# E3 ligase mediated control of Foxp3 expression and immune suppression-mechanisms and potential as immunotherapeutic target

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2020 · $413,515

## Abstract

Project Summary
Tumor cells exploit mechanisms of immune regulation to evade detection and eradication by host defenses.
Foxp3+CD4+CD25+ regulatory T cell (Treg)-mediated immune suppression is crucial for immune evasion by
tumor cells and an obstacle for successful tumor immunotherapy. Hence, the ability to disrupt Treg function is
of major therapeutic significance. Recent work by us and others revealed that the key Treg transcription factor,
Foxp3 is subject to polyubiquitination-dependent posttranslational regulation. Particularly, we found the E3
ubiquitin ligase Stub1, which is induced in response to a range of stress signals, facilitates the degradation of
Foxp3 providing a potential target for dynamic modulation of Treg suppression. In the current proposal, we are
seeking to: 1) Dissect molecular signaling pathways involved in Stub1 expression and its post-translational
modification; 2) Understand the consequences of physiological Stub1 induction and genetic deletion for Treg
cell homeostasis, differentiation and function; and 3) Test pharmacological activators of Foxp3 ubquitination as
novel immunotherapic strategies to undermine immune suppression in the cancer setting. These studies will
expand our understanding of the mechanisms behind posttranslational Foxp3 regulation. Specifically, we will
further explore pathways determining Stub1 activity and expression, including the previously unappreciated
phosphorylation of the ligase by the kinase GS3Kβ. To this end, we will utilize biochemical approaches and
well-characterized models of in vitro and in vivo Treg function to establish the consequences of ablating these
pathways. Furthermore, pharmacological modifiers of the Stub1/Ubiquitin-dependent pathway for Foxp3
degradation (identified in a drug screen and previous studies) will be tested for efficacy as breakers of immune
suppression - a major obstacle for anti-cancer immunotherapy. This vetting will be carried out in an aggressive
murine melanoma model (in vivo) as well as in ex vivo studies of human leukocytes obtained from healthy
donors and advanced cancer patients. In so doing we will determine the potential therapeutic application of
modulating Stub1 activity to boost anti-tumor immunity. Our experiments may reveal novel modes of regulating
Stub1 activity and Foxp3 protein downregulation. Detailed assessment of physiological Stub1 induction and its
impact on Foxp3 and Treg function is predicted to demonstrate a potent therapeutic application. Use of Stub1-
activators in combination with proven checkpoint targeting agents may yield even better anti-tumor efficacy.

## Key facts

- **NIH application ID:** 9849731
- **Project number:** 5R01AI137046-03
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** DREW M. PARDOLL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $413,515
- **Award type:** 5
- **Project period:** 2018-02-01 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9849731

## Citation

> US National Institutes of Health, RePORTER application 9849731, E3 ligase mediated control of Foxp3 expression and immune suppression-mechanisms and potential as immunotherapeutic target (5R01AI137046-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9849731. Licensed CC0.

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