Safety and efficacy of SNK01 (autologous natural killer cells) in combination with cytotoxic chemotherapy and/or cetuximab after failure of prior tyrosine kinase inhibitor in non-small cell lung cancer: non-clinical mouse model and phase I/IIa clinical study.

BACKGROUND: Choosing treatments for epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) patients with osimertinib resistance is challenging. We evaluated the safety and efficacy of SNK01 (autologous natural killer (NK) cells) in combination with cytotoxic chemotherapy and/or cetuximab (an anti-EGFR monoclonal antibody) in treating EGFR-mutated NSCLC in this non-clinical and phase I/IIa clinical trial. METHODS: We developed a cell line-derived xenograft-humanized mouse model with an osimertinib-resistant lung cancer cell line. The mice were divided into four groups based on treatment (no treatment, cetuximab, SNK01, and combination groups) and treated weekly for 5 weeks. In the clinical study, 12 patients with EGFR-mutated NSCLC who failed prior tyrosine kinase inhibitor (TKI) received SNK01 weekly in combination with gemcitabine/carboplatin (n=6) or cetuximab/gemcitabine/carboplatin (n=6) and dose escalation of SNK01 following the "3+3" design. RESULTS: In the non-clinical study, an increase in NK cells in the blood and enhanced NK cell tumor infiltration were observed in the SNK01 group. The volume of tumor extracted after treatment was the smallest in the combination group. In the clinical study, 12 patients (median age, 60.9 years; all adenocarcinoma cases) received SNK01 weekly for 7-8 weeks (4×109 cells/dose (n=6); 6×109 cells/dose (n=6)). The maximum feasible dose of SNK01 was 6×109 cells/dose without dose-limiting toxicity. Efficacy outcomes showed an objective response rate of 25%, disease control rate of 100%, and median progression-free survival of 143 days. CONCLUSION: SNK01 in combination with cytotoxic chemotherapy, including cetuximab, for EGFR-mutated NSCLC with TKI resistance was safe and exerted a potential antitumor effect. TRIAL REGISTRATION NUMBER: NCT04872634.

PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development.

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo.

HDAC5-mediated exosomal Maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma.

BACKGROUND: Tumor-derived exosomes are critical elements of the cell-cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes. METHODS: We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components. RESULTS: We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size. CONCLUSIONS: Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity. Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma.

Identification of exosomal microRNA panel as diagnostic and prognostic biomarker for small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) has an exceptionally poor prognosis; as most of the cases are initially diagnosed as extensive disease with hematogenous metastasis. Therefore, the early diagnosis of SCLC is very important and may improve its prognosis. METHODS: To investigate the feasibility of early diagnosis of SCLC, we examined exosomal microRNAs (miRNAs) present in serum obtained from patients with SCLC. First, exosomes were isolated in serum from patients with SCLC and healthy individuals and were characterized using particle size and protein markers. Additionally, miRNA array was performed to define SCLC-specific exosomal miRNAs. Second, the obtained miRNAs were further validated employing a large cohort. Finally, the ability to diagnose SCLC was estimated by area under the curve (AUC), and intracellular mRNA change patterns were verified through validated miRNAs. RESULTS: From the miRNA array results, we selected 51-miRNAs based on p-values and top 10 differentially expressed genes, and 25-miRNAs were validated using quantitative reverse transcription-polymerase chain reaction. The 25-miRNAs were further validated employing a large cohort. Among them, 7-miRNAs showed significant differences. Furthermore, 6-miRNAs (miR-3565, miR-3124-5p, miR-200b-3p, miR-6515, miR-3126-3p and miR-9-5p) were up-regulated and 1-miRNA (miR-92b-5p) was down-regulated. The AUC value of each miRNA sets between 0.64 and 0.76, however the combined application of 3-miRNAs (miR-200b-3p, miR-3124-5p and miR-92b-5p) remarkably improved the diagnostic value (AUC = 0.93). Gene ontology analysis revealed that the 3-miRNA panel is linked to various oncogene pathways and nervous system development. When the 3-miRNAs were introduced to cells, the resulting changes in total mRNA expression strongly indicated the presence of lung diseases, including lung cancer. In addition, the 3-miRNA panel was significantly associated with a poorer prognosis, although individual miRNAs have not been validated as prognostic markers. CONCLUSION: Our study identified SCLC-specific exosomal miRNAs, and the 3-miRNAs panel (miR-200b-3p, miR-3124-5p and miR-92b-5p) may serve as a diagnostic and prognostic marker for SCLC.

AXL receptor tyrosine kinase inhibition improves the anti-tumor effects of CD8+ T cells by inducing CD103+ dendritic cell-mediated T cell priming.

