Down-regulation of RCC1 sensitizes immunotherapy by up-regulating PD-L1 via p27kip1/CDK4 axis in non-small cell lung cancer

In recent years, although Immune Checkpoint Inhibitors (ICIs) significantly improves survival both in local advanced stage and advanced stage of non-small cell lung cancer (NSCLC), the objective response rate of ICI monotherapy is still only about 20%. Thus, to identify the mechanisms of ICI resistance is critical to increase the efficacy of ICI treatments. By bioinformatics analysis, we found that the expression of regulator of chromosome condensation 1 (RCC1) in lung adenocarcinoma was significantly higher than that in normal lung tissue in TCGA and Oncomine databases. The survival analysis showed that high expression RCC1 was associated with the poor prognosis of NSCLC. And the expression of RCC1 was inversely related to the number of immune cell infiltration. In vitro, knockdown of RCC1 not only significantly inhibited the proliferation of lung adenocarcinoma cells but also increased the expression levels of p27^kip1 and PD-L1, and decreased the expression level of CDK4 and p-Rb. In vivo, knockdown of RCC1 significantly slowed down the growth rate of tumour, and further reduced the volume and weight of tumour model after treated by PD-L1 monoclonal antibody. Therefore, RCC1 could up-regulate the expression level of PD-L1 by regulating p27^kip1/CDK4 pathway and decrease the resistance to ICIs. And this study might provide a new way to increase the efficacy of PD-L1 monoclonal antibody by inhibiting RCC1.     

PD-1/PD-L1免疫检查点抗体药物制剂稳定性开发

近年来抗体药物在生物医药领域发展迅速。随着抗体疗法种类的不断增加和PD-1/PD-L1靶点蛋白结构的确证,临床上,越来越多针对PD-1/PD-L1免疫检查点的单克隆抗体药物被不断开发并应用于治疗死亡率高、治愈率低的多种癌症中。但是抗体药物制剂开发水平还需进一步提高,一方面同一靶点的抗体产品同质化严重,另一方面抗体药物的理化性质比小分子药物复杂,因此需要针对不同单克隆抗体的药物特性,筛选出适应于临床应用的稳定蛋白制剂处方。概括了不同抗体药物制剂处方成分(缓冲液成分、药物辅料)的作用,结合PD-1/PD-L1靶点介绍了抗体药物制剂稳定性开发的一般策略以及CDE相关的审评要点。     

PD-1/PD-L1免疫检查点抗体药物制剂稳定性开发

近年来抗体药物在生物医药领域发展迅速。随着抗体疗法种类的不断增加和PD-1/PD-L1靶点蛋白结构的确证,临床上,越来越多针对PD-1/PD-L1免疫检查点的单克隆抗体药物被不断开发并应用于治疗死亡率高、治愈率低的多种癌症中。但是抗体药物制剂开发水平还需进一步提高,一方面同一靶点的抗体产品同质化严重,另一方面抗体药物的理化性质比小分子药物复杂,因此需要针对不同单克隆抗体的药物特性,筛选出适应于临床应用的稳定蛋白制剂处方。概括了不同抗体药物制剂处方成分(缓冲液成分、药物辅料)的作用,结合PD-1/PD-L1靶点介绍了抗体药物制剂稳定性开发的一般策略以及CDE相关的审评要点。     

PD-1/PD-L1与Treg细胞相关性的研究新进展

PD-1和PD-L属于B7家族的共刺激分子,介导免疫反应的负性调节信号。Treg细胞是一个具有免疫调节作用的T细胞亚群,在机体的免疫耐受和免疫稳定中具有重要作用。本文就PD-1/PD-L1与Treg细胞的免疫调节作用及相关性研究进展作简要综述。     

CDK4/6信号通路靶向治疗剂在癌症治疗中的研究进展

多数癌症的发生发展都具有细胞周期高度活化的特性.细胞周期蛋白依赖性激酶4/6(CDK4/6)不仅在细胞有丝分裂中发挥了巨大作用,而且参与了衰老、凋亡和组蛋白调节等诸多生物学过程,并在多种癌症的发生发展中被异常激活.FDA批准了 Palbociclib、Ribociclib和Abemaciclib等3种靶向CDK4/6的...     

