CDK4/6 signaling attenuates the effect of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

Background: Epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion and exon 21 L858R, are driver oncogenes of non-small cell lung cancer (NSCLC), with EGFR tyrosine kinase inhibitors (TKIs) being effective against EGFR-mutant NSCLC. However, the efficacy of EGFR-TKIs is transient and eventually leads to acquired resistance. Herein, we focused on the significance of cell cycle factors as a mechanism to attenuate the effect of EGFR-TKIs in EGFR-mutant NSCLC before the emergence of acquired resistance. Methods: Using several EGFR-mutant cell lines, we investigated the significance of cell cycle factors to attenuate the effect of EGFR-TKIs in EGFR-mutant NSCLC. Results: In several EGFR-mutant cell lines, certain cancer cells continued to proliferate without EGFR signaling, and the cell cycle regulator retinoblastoma protein (RB) was not completely dephosphorylated. Further inhibition of phosphorylated RB with cyclin-dependent kinase (CDK) 4/6 inhibitors, combined with the EGFR-TKI osimertinib, enhanced G0/G1 cell cycle accumulation and growth inhibition of the EGFR-mutant NSCLC in both in vitro and in vivo models. Furthermore, residual RB phosphorylation without EGFR signaling was maintained by extracellular signal-regulated kinase (ERK) signaling, and the ERK inhibition pathway showed further RB dephosphorylation. Conclusions: Our study demonstrated that the CDK4/6-RB signal axis, maintained by the MAPK pathway, attenuates the efficacy of EGFR-TKIs in EGFR-mutant NSCLC, and targeting CDK4/6 enhances this efficacy. Thus, combining CDK4/6 inhibitors and EGFR-TKI could be a novel treatment strategy for TKI-naïve EGFR-mutant NSCLC.

Chemo-preventive effects and antitumorigenic mechanisms of beer and nonalcoholic beer toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) - induced lung tumorigenesis in A/J mice.

We investigated the chemopreventive effects of beer, nonalcoholic beers (NABs), and beer-components (glycine betaine (GB)) on NNK-induced lung tumorigenesis in A/J mice, and the possible mechanisms underlying the antitumorigenic effects of beer, NABs, and beer-components. Beer, NABs, and GB reduced NNK-induced lung tumorigenesis. We investigated the antimutagenicity of beer, NABs and beer-components (GB and pseudouridine (PU)) toward the mutagenicity of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Beer, NABs, and beer components were antimutagenic toward MNNG and NNK in the Ames test using S. typhimurium TA1535. In contrast, MNNG and NNK mutagenicity detected in S. typhimurium YG7108, a strain lacking O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of beer, NABs, or beer components, suggesting that they may mediate its antimutagenic effect by enhancing DNA damage repair. Phosphorylation of Akt and STAT3, with or without epidermal growth factor stimulation, in lung epithelial-like A549 cells were significantly decreased following beer, NABs, GB and PU. They targeted both the initiation and growth/progression steps of carcinogenesis, specifically via antimutagenesis, stimulation of alkyl DNA-adduct repair, and suppression of Akt- and STAT3- mediated growth signaling. GB and PU may contribute, in part, to the biological effects of beer and NABs via the suppression of Akt and STAT3 phosphorylation.

PD-1 blockade augments CD8+ T cell dependent antitumor immunity triggered by Ad-SGE-REIC in Egfr-mutant lung cancer.

OBJECTIVES: No immunotherapeutic protocol has yet been established in never-smoking patients with lung cancer harboring driver oncogenic mutations, such as epidermal growth factor receptor (EGFR) mutations. The immunostimulatory effect of Ad-REIC, a genetically engineered adenovirus vector expressing a tumor suppressor gene, reduced expression in immortalized cells (REIC), has been investigated in clinical trials for various solid tumors. However, the immunostimulatory effect of the Ad-REIC in EGFR-mutant lung cancer with a non-inflamed tumor microenvironment (TME) has not been explored. MATERIALS AND METHODS: We used a syngeneic mouse model developed by transplanting Egfr-mutant lung cancer cells into single or double flanks of C57BL/6J mice. Ad-SGE-REIC, a 2nd-generation vector with an enhancer sequence, was injected only into the tumors from one flank, and its antitumor effects were assessed. Tumor-infiltrating cells were evaluated using immunohistochemistry or flow cytometry. The synergistic effects of Ad-SGE-REIC and PD-1 blockade were also examined. RESULTS: Injection of Ad-SGE-REIC into one side of the tumor induced not only a local antitumor effect but also a bystander abscopal effect in the non-injected tumor, located on the other flank. The number of PD-1+CD8+ T cells increased in both injected and non-injected tumors. PD-1 blockade augmented the local and abscopal antitumor effects of Ad-SGE-REIC by increasing the number of CD8+ T cells in the TME of Egfr-mutant tumors. Depletion of CD8+ cells reverted the antitumor effect, suggesting they contribute to antitumor immunity. CONCLUSION: Ad-SGE-REIC induced systemic antitumor immunity by modifying the TME status from non-inflamed to inflamed, with infiltration of CD8+ T cells. Additionally, in Egfr-mutant lung cancer, this effect was enhanced by PD-1 blockade. These findings pave the way to establish a novel combined immunotherapy strategy with Ad-SGE-REIC and anti-PD-1 antibody for lung cancer with a non-inflamed TME.

CD8+ T-cell Responses Are Boosted by Dual PD-1/VEGFR2 Blockade after EGFR Inhibition in Egfr-Mutant Lung Cancer.

Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in nonsmoking-related, non-small cell lung cancer (NSCLC). EGFR-mutant NSCLC has a noninflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune-checkpoint inhibitors, such as antiprogrammed cell death-1 (anti-PD-1), have weak antitumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by the sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, the simultaneous triple blockade had no such effect. The PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pretreatment with the EGFR-TKI, suggesting that the treatment schedule is crucial for the efficacy of the dual blockade therapy. Pretreatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing the expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a noninflamed TME.

