CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy

Sep 23, 2026

Uploaded by Patrick

Macrophages eliminate cancer cells through CD47/SIRPα-mediated phagocytosis, positioning the CD47 axis as an innate immune checkpoint for immunotherapy. Despite frequent CD47 overexpression, how CD47 protein stability is regulated via protein stability control remains unclear. Using USP2/SPOP mechanistic assays and multiple human cancer cell lines, the study tests USP2 inhibition across mouse tumor models and evaluates anti–PD-1 efficacy, immune phenotypes, and survival.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages17

Reading Time43 min

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Abstract

Background

Experimental design

Results

Conclusion

Introduction

Adaptive immune checkpoint therapies

Innate immune checkpoints and the CD47/SIRPα axis

What is already known on this topic

What this study adds

How this study might affect research, practice or policy

Open access Original research CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy Peiqiang Yan,1 Xia Bu,2 Tao Hou,1 Li Chen,1 Guoxuan Zhong,3 Daoyuan Huang,1 Jingchao Wang,1 Yihang Qi,1 Weiwei Jiang,1 Zhe Li,3 Xutong Xue,4 Yang Gao,5 Jing Liu,5 Hiroyuki Inuzuka,1 Gordon J Freeman ,2 Wenyi Wei ,1 Xiaoming Dai1,3,6 To cite: Yan P, Bu X, Hou T, et al. CD47 destabilization via manipulating the SPOP-USP2 axis augments macrophage phagocytosis and cancer immunotherapy. Journal for ImmunoTherapy of Cancer 2026;14:e013498 . doi:10 . 1136/ jitc-2025-013498 ► Additional supplemental material is published online only. To view, please visit the journal online ([https://doi.org/10.1136/](https://doi.org/10.1136/)[ ](https://doi.org/10.1136/)[jitc-2025-013498](jitc-2025-013498)) . PY, XB and TH contributed equally. Accepted 07 January 2026 © Author(s) (or their employer(s)) 2026. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group. For numbered affiliations see end of article. Correspondence to Dr Wenyi Wei; [wwei2@bidmc.harvard.edu](wwei2@bidmc.harvard.edu) Dr Xiaoming Dai; [xdai@nju.edu.cn](xdai@nju.edu.cn) Dr Gordon J Freeman; gordon_freeman@dfci. harvard. edu ABSTRACT Background Macrophages can eliminate cancer cells through phagocytosis via the CD47/signal regulatory protein α axis, which provides promising targets for cancer immunotherapy as innate immune checkpoints. Although CD47 is overexpressed in multiple cancer types, it remains largely unknown whether and how CD47 can be targeted by manipulating its protein stability. Experimental design Multiple human cancer cell lines were used to identify the function of the ubiquitinspecific protease 2 (USP2) /speckle-type POZ protein (SPOP) axis and the USP2 inhibitor on CD47 protein stability by immunoblot and immunoprecipitation, real-time quantitative PCR, in vitro deubiquitination assay, cell fractionation assay, flow cytometry, and phagocytosis assay. We investigated the antitumor immune response and immunotherapy effects of the USP2 inhibitor using multiple syngeneic and orthotopic mouse tumor models, bioluminescence imaging, immune cell depletion, tumor-infiltrating lymphocyte (TIL) isolation, and flow cytometry. Results Here, we report that ML364, an inhibitor of the USP2 deubiquitinase, reduces the protein abundance of CD47 . Mechanistically, USP2 deubiquitinates and protects CD47 from proteasome-mediated degradation. Furthermore, we reveal that USP2 itself can be ubiquitinated by the SPOP ubiquitin E3 ligase, which leads to USP2 degradation and decreased CD47 protein abundance. Functionally, ML364 promotes macrophage phagocytosis of cancer cells by reducing the expression of CD47 and enhances the efficacy of anti-programmed cell death protein-1 (PD-1) immunotherapy, thereby inhibiting tumor growth and improving the overall survival rate in multiple syngeneic and orthotopic mouse tumor models. Bioinformatic analyses indicate that low USP2 expression or high SPOP expression predicts a better response to anti-PD-1 treatment. Conclusion Hence, our findings reveal a pivotal role of the SPOP/USP2 axis in regulating CD47 protein stability and advocate for combining USP2 inhibitors with anti-PD-1 immunotherapy to combat cancer. WHAT IS ALREADY KNOWN ON THIS TOPIC | ⇒ Although CD47 plays an important role in the immune evasion of cancer cells, the exact molecular mechanisms by which CD47 protein stability is regulated remain largely unknown.
WHAT THIS STUDY ADDS | | --- | | ⇒ Here, we report that ubiquitin-specific protease 2 (USP2) deubiquitinates and protects CD47 from degradation. USP2 itself can be ubiquitinated by the speckle-type POZ protein (SPOP) E3 ligase, leading to USP2 degradation and decreased CD47 expression. The USP2 inhibitor, ML364, promotes macrophage phagocytosis of cancer cells, enhances the efficacy of a

FAQs

What is the main role of CD47 in cancer immunotherapy according to the document?

CD47 helps cancer cells evade macrophage-mediated phagocytosis by engaging the CD47/SIRPα (don’t eat me) signaling axis. This makes CD47 a promising innate immune checkpoint target.

How does the USP2/SPOP axis influence CD47 protein stability?

USP2 deubiquitinates and protects CD47 from proteasome-mediated degradation. SPOP-mediated ubiquitination can also target USP2 for degradation, decreasing CD47 protein abundance.

What is the effect of the USP2 inhibitor ML364 on tumor immunity?

ML364 reduces CD47 protein abundance, enhances macrophage phagocytosis of cancer cells, and improves the efficacy of anti–PD-1 immunotherapy, leading to suppressed tumor growth and better survival in multiple mouse tumor models.

Sep 23, 2026

Uploaded by Patrick