Clinico-Immunological Effects of a Single-Agent CDK4/6 Inhibitor in Advanced HR+/HER2− Breast Cancer Based on a Window of Opportunity Study

Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6 i), abemaciclib, palbociclib, and ribociclib, have been FDA-approved for the treatment of hormone receptor-positive (HR+), HER2−negative (HER2−) advanced breast cancer (aBC). This targeted therapy has revived hope in those aBC patients who did not respond to standard therapies. Interestingly, when administered as a single agent, CDK4/6 modulated several peripheral blood cells after a short-course treatment of 28 days. However, the impact of these immune effects has yet to be thoroughly investigated. Methods: We administered abemaciclib, palbociclib, and ribociclib monotherapy to 23 patients with HR+/HER2− metastatic breast cancer. The aim is to investigate the impact of on-treatment modifications on peripheral blood cells and their composite scores in patients after a 28-day course of CDK4/6 i alone. Results: In the current study, we observed a significant decrease in neutrophils (p-value < 0.001) for patients treated with abemaciclib, palbociclib, and ribociclib. An overall decrease of T_regs was observed and potentially linked to palbociclib treatment. The neutrophile to lymphocyte (N/L) ratio was also decreased overall and potentially linked to abemaciclib and palbociclib treatment. Platelets were decreased in patients administered with abemaciclib. Notably, the radiometabolic response was available only for those patients treated with ribociclib and abemaciclib, and only those lesions treated with ribociclib reached statistical relevance. Conclusions: Our study strongly supports the notion that CDK4/6 inhibitors induce tumour immune modulation. N/L ratio and platelet levels decreased due to treatment. Future studies should test whether patients would benefit from immunomodulators in association with CDK4/6 agents in a larger clinical trial. Moreover, the CDK4/6-induced immune modulation could also be considered a potential predictive clinical factor in HR+/HER2− advanced breast cancer.     

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3⁺, CD4⁺ and CD8⁺ T cells in spleens and lower PD-1 expression in CD4⁺ and CD8⁺ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.     

宫颈癌组织中PD-L1的表达及临床意义分析

目的:探讨宫颈癌组织中程序性死亡因子配体(Programmed death ligand 1,PD-L1)的表达及临床意义,为宫颈癌免疫治疗提供理论和实验基础。方法:采用免疫组织化学染色的方法检测10例正常宫颈组织、71例宫颈上皮内瘤变(Cervical intraepithelial neoplasias,CIN)组织和89例宫颈癌组织中PD-L1蛋白的表达,分析PD-L1的表达与宫颈癌患者临床病理参数(临床分期、病理类型、年龄、淋巴转移)的相关性。结果:正常宫颈组织中无PD-L1表达,CIN中PD-L1表达的免疫评分低,呈弱阳性;宫颈癌中PD-L1表达的免疫评分高,呈强阳性。CIN和宫颈癌组织中PD-L1的阳性表达率分别为42%(30/41)和75%(66/89),其差异有统计学意义(P0.05)。PD-L1的表达与宫颈癌患者年龄、病理分级、淋巴转移无关(P0.05),而与患者临床分期显著相关(P=0.0021)。结论:PD-L1的表达上调可能参与了宫颈癌的发生和发展。     

不同检测策略在预测非小细胞肺癌的PD1/PD-L1抑制剂临床疗效中的应用

近10年来,程序性死亡因子1(programmed death-1,PD-1)及其配体(programmed death ligand-1,PD-L1)的抑制剂在非小细胞肺癌(non-small cell lung cancer,NSCLC)的临床治疗中取得了重大突破,有望改变晚期NSCLC的治疗方式。然而,PD-1/PD-L1抑制剂在对NSCLC的治疗中需要借助有效的生物标志物以寻找受益人群(约20%～40%)。目前,临床上主要的判断标准是PD-L1的表达水平。本文综述了近年来在NSCLC中,与预测PD-1/PD-L1抑制剂疗效的PD-L1表达相关的检测方法,包括免疫组化、基于DNA/RNA水平检测、可溶性PD-L1的检测、正电子发射断层显像(positron emission tomography,PET)技术、多重免疫组化技术、流式细胞术和液体活检技术等,着重探讨了不同检测策略在评价PD-L1表达上的最新进展及应用前景,从而推动其在NSCLC免疫治疗中的临床应用。     

