Effect of Stereotactic Body Radiation Therapy on Diverse Organ Lesions in Advanced Non-Small Cell Lung Cancer Patients Receiving Immune Checkpoint Inhibitors.

OBJECTIVE: The combination of stereotactic body radiation therapy (SBRT) and immune checkpoint inhibitors (ICIs) is actively being explored in advanced non-small-cell lung cancer (NSCLC) patients. However, little is known about the optimal fractionation and radiotherapy target lesions in this scenario. This study investigated the effect of SBRT on diverse organ lesions and radiotherapy dose fractionation regimens on the prognosis of advanced NSCLC patients receiving ICIs. METHODS: The medical records of advanced NSCLC patients consecutively treated with ICIs and SBRT were retrospectively reviewed at our institution from Dec. 2015 to Sep. 2021. Patients were grouped according to radiation sites. Progression-free survival (PFS) and overall survival (OS) were recorded using the Kaplan-Meier method and compared between different treatment groups using the log-rank (Mantel-Cox) test. RESULTS: A total of 124 advanced NSCLC patients receiving ICIs combined with SBRT were identified in this study. Radiation sites included lung lesions (lung group, n=43), bone metastases (bone group, n=24), and brain metastases (brain group, n=57). Compared with the brain group, the mean PFS (mPFS) in the lung group was significantly prolonged by 13.3 months (8.5 months vs. 21.8 months, HR=0.51, 95%CI: 0.28-0.92, P=0.0195), and that in the bone group prolonged by 9.5 months with a 43% reduction in the risk of disease progression (8.5 months vs. 18.0 months, HR=0.57, 95%CI: 0.29-1.13, P=0.1095). The mPFS in the lung group was prolonged by 3.8 months as compared with that in the bone group. The mean OS (mOS) in the lung and bone groups was longer than that of the brain group, and the risk of death decreased by up to 60% in the lung and bone groups as compared with that of the brain group. When SBRT was concurrently given with ICIs, the mPFS in the lung and brain groups were significantly longer than that of the bone group (29.6 months vs. 16.5 months vs. 12.1 months). When SBRT with 8-12 Gy per fraction was combined with ICIs, the mPFS in the lung group was significantly prolonged as compared with that of the bone and brain groups (25.4 months vs. 15.2 months vs. 12.0 months). Among patients receiving SBRT on lung lesions and brain metastases, the mPFS in the concurrent group was longer than that of the SBRT→ICIs group (29.6 months vs. 11.4 months, P=0.0003 and 12.1 months vs. 8.9 months, P=0.2559). Among patients receiving SBRT with <8 Gy and 8-12 Gy per fraction, the mPFS in the concurrent group was also longer than that of the SBRT→ICIs group (20.1 months vs. 5.3 months, P=0.0033 and 24.0 months vs. 13.4 months, P=0.1311). The disease control rates of the lung, bone, and brain groups were 90.7%, 83.3%, and 70.1%, respectively. CONCLUSION: The study demonstrated that the addition of SBRT on lung lesions versus bone and brain metastases to ICIs improved the prognosis in advanced NSCLC patients. This improvement was related to the sequence of radiotherapy combined with ICIs and the radiotherapy fractionation regimens. Dose fractionation regimens of 8-12 Gy per fraction and lung lesions as radiotherapy targets might be the appropriate choice for advanced NSCLC patients receiving ICIs combined with SBRT.

Targeting polymerase θ impairs tumorigenesis and enhances radiosensitivity in lung adenocarcinoma.

Radioresistance remains a major obstacle to efficacious radiotherapy in non-small-cell lung cancer (NSCLC). DNA replication proteins are novel targets for radiosensitizers. POLQ is a DNA polymerase involved in DNA damage response and repair. We found that POLQ is overexpressed in NSCLC and is clinically correlated with high tumor stage, poor prognosis, increased tumor mutational burden, and ALK and TP5 mutation status; POLQ inhibition impaired lung tumorigenesis. Notably, POLQ expression was higher in radioresistant lung cancer cells than in wild-type cancer cells. Moreover, POLQ expression was further increased in radioresistant cells after radiation. Enhanced radioresistance is through a prolonged G2/M phase and faster repair of DNA damage, leading to reduced radiation-induced apoptosis. Novobiocin (NVB), a POLQ inhibitor, specifically targeted cancer cells. Genetic knockdown of POLQ or pharmacological inhibition by NVB decreased radioresistance in lung adenocarcinoma while causing little toxicity to normal pulmonary epithelial cells. In conclusion, POLQ is a promising and practical cancer-specific target to impair tumorigenesis and enhance radiosensitivity in NSCLC.

Activation of NLRP3 inflammasome in lung epithelial cells triggers radiation-induced lung injury.

