Identification of a FoxP3-specific small molecule degrader with potent antitumor immunotherapeutic efficacy 2309510
T regulatory cell (Treg) infiltration and accumulation within solid tumors leads to tumor immune evasion and is a major barrier to immunotherapy efficacy. Thus, immunotherapeutic strategies aimed at reducing Treg abundance and/or suppressive function hold significant promise for cancer treatment. Efforts to modulate Tregs have largely relied on non-specific molecular or cellular approaches; however, targeting Forkhead box protein 3 (FoxP3), the master transcription factor for Tregs holds significant promise. Here, we report the first FoxP3-specific small molecule degrader which promotes proteasomal degradation of FoxP3 and partially reduces Treg suppressive function. To identify potential hits, we used high content imaging to screen a 640 small, electrophilic compound library. The top hits were resynthesized and validated by flow cytometry in human Treg-like MT-2 cells, leading to identification of our lead compound, termed FD03. To explore the mechanism by which FD03 mediated FoxP3 reduction, we employed co-immunoprecipitation and immunoblotting techniques. We then evaluated therapeutic efficacy of our compound in ex vivo and in vivo settings using flow cytometry as a readout. After identifying FD03 as the lead compound, we further characterized its efficacy finding that FD03 had a EC50 of ∼5-10 uM in primary murine and human Tregs. Mechanistically, FD03 degrades FoxP3 in part through facilitating FoxP3 interaction with its E3 ligase, STUB1, to promote FoxP3 ubiquitination-mediated proteasomal degradation. Finally, we characterized the therapeutic potential of FD03: we illustrated that FD03 can reduce Treg suppressive function. Importantly, we demonstrated that FD03 treatment in tumor-bearing mice can decrease tumor burden and skew the immune cell landscape towards an inflammatory phenotype. Overall, we demonstrated proof-of-concept and feasibility of targeting FoxP3, which has been known to be “undruggable”, in a chemical manner by small molecules. 1F31CA287701-01, T32GM15538-10, 1T32GM149439-01 Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)