AXL is a member of TAM receptor family and has been highlighted as a potential target for cancer treatment. Accumulating evidence has uncovered the critical role of the AXL signaling pathway in tumor growth, metastasis, and resistance against anti-cancer drugs, as well as its association with cancer immune escape. However, the function of AXL as a manipulator of the immune system in the tumor microenvironment (TME) remains unclear. Therefore, in this study, we investigated the impact of AXL on immune cells in the TME of a syngeneic tumor model using AXL knockout (AXL-/-) mice. Compared to AXL wild-type (AXL+/+) mice, tumor growth was significantly suppressed in AXL-/- mice, and an induced population of tumor-infiltrated CD8+ T cells and CD103+ dendritic cells (DCs) was observed. The change of CD8+ T cells and CD103+ DCs was also confirmed in tumor-draining lymph nodes (TdLN). In addition, the clonal expansion of OVA-specific CD8+ T cells was dominant in AXL-/- mice. Finally, anti-PD-1 treatment evidenced synergistic anti-cancer effects in AXL-/- mice. Overall, our data indicate that AXL signaling may inhibit the clonal expansion of tumor-specific CD8+ T cells through the regulation of the migration of CD8+ T cells and DCs in TME. Thus, AXL may be a powerful molecular target to improve anti-cancer effects through single or combined therapy with immune checkpoint inhibitors (ICI).

Generation of genetically engineered mice for lung cancer with mutant EGFR.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFRL858R/T790M/C797S and firefly luciferase using Cas9-mediated homology-independent targeted integration. Using a lung-specific Sftpc-CreERT2 mouse line, we induced expression of both the human EGFRL858R/T790M/C797S transgene and firefly luciferase in the lungs of adult mice. The expression of these genes and lung cancer occurrence was monitored using an in vivo imaging system and magnetic resonance imaging, respectively. Overall, our mouse model can be utilized to develop new drugs for overcoming C797S-mediated resistance to osimertinib; further, such knock-in systems for expressing oncogenes may be applied to study tumorigenesis and the development of other targeted agents.

The microbiome of lung cancer tissue and its association with pathological and clinical parameters.

Lung cancer is the primary cause of cancer-related deaths worldwide. Recently, although the microbiome has emerged as the key modulator of the carcinogenesis, it has not been evaluated in lung cancer. Here, we evaluated the microbial composition of lung cancer tissues according to the histologic type and genetic mutation, compared it with that of the adjacent normal lung tissues, and investigated the association between the lung microbiome and clinical parameters. We collected lung tissue samples from 162 patients with non-small cell lung cancer (NSCLC, 162 cancer and 54 adjacent normal tissues), surgically resected between January 2018 and December 2019, and analyzed their microbiome using 16S rRNA gene amplicon sequencing, the QIIME2 pipeline, and statistical analyses. NSCLC tissues had significantly lower alpha diversity than the normal tissues, and their microbial composition differed according to the histologic type and cancer genetic mutation. The genera Romboutsia, Novosphingobium, Acinetobacter, and Prevotella were significantly overrepresented in NSCLC tissues. Alpha diversity steadily declined from a normal to a more advanced stage, and microbial compositional differences were noted along with recurrence. Stenotrophomonas was the most predominant genus in the NSCLC tissues of patients with recurrence. The pathways related to the tricarboxylic acid cycle and L-glutamate and L-glutamine biosynthesis were predominant in adenocarcinoma, whereas those related to purine and pyrimidine nucleotide degradation and formaldehyde assimilation were predominant in squamous cell carcinoma. Our findings suggest that the altered lung cancer microbial composition might be associated with cancer initiation and/or progression.

The reversible fourth-generation EGFR tyrosine kinase inhibitor OBX02-011 overcomes C797S-mediated resistance in lung cancer.

Osimertinib is an irreversible third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that was initially developed to overcome the EGFR T790M mutation and is used as a standard therapy in patients with advanced non-small cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the remarkable initial efficacy, osimertinib, like other EGFR-TKIs, is limited by the emergence of acquired resistance. As the EGFR mutation C797S has been identified as a key driver of acquired resistance to osimertinib, development of a drug that targets this clinically relevant mutation could help improve patient outcomes. Here, we report the discovery and preclinical efficacy of OBX02-011, a reversible fourth-generation EGFR TKI that overcomes the EGFR C797S mutation. Compared to approved EGFR TKIs, OBX02-011 showed potent anticancer effects and inhibited EGFR-related signaling in various models, including those harboring the EGFR C797S mutation. Additionally, in transgenic mouse models (EGFRL858R/T790M/C797S), OBX02-011 treatment effectively inhibited tumor growth and EGFR activity, leading to enhanced survival. Collectively, these results suggest that OBX02-011 may be a promising new EGFR TKI to overcome C797S-mediated resistance in NSCLC.

