Post-Doctoral Research Fellow
University of Pennsylvania, United States
Disclosure(s): No financial relationships to disclose
Laura Wang, BS
Laboratory Technician
University of Pennsylvania, United States
Disclosure information not submitted.
Kevin Do, BS
Laboratory Technician
University of Pennsylvania, United States
Disclosure information not submitted.
Shan Zeng, PhD
Senior Scientist
University of Pennsylvania, United States
Disclosure information not submitted.
Ferdinando Rossi, PhD
Senior Scientist
University of Pennsylvania, United States
Disclosure information not submitted.
Ronald P. DeMatteo, MD, FACS (he/him/his)
Chair, Department of Surgery
The Hospital of the University of Pennsylvania
Philadelphia, Pennsylvania, United States
Disclosure information not submitted.
GIST is the most common human sarcoma, and CD40 is a type of tumor necrosis factor (TNF) receptor expressed on antigen-presenting cells in the tumor microenvironment. Previously, we showed that CD40 activation enhanced the effect of imatinib in GIST mice via immune cell activation. Here, we sought to determine whether CD40 agonism has direct effects on tumor cells.
Methods:
The T1 human GIST cell line was analyzed for CD40 expression by flow cytometry. Cells were then treated with imatinib, an agonistic CD40 monoclonal antibody, both, or isotype control. After 24 hours, western blot and RT-PCR were performed. Cell viability assays were performed at 72 hours after treatment.
Results:
Flow cytometry revealed high expression of CD40 on T1 cells (91.5%). CD40 agonism alone decreased the viability of T1 cells by 25% (n=5, SEM=0.2, p=0.01), and when combined with imatinib, decreased the viability by 75% (n=5, SEM=0.02, p< 0.0001). On western blot analysis, anti-CD40 treatment alone decreased Kit signaling in T1 cells evidenced by decreases in the phosphorylated forms of Kit (p-Kit), AKT serine/threonine kinase (p-AKT), ribosomal protein S6 (p-S6), extracellular signal-regulated kinase (p-ERK), and the transcription factor STAT3 (p-STAT3). These decreased signals were even more pronounced with combination therapy. Anti-CD40 increased the phosphorylated transcription factor nuclear factor kappa B (p-NFkB p65), one of the many downstream targets of CD40, and the immunosuppressive molecule, indoleamine 2,3-dioxygenase (Ido). RT-PCR of T1 cells treated with anti-CD40 alone showed an increase in Ido RNA expression (p=0.0001). This direct compensatory anti-inflammatory response was supported by increased RNA expression of transforming growth factor beta (TGF-b) and its activating target, STAT3.
Conclusions:
As anti-CD40 is known to work mostly on immune cells in other tumor models, our findings highlight direct tumor effects of CD40 agonism in GIST including decreasing cell viability, decreasing Kit signaling, and stimulating tumor cell defense anti-inflammatory responses.