BACKGROUND: Radiation-induced lung injury (RILI) is the most common and serious complication of chest radiotherapy. However, reported radioprotective agents usually lead to radiation resistance in tumor cells. The key to solving this problem is to distinguish between the response of tumor cells and normal lung epithelial cells to radiation damage. METHODS: RNA-Seq was used to recognize potential target of alleviating the progression of RILI as well as inhibiting tumor growth. The activation of NLRP3 inflammasome in lung epithelial cells was screened by qRT-PCR, western blotting, immunofluorescence, and ELISA. An in vivo model of RILI and in vitro conditioned culture model were constructed to evaluate the effect of NLRP3/interleukin-1ß on fibroblasts activation. ROS, ATP, and (NADP)+/NADP(H) level in lung epithelial cells was detected to explore the mechanism of NLRP3 inflammasome activation. The lung macrophages of the mice were deleted to evaluate the role of lung epithelial cells in RILI. Moreover, primary cells were extracted to validate the results obtained from cell lines. RESULTS: NLRP3 activation in epithelial cells after radiation depends on glycolysis-related reactive oxygen species accumulation. DPYSL4 is activated and acts as a negative regulator of this process. The NLRP3 inflammasome triggers interleukin-1ß secretion, which directly affects fibroblast activation, proliferation, and migration, eventually leading to lung fibrosis. CONCLUSIONS: Our study suggests that NLRP3 inflammasome activation in lung epithelial cells is essential for radiation-induced lung injury. These data strongly indicate that targeting NLRP3 may be effective in reducing radiation-induced lung injury in clinical settings.

PD-1+ mast cell enhanced by PD-1 blocking therapy associated with resistance to immunotherapy.

BACKGROUND: Programmed cell death protein 1 (PD-1) antibody has been approved for a variety of tumors, but its effective rate is unsatisfactory. New evidence suggests that mast cells are an important component of the tumor microenvironment and are associated with resistance to immunotherapy, but the underlying mechanism is not clear. METHODS: Bioinformatics analysis of patients with melanoma in TCGA-SKCM and GSE91061 was used to determine the prognostic value of mast cells and their association with anti-PD-1 immunotherapy. HMC-1 cells (mast cell line) and bone marrow-derived mast cells (BMMCs) were used to verify the effect of PD-1 antibody and cromolyn sodium in vitro. The mouse subcutaneous melanoma model was used to verify the effect of the PD-1 antibody on mast cells in vivo. RESULTS: Bioinformatics analysis showed that mast cells were a poor prognostic factor associated with resistance to anti-PD-1 immunotherapy. PD-1 was expressed on the mast cell membrane. The PD-1 antibody promoted the release of histamine and cytokines from mast cells via the PI3K/AKT pathway and calcium signaling pathway. The activation of mast cells induced by PD-1 antibody could be partially inhibited by cromolyn sodium. In vivo, cromolyn sodium increased the efficacy of PD-1 antibody and decreased the infiltration of mast cells and the density of microvessels. CONCLUSION: PD-1+ mast cell activated by PD-1 antibody plays a negative role in the tumor microenvironment via the enhanced function of releasing histamine and cytokines. Inhibition of mast cell may provide a new solution to solve the low response rate of anti-PD-1 immunotherapy.

NLRP6 is required for cancer-derived exosome-modified macrophage M2 polarization and promotes metastasis in small cell lung cancer.

Metastasis remains the primary cause of small cell lung cancer (SCLC)-related deaths. Growing evidence links tumor metastasis with a pre-metastatic microenvironment characterized by an anti-inflammatory response, immunosuppression, and the presence of tumor-derived exosomes. To clarify the relationships among these factors in SCLC, we analyzed SCLC patient samples as well as a mouse model. Among the infiltrating immune cells, our study focused on the tumor-associated macrophages (TAMs), that are well-known to promote tumor progression and metastasis. We found that high expression of the alternatively activated (M2) TAM marker, CD206+ was associated clinically with a poorer prognosis and metastasis state in patients with SCLC. Moreover, infiltrating macrophages (MØ) were found in the metastatic foci of an SCLC mouse model. Additionally, we observed dominant switching to M2 phenotype, accompanied by increased NLRP6 expression. Since tumor-derived exosomes are the key links between the tumor and its immune microenvironment, we further investigated whether SCLC-derived exosomes contributed to the MØ phenotype switch. Our findings showed for the first time that SCLC-derived exosomes induce the M2 switch via the NLRP6/NF-κB pathway, and thus, promote SCLC metastasis in vitro and in vivo. Collectively, these results indicate a novel mechanism by which SCLC-derived exosomes induce immunosuppression of distant MØ to promote systemic metastasis by activating NLRP6. Here, we highlight the close relationship between the tumor-derived exosomes, inflammasomes and immune microenvironment in SCLC metastasis.

Activated amino acid response pathway generates apatinib resistance by reprograming glutamine metabolism in non-small-cell lung cancer.