Sertad1 Induces Neurological Injury after Ischemic Stroke via the CDK4/p-Rb Pathway

SERTA domain-containing protein 1 (Sertad1) is upregulated in the models of DNA damage and Alzheimer’s disease, contributing to neuronal death. However, the role and mechanism of Sertad1 in ischemic/hypoxic neurological injury remain unclear. In the present study, our results showed that the expression of Sertad1 was upregulated in a mouse middle cerebral artery occlusion and reperfusion model and in HT22 cells after oxygen-glucose deprivation/reoxygenation (OGD/R). Sertad1 knockdown significantly ameliorated ischemia-induced brain infarct volume, neurological deficits and neuronal apoptosis. In addition, it significantly ameliorated the OGD/R-induced inhibition of cell viability and apoptotic cell death in HT22 cells. Sertad1 knockdown significantly inhibited the ischemic/hypoxic-induced expression of p-Rb, B-Myb, and Bim in vivo and in vitro. However, Sertad1 overexpression significantly exacerbated the OGD/R-induced inhibition of cell viability and apoptotic cell death and p-Rb, B-Myb, and Bim expression in HT22 cells. In further studies, we demonstrated that Sertad1 directly binds to CDK4 and the CDK4 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"ON123300","term_id":"2214659616"}}ON123300 restores the effects of Sertad1 overexpression on OGD/R-induced apoptotic cell death and p-Rb, B-Myb, and Bim expression in HT22 cells. These results suggested that Sertad1 contributed to ischemic/hypoxic neurological injury by activating the CDK4/p-Rb pathway.     

Mechanisms of abemaciclib,a CDK4/6 inhibitor,induced apoptotic cell death in prostate cancer cells in vitro

The therapeutic effects of abemaciclib (ABE), an inhibitor of cyclin- dependent kinases (CDK) 4/6, on the proliferation of two types of prostate cancer (PC) cells were revealed. In this study, in vitro cytotoxic and apoptotic effects of ABE on metastatic castration-resistant prostate cancer (mCRPC) androgen receptor (AR) negative PC-3 and AR mutant LNCaP PC cells were analyzed with WST-1, Annexin V, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential, RT-PCR, western blot, and apoptosis protein array. ABE considerably inhibited the growth of PC cells in a dose-dependent manner (p<0.01) and caused significant apoptotic cell death through the suppression of CDK4/6-Cyclin D complex, ROS generation and depolarization of mitochondria membrane potential. However, PC-3 cells were more sensitive to ABE than LNCaP cells. Furthermore, the expression levels of several pro-apoptotic and cell cycle regulatory proteins were upregulated by ABE in especially PC-3 cells with the downregulation of apoptotic inhibitor proteins. Our results suggest that ABE inhibits PC cell growth and promotes apoptosis and thus ABE treatment may be a promising treatment strategy in especially mCRPC. Further preclinical and clinical studies should be performed to clarify the clinical use of ABE for the treatment of PC.     

Tumor cell-intrinsic CD274/PD-L1: A novel metabolic balancing act with clinical potential

Tumor expression of the immune co-signaling molecule CD274/PD-L1 was originally described as impeding antitumor immunity by direct engagement of its receptor, PDCD1/PD-1, on antitumor T cells. Melanoma-intrinsic PDCD1 was recently shown to promote tumor growth and MTOR signals in cooperation with tumor CD274, and sarcoma-intrinsic CD274 signaling promotes glucose metabolism to impede antitumor immunity. Our recent report shows that tumor cell-intrinsic CD274 promotes MTORC1 signaling in mouse melanoma and mouse and human ovarian cancer, inhibits autophagy and sensitizes some tumors to clinically available pharmacological autophagy inhibitors and confers resistance to MTOR inhibitors. Tumor CD274 could be a biomarker of autophagy or MTOR inhibitor response in selected tumors, and these inhibitors could improve anti-CD274 or anti-PDCD1 cancer immunotherapy. As we found that distinct tumor types exhibit this CD274-driven phenotype, it could be widely applicable.     

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.     

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.     

