Regulation and Function of the PD-L1 Checkpoint

Programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) is a negative modulatory signaling pathway for activation of T cell. It is acknowledged that PD-1/PD-L1 axis plays a crucial role in the progression of tumor by altering status of immune surveillance. As one of the most promising immune therapy strategies, PD-1/PD-L1 inhibitor is a breakthrough for the therapy of some refractory tumors. However, response rate of PD-1/PD-L1 inhibitors in overall patients is unsatisfactory, which limits the application in clinical practice. Therefore, biomarkers which could effectively predict the efficacy of PD-1/PD-L1 inhibitors are crucial for patient selection. Biomarkers reflecting tumor immune microenvironment and tumor cell intrinsic features, such as PD-L1 expression, density of tumor infiltrating lymphocyte (TIL), tumor mutational burden, and mismatch-repair (MMR) deficiency, have been noticed to associate with treatment effect of anti-PD-1/anti-PD-L1 therapy. Furthermore, gut microbiota, circulating biomarkers, and patient previous history have been found as valuable predictors as well. Therefore establishing a comprehensive assessment framework involving multiple biomarkers would be meaningful to interrogate tumor immune landscape and select sensitive patients.

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Biomarkers for predicting efficacy of PD-1/PD-L1 inhibitors

Immune checkpoint blockade therapy has become a major weapon in fighting cancer. Antibody drugs, such as anti-PD-1 and anti-PD-L1, demonstrate obvious advantages such as broad applicability across cancer types and durable clinical response when treatment is effective. However, the overall response rates are still unsatisfying, especially for cancers with low mutational burden. Moreover, adverse effects, such as autoimmune symptoms and tumor hyperprogression, present a significant downside in some clinical applications. These challenges reflect the urgent need to fully understand the basic biology of immune checkpoints. In this review, we discuss regulation of immune checkpoint signaling at multiple levels to provide an overview of our current understanding of checkpoint biology. Topics include the regulation of surface expression levels for known immune checkpoint proteins via surface delivery, internalization, recycling, and degradation. Upon reaching the surface, checkpoints engage in both conventional trans and also cis interactions with ligands to induce signaling and regulate immune responses. Novel therapeutic strategies targeting these pathways in addition to classical checkpoint blockade have recently emerged and been tested in preclinical models, providing new avenues for developing next-generation immunotherapies.

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Immune checkpoint signaling and cancer immunotherapy.

Mechanisms Controlling PD-L1 Expression in Cancer

Combination Cancer Therapy with Immune Checkpoint Blockade: Mechanisms and Strategies.

Treatments that target immune checkpoints, such as the one mediated by programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been approved for treating human cancers with durable clinical benefit. However, many patients with cancer fail to respond to compounds that target the PD-1 and PD-L1 interaction, and the underlying mechanism(s) is not well understood. Recent studies revealed that response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumour cells. Hence, it is important to understand the mechanistic pathways that control PD-L1 protein expression and stability, which can offer a molecular basis to improve the clinical response rate and efficacy of PD-1-PD-L1 blockade in patients with cancer. Here we show that PD-L1 protein abundance is regulated by cyclin D-CDK4 and the cullin 3-SPOP E3 ligase via proteasome-mediated degradation. Inhibition of CDK4 and CDK6 (hereafter CDK4/6) in vivo increases PD-L1 protein levels by impeding cyclin D-CDK4-mediated phosphorylation of speckle-type POZ protein (SPOP) and thereby promoting SPOP degradation by the anaphase-promoting complex activator FZR1. Loss-of-function mutations in SPOP compromise ubiquitination-mediated PD-L1 degradation, leading to increased PD-L1 levels and reduced numbers of tumour-infiltrating lymphocytes in mouse tumours and in primary human prostate cancer specimens. Notably, combining CDK4/6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumour regression and markedly improves overall survival rates in mouse tumour models. Our study uncovers a novel molecular mechanism for regulating PD-L1 protein stability by a cell cycle kinase and reveals the potential for using combination treatment with CDK4/6 inhibitors and PD-1-PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human cancers.

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Cyclin D–CDK4 kinase destabilizes PD-L1 via cullin 3–SPOP to control cancer immune surveillance

An Amendment to this paper has been published and can be accessed via a link at the top of the paper. The original Letter has not been corrected.

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Author Correction: Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance.

// Yuyong Tan 1, 2 , Yanpeng Ci 2, 3 , Xiangpeng Dai 2 , Fei Wu 2, 4 , Jianping Guo 2 , Deliang Liu 1 , Brian J. North 2 , Jirong Huo 1 and Jinfang Zhang 2 1 Department of Gastroenterology, The Second Xiangya Hospital of Central South University, Changsha 410011, P.R. China 2 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA 3 School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, P.R. China 4 Department of Urology, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China Correspondence to: Brian J. North, email: bnorth@bidmc.harvard.edu Jirong Huo, email: hjr198@hotmail.com Jinfang Zhang, email: jzhang17@bidmc.harvard.edu Keywords: HDAC6, SPOP, Cullin 3, ubiquitination, tumorigenesis Received: March 29, 2017     Accepted: April 15, 2017     Published: May 24, 2017 ABSTRACT The histone deacetylase 6 (HDAC6) plays critical roles in human tumorigenesis and metastasis. As such, HDAC6-selective inhibitors have entered clinical trials for cancer therapy. However, the upstream regulator(s), especially ubiquitin E3 ligase(s), responsible for controlling the protein stability of HDAC6 remains largely undefined. Here, we report that Cullin 3 SPOP earmarks HDAC6 for poly-ubiquitination and degradation. We found that the proteasome inhibitor MG132, or the Cullin-based E3 ligases inhibitor MLN4924, but not the autophagosome-lysosome inhibitor bafilomycin A1, stabilized endogenous HDAC6 protein in multiple cancer cell lines. Furthermore, we demonstrated that Cullin 3-based ubiquitin E3 ligase(s) primarily reduced the stability of HDAC6. Importantly, we identified SPOP, an adaptor protein of Cullin 3 family E3 ligases, specifically interacted with HDAC6, and promoted its poly-ubiquitination and subsequent degradation in cells. Notably, cancer-derived SPOP mutants disrupted their binding with HDAC6 and thereby failed to promote HDAC6 degradation. More importantly, increased cellular proliferation and migration in SPOP -depleted HCT116 colon cancer cells could be partly reversed by additional depletion of HDAC6 , suggesting that HDAC6 is a key downstream effector for SPOP tumor suppressor function. Together, our data identify the tumor suppressor SPOP as an upstream negative regulator for HDAC6 stability, and SPOP loss-of-function mutations might lead to elevated levels of the HDAC6 oncoprotein to facilitate tumorigenesis and metastasis in various human cancers.

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Yanpeng Ci

Papers

Cyclin D–CDK4 kinase destabilizes PD-L1 via cullin 3–SPOP to control cancer immune surveillance

Author Correction: Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance.

Cullin 3SPOP ubiquitin E3 ligase promotes the poly-ubiquitination and degradation of HDAC6.