The efficacy of apatinib has been confirmed in the treatment of solid tumors, including non-small-cell lung cancer (NSCLC). However, the direct functional mechanisms of tumor lethality mediated by apatinib and the precise mechanisms of drug resistance are largely unknown. In this study, we demonstrated that apatinib could reprogram glutamine metabolism in human NSCLC via a mechanism involved in amino acid metabolic imbalances. Apatinib repressed the expression of GLS1, the initial and rate-limiting enzyme of glutamine catabolism. However, the broken metabolic balance led to the activation of the amino acid response (AAR) pathway, known as the GCN2/eIF2α/ATF4 pathway. Moreover, activation of ATF4 was responsible for the induction of SLC1A5 and ASNS, which promoted the consumption and metabolization of glutamine. Interestingly, the combination of apatinib and ATF4 silencing abolished glutamine metabolism in NSCLC cells. Moreover, knockdown of ATF4 enhanced the antitumor effect of apatinib both in vitro and in vivo. In summary, this study showed that apatinib could reprogram glutamine metabolism through the activation of the AAR pathway in human NSCLC cells and indicated that targeting ATF4 is a potential therapeutic strategy for relieving apatinib resistance.

Targeting KDM4C enhances CD8+ T cell mediated antitumor immunity by activating chemokine CXCL10 transcription in lung cancer.

BACKGROUND: Although immune checkpoint blockade (ICB) has been proven to achieve a persistent therapeutic response in various tumor types, only 20%-40% of patients benefit from this treatment. Radiotherapy (RT) can enhance tumor immunogenicity and improve the ICB response, but the outcome achieved by combining these two modalities remains clinically unsatisfactory. We previously uncovered that lysine-specific demethylase 4C (KDM4C) is a regulator of radiosensitivity in lung cancer. However, the role of KDM4C in antitumor immunity has not yet been investigated. METHODS: Infiltrating immune cells in our mouse tumor model were screened by flow cytometry. An in vivo subcutaneous transplanted tumor model and in vitro conditioned culture model were constructed to detect the quantitative and functional changes in CD8+ T cells. RNA sequencing and chromatin immunoprecipitation-PCR assays were used to explore the downstream regulatory mechanism of KDM4C in antitumor immunity. A C57BL/6 mouse tumor model was developed to evaluate the efficacy and safety of a triple therapy (the KDM4C-specific inhibitor SD70 plus RT and an anti-PD-L1 antibody) in lung cancer in vivo. RESULTS: Genetical or pharmacological inhibition of KDM4C specifically increased CD8+ T cell infiltration; promoted the proliferation, migration and activation of CD8+ T cells; and alleviated CD8+ T cell exhaustion in mouse tumor tissues. Mechanistically, KDM4C inhibition increased the binding of H3K36me3 to the CXCL10 promoter region, thus inducing CXCL10 transcription and enhancing the CD8+ T cell mediated antitumor immune response. More importantly, among the tested regimens, the triple therapy achieved the best therapeutic efficacy with tolerable toxicity in lung cancer. CONCLUSIONS: Our data reveal a crucial role for KDM4C in antitumor immunity in lung cancer and indicate that targeting KDM4C in combination with radioimmunotherapy might be a promising synergistic strategy in lung cancer.

REV1 promotes lung tumorigenesis by activating the Rad18/SERTAD2 axis.

REV1 is the central member of the family of TLS polymerases, which participate in various DNA damage repair and tolerance pathways and play a significant role in maintaining genomic stability. However, the role of REV1 in tumors is rarely reported. In this study, we found that the expression of REV1 was significantly upregulated in lung cancer tissues compared with matched adjacent tissues and was associated with poor prognosis. Functional experiments demonstrated that REV1 silencing decreased the growth and proliferation capacity of lung cancer cells. Mechanistically, REV1 upregulated the expression of SERTAD2 in a Rad18-dependent manner, thereby promoting lung carcinogenesis. A novel REV1 inhibitor, JH-RE-06, suppressed lung tumorigenesis in vivo and in vitro and was shown to be safe and well tolerated. Our study confirmed that REV1 is a potential diagnostic marker and therapeutic target for lung cancer and that JH-RE-06 may be a safe and efficient therapeutic agent for NSCLC.