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients.

A case of interstitial pneumonia associated with systemic sclerosis and primary peritoneal serous carcinoma successfully treated with cyclophosphamide.

A 62-year-old woman with edema and color changes in her fingers underwent computed tomography (CT); slight interstitial changes were detected in the lungs with multiple tumors in the anterior and hilar region of the liver. Based on the blood test findings, she was diagnosed with interstitial pneumonia associated with systemic sclerosis. Ultrasound-guided biopsy from the hepatic hilar lymph node revealed poorly differentiated serous adenocarcinoma cells. High serum CA-125 levels suggested primary peritoneal serous carcinoma (PPSC). Owing to increased interstitial shadows on chest CT images and worsening respiratory distress, intravenous cyclophosphamide and oral prednisolone treatment was started. The skin-related symptoms, respiratory distress, and interstitial shadows improved, and the tumor size reduced. Eighteen months later, the patient has had no exacerbation of interstitial pneumonia, and the PPSC is well controlled.

VEGFR2 blockade augments the effects of tyrosine kinase inhibitors by inhibiting angiogenesis and oncogenic signaling in oncogene-driven non-small-cell lung cancers.

Molecular agents targeting the epidermal growth factor receptor (EGFR)-, anaplastic lymphoma kinase (ALK)- or c-ros oncogene 1 (ROS1) alterations have revolutionized the treatment of oncogene-driven non-small-cell lung cancer (NSCLC). However, the emergence of acquired resistance remains a significant challenge, limiting the wider clinical success of these molecular targeted therapies. In this study, we investigated the efficacy of various molecular targeted agents, including erlotinib, alectinib, and crizotinib, combined with anti-vascular endothelial growth factor receptor (VEGFR) 2 therapy. The combination of VEGFR2 blockade with molecular targeted agents enhanced the anti-tumor effects of these agents in xenograft mouse models of EGFR-, ALK-, or ROS1-altered NSCLC. The numbers of CD31-positive blood vessels were significantly lower in the tumors of mice treated with an anti-VEGFR2 antibody combined with molecular targeted agents compared with in those of mice treated with molecular targeted agents alone, implying the antiangiogenic effects of VEGFR2 blockade. Additionally, the combination therapies exerted more potent antiproliferative effects in vitro in EGFR-, ALK-, or ROS1-altered NSCLC cells, implying that VEGFR2 inhibition also has direct anti-tumor effects on cancer cells. Furthermore, VEGFR2 expression was induced following exposure to molecular targeted agents, implying the importance of VEGFR2 signaling in NSCLC patients undergoing molecular targeted therapy. In conclusion, VEGFR2 inhibition enhanced the anti-tumor effects of molecular targeted agents in various oncogene-driven NSCLC models, not only by inhibiting tumor angiogenesis but also by exerting direct antiproliferative effects on cancer cells. Hence, combination therapy with anti-VEGFR2 antibodies and molecular targeted agents could serve as a promising treatment strategy for oncogene-driven NSCLC.

Rapid Disease Progression of Advanced Non-small Cell Lung Cancer Five Months after Cessation of Pembrolizumab.

We report a case of late-onset hyperprogressive disease after cessation of a PD-1 inhibitor. A male was diagnosed with metastatic lung adenocarcinoma with little progression for 2 months before treatment. He received pembrolizumab as a second-line treatment and was subsequently prescribed docetaxel for 3 months until a slight increase in pleural effusion. At the time of progression to docetaxel, he commenced prednisolone because of immune-system-related diarrhea. After that, his general condition rapidly worsened with severe fatigue and hypoxia. Computed tomography revealed a massive increase of pleural effusion and replacement of almost the entire liver with cancer over a period of 5 weeks.

Granulation Tissue-induced Pseudo-relapse During Nivolumab Treatment in Advanced Non-small Cell Lung Cancer.

Atypical tumor responses such as pseudo-progression or hyper-progression sometimes occur during immune check point inhibitor therapy. Distinct from both responses, we experienced a case of non-small cell lung cancer (NSCLC) with a pseudo-relapse, in which development of granulation mimicked cancer relapse during nivolumab therapy. A male with advanced NSCLC started nivolumab as a second-line therapy. After 15 cycles of nivolumab with a complete response, tumor markers started increasing and positron-emission computed tomography indicated a hot spot in the sigmoid colon. Laparoscopic segmental sigmoid colon resection revealed granulation tissue without any relapse of malignant cells. The results showed that even if radiographical tumor progression is found during immune therapy, histological confirmation should be considered.

Efficacy of afatinib treatment for lung adenocarcinoma harboring exon 18 delE709_T710insD mutation.

Exon 18 delE709_T710insD is an extremely rare mutation in epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC); the efficacy of EGFR tyrosine kinase inhibitors against this mutation remains unclear. In this case report, we report a case of NSCLC harboring EGFR exon 18 delE709_T710insD that was not detected by a commercially available assay, but was detected by a next-generation sequencing cancer panel. A 56-year old female patient with advanced NSCLC was diagnosed as EGFR-mutation-negative using the PNAClamp method. ALK rearrangement was also absent and she received cytotoxic chemotherapies. Clinical characteristics, including adenocarcinoma histology and no history of smoking, implied the presence of a driver mutation, so a next-generation-sequencing Oncomine® Cancer Research Panel was conducted in the patient's clinical course and the EGFR exon 18 delE709_T710insD mutation was detected. The patient started afatinib as sixth-line treatment and her pulmonary lesion significantly decreased in size. Afatinib was continued for 7 months until disease progressed.