The G1 phase optical reporter serves as a sensor of CDK4/6 inhibition in vivo

Visualization of cell-cycle G1 phase for monitoring the early response of cell cycle specific drug remains challenging. In this study, we developed genetically engineered bioluminescent reporters by fusing full-length cyclin E to the C-terminal luciferase (named as CycE-Luc and CycE-Luc2). Next, HeLa cell line or an ER-positive breast cancer cell line MCF-7 was transfected with these reporters. In cellular assays, the bioluminescent signal of CycE-Luc and CycE-Luc2 was accumulated in the G1 phase and decreased after exiting from the G1 phase. The expression of CycE-Luc and CycE-Luc2 fusion protein was regulated in a cell cycle-dependent manner, which was mediated by proteasome ubiquitination and degradation. Next, our in vitro and in vivo experiment confirmed that the cell cycle arrested by anti-cancer agents (palbociclib or 5-FU) was monitored quantitatively and dynamically by bioluminescent imaging of these reporters in a real-time and non-invasive manner. Thus, these optical reporters could reflect the G1 phase alternation of cell cycle, and might become a future clinically translatable approach for predicting and monitoring response to palbociclib in patients with ER-positive breast cancer.     

Baseline monocyte and its classical subtype may predict efficacy of PD-1/PD-L1 inhibitor in cancers

Background: Programmed death 1 (PD-1)/ programmed death-ligand 1 (PD-L1) inhibitor is one of the most popular immune therapies. Biomarkers for predicting response are highly needed, but no biomarkers are widely used till now.Patients and methods: From February 2018 to April 2019, pan-cancer patients treated with PD-1 or PD-L1 inhibitor as a single agent in our center were included. The benefit group included patients with partial response, complete response and stable disease, while the patients with progressive disease were classified into the nonbenefit group, according to the RECIST 1.1 criteria. Baseline peripheral blood was sampled to determine absolute monocyte count (AMC) and/or classical monocyte frequency (CMF) of peripheral blood mononuclear cells. Then, the association of the above-mentioned two biomarkers with response or progression-free survival (PFS) was evaluated.Results: In total, 107 patients enrolled in the present study. The nonbenefit group had significantly larger number of AMC than benefit group (P<0.001), and patients with higher AMC had decreased PFS time (P=0.001). Of 39 patients tested for CMF, the nonbenefit group had significantly higher CMF than benefit group (P=0.002), and patients with higher CMF had significantly decreased PFS time (P=0.002). The sensitivity of AMC and CMF was 87.9% and 85.7%, respectively, and the specificity was 44.9% and 61.1%, respectively. Multivariate analysis showed high baseline CMF and AMC were both significantly associated with decreased PFS time.Conclusion: Baseline CMF and baseline AMC can be potential pan-cancer biomarkers to predict efficacy of PD-1/PD-L1 inhibitors, especially in the PD-L1 subgroup.     

PD-1/PD-L1抑制剂对肺鳞状细胞癌患者调节性T细胞及其亚型的影响

摘要 目的：探讨PD-1/PD-L1抑制剂对肺鳞状细胞癌（鳞癌）患者调节性T细胞（Regulatory T cell，Treg细胞）及其亚型的影响。方法：2019年1月至2020年12月来我院就诊的肺鳞癌初诊患者纳入本项研究；患者均接受Nivolumab治疗，根据治疗效果不同，将患者分为缓解组（n=20）和无效组（n=20）；流式细胞术检测外周血总Treg细胞、PD-1+Treg细胞、CD150+Treg细胞、CTLA-4+Treg细胞、LAG-3+Treg细胞的比例。结果：缓解组外周血总Treg细胞、CTLA-4+Treg细胞和LAG-3+Treg细胞的比例分别为4.052±0.231%、1.616±0.099%和0.328±0.021%，无效组的比例分别为5.532±0.395%、3.277±0.224%和0.857±0.108%，差异均具有显著的统计学意义（t=3.239，P=0.009；t=6.776，P＜0.001；t=4.807，P＜0.001）；缓解组外周血PD-1+Treg细胞和CD150+Treg细胞的比例分别为0.195±0.016%和1.530±0.113%，无效组的比例分别为0.203±0.018%和1.787±0.157%，差异均不具有统计学意义（t=0.318，P=0.757；t=1.329，P=0.214）。结论：PD-1/PD-L1抑制剂治疗无效的肺鳞癌患者外周血总Treg细胞、CTLA-4+Treg细胞和LAG-3+Treg细胞的比例均显著升高。