Targeting ADRB2 enhances sensitivity of non-small cell lung cancer to VEGFR2 tyrosine kinase inhibitors.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) tyrosine kinase inhibitors (TKIs) have achieved remarkable clinical progress in the treatment of non-small-cell lung cancer; however, resistance has limited their therapeutic efficacy. Therefore, understanding the mechanisms of VEGF-TKI and ICI resistance will help to develop effective treatment strategies for patients with advanced NSCLC. Our results suggested that treatment with VEGFR2-TKIs upregulated ADRB2 expression in NSCLC cells. Propranolol, a common ADRB2 antagonist, significantly enhanced the therapeutic effect of VEGFR2-TKIs by inhibiting the ADRB2 signaling pathway in NSCLC cells in vitro and in vivo. Mechanically, the treatment-induced ADRB2 upregulation and the enhancement of ADRB2/VEGFR2 interaction caused resistance to VEGFR2-TKIs in NSCLC. And the inhibition of the ADRB2/CREB/PSAT1 signaling pathway sensitized cells to VEGFR2-TKIs. We demonstrated that ADRB2 signaling is crucial in mediating resistance to VEGFR2-TKIs and provided a novel promising combinatory approach to enhance the antitumor effect of VEGFR2-TKIs in NSCLC combining with propranolol.

BRD4-IRF1 axis regulates chemoradiotherapy-induced PD-L1 expression and immune evasion in non-small cell lung cancer.

BACKGROUND: Chemoradiotherapy-induced PD-L1 upregulation leads to therapeutic resistance and treatment failure. The PD-1/PD-L1 blocking antibodies sensitize cancers to chemoradiotherapy by blocking extracellular PD-1 and PD-L1 binding without affecting the oncogenic function of intracellular PD-L1. Reversing the chemoradiation-induced PD-L1 expression could provide a new strategy to achieve a greater anti-tumour effect of chemoradiotherapy. Here, we aimed to identify candidate small molecular inhibitors that might boost the anti-tumour immunity of chemoradiotherapy by decreasing treatment-induced PD-L1 expression in non-small cell lung cancer (NSCLC). METHODS: A drug array was used to recognize compounds that can suppress the cisplatin-induced and radiation-induced PD-L1 expression in NSCLC via the flow cytometry-based assay. We examined whether and how targeting bromodomain containing 4 (BRD4) inhibits chemoradiation-induced PD-L1 expression and evaluated the effect of BRD4 inhibition and chemoradiation combination in vivo. RESULTS: BRD4 inhibitors JQ1 and ARV-771 were identified as the most promising drugs both in the cisplatin and radiation screening projects in two NSCLC cell lines. Targeting BRD4 was supposed to block chemoradiotherapy inducible PD-L1 expression by disrupting the recruitment of BRD4-IRF1 complex to PD-L1 promoter. A positive correlation between BRD4 and PD-L1 expression was observed in human NSCLC tissues. Moreover, BRD4 inhibition synergized with chemoradiotherapy and PD-1 blockade to show a robust anti-tumour immunity dependent on CD8+ T cell through limiting chemoradiation-induced tumour cell surface PD-L1 upregulation in vivo. Notably, the BRD4-targeted combinatory treatments did not show increased toxicities. CONCLUSION: The data showed that BRD4-targeted therapy synergized with chemoradiotherapy and anti-PD-1 antibody by boosting anti-tumour immunity in NSCLC.

CDK7 inhibitor THZ1 enhances antiPD-1 therapy efficacy via the p38α/MYC/PD-L1 signaling in non-small cell lung cancer.

BACKGROUND: The cyclin-dependent kinase 7 (CDK7) subunit of TFIIH regulates RNA polymerase-II-based transcription and promotes tumor progression. However, the mechanisms involved in CDK7-mediated immune evasion are unclear in non-small cell lung cancer (NSCLC). METHODS: RNA silencing and pharmacologic inhibitors were used to evaluate the functions of CDK7/p38α/MYC/PD-L1 axis in cancer cell proliferation and antiPD-1 therapy resistance. Flow cytometry was performed to detect the status of the immune microenvironment after CDK7 inhibition and antiPD-1 therapy in vivo. CD8 depletion antibodies were used to assess the role of CD8+ T cells in combined CDK7 and PD-1 blockade. The associations among CDK7, p38α, MYC, PD-L1, infiltrating T cells, and survival outcomes were validated in two tissue microarrays and public transcriptomic data of NSCLC. RESULTS: High CDK7 mRNA and protein levels were identified to be associated with poor prognosis in NSCLC. CDK7 silencing and CDK7 inhibitor THZ1 elicited apoptosis and suppressed tumor growth. Moreover, CDK7 ablation specifically suppressed p38α/MYC-associated genes, and THZ1 inhibited MYC transcriptional activity through downregulating p38α. CDK7 inhibition sensitized NSCLC to p38α inhibitor. Further, THZ1 suppressed PD-L1 expression by inhibiting MYC activity. THZ1 boosted antitumor immunity by recruiting infiltrating CD8+ T cells and synergized with antiPD-1 therapy. The CDK7/MYC/PD-L1 signature and infiltrating T cell status collectively stratified NSCLC patients into different risk groups. CONCLUSION: These data suggest that the combined CDK7 inhibitor THZ1 and antiPD-1 therapy can be an effective treatment in NSCLC.