A Phase I/IIa Randomized Trial Evaluating the Safety and Efficacy of SNK01 Plus Pembrolizumab in Patients with Stage IV Non-Small Cell Lung Cancer.

PURPOSE: The aim of this study is to evaluate the safety and efficacy of ex vivo activated and expanded natural killer (NK) cell therapy (SNK01) plus pembrolizumab in a randomized phase I/IIa clinical trial. MATERIALS AND METHODS: Overall, 18 patients with advanced non-small cell lung cancer (NSCLC) and a programmed death ligand 1 tumor proportion score of 1% or greater who had a history of failed frontline platinum-based therapy were randomized (2:1) to receive pembrolizumab every 3 weeks +/- 6 weekly infusions of SNK01 at either 2×109 or 4×109 cells per infusion (pembrolizumab monotherapy vs. SNK01 combination). The primary endpoint was safety, whereas the secondary endpoints were the objective response rate (ORR), progression-free survival (PFS), overall survival, and quality of life. RESULTS: Since no dose-limiting toxicity was observed, the maximum tolerated dose was determined as SNK01 4×109 cells/dose. The safety data did not show any new safety signals when SNK01 was combined with pembrolizumab. The ORR and the 1-year survival rate in the NK combination group were higher than those in patients who underwent pembrolizumab monotherapy (ORR, 41.7% vs. 0%; 1-year survival rate, 66.7% vs. 50.0%). Furthermore, the median PFS was higher in the SNK01 combination group (6.2 months vs. 1.6 months, p=0.001). CONCLUSION: Based on the findings of this study, the NK cell combination therapy may consider as a safe treatment method for stage IV NSCLC patients who had a history of failed platinum-based therapy without an increase in adverse events.

BIX01294 inhibits EGFR signaling in EGFR-mutant lung adenocarcinoma cells through a BCKDHA-mediated reduction in the EGFR level.

BIX01294 (BIX), an inhibitor of the G9a histone methyltransferase, has been reported to have antitumor activity against a variety of cancers. However, the molecular mechanisms underlying its anticancer effects, particularly those against lung cancer, remain unclear. Here, we report that BIX induces apoptotic cell death in EGFR-mutant non-small cell lung cancer (NSCLC) cells but not in their wild-type counterparts. Treatment with BIX resulted in a significant reduction in the EGFR level and inhibition of EGFR signaling only in EGFR-mutant NSCLC cells, leading to apoptosis. BIX also inhibited mitochondrial metabolic function and decreased the cellular energy levels that are critical for maintaining the EGFR level. Furthermore, BIX transcriptionally downregulated the transcription of branched-chain α-keto acid dehydrogenase (BCKDHA), which is essential for fueling the tricarboxylic acid (TCA) cycle. Interestingly, this BCKDHA downregulation was due to inhibition of Jumanji-domain histone demethylases but not the G9a histone methyltransferase. We observed that KDM3A, a Jumonji histone demethylase, epigenetically regulates BCKDHA expression by binding to the BCKDHA gene promoter. BIX exposure also led to a significant decrease in the EGFR level, causing apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Taken together, our current data suggest that BIX triggers apoptosis only in EGFR-mutant NSCLC cells via inhibition of BCKDHA-mediated mitochondrial metabolic function.

Contribution of p53 in sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer.

The emergence of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) with activating EGFR mutations is a major hindrance to treatment. We investigated the effects of p53 in primary sensitivity and acquired resistance to EGFR-TKIs in NSCLC cells. Changes in sensitivity to EGFR-TKIs were determined using p53 overexpression or knockdown in cells with activating EGFR mutations. We investigated EMT-related molecules, morphologic changes, and AXL induction to elucidate mechanisms of acquired resistance to EGFR-TKIs according to p53 status. Changes in p53 status affected primary sensitivity as well as acquired resistance to EGFR-TKIs according to cell type. Firstly, p53 silencing did not affect primary and acquired resistance to EGFR-TKIs in PC-9 cells, but it led to primary resistance to EGFR-TKIs through AXL induction in HCC827 cells. Secondly, p53 silencing in H1975 cells enhanced the sensitivity to osimertinib through the emergence of mesenchymal-to-epithelial transition, and the emergence of acquired resistance to osimertinib in p53 knockout cells was much slower than in H1975 cells. Furthermore, two cell lines (H1975 and H1975/p53KO) demonstrated the different mechanisms of acquired resistance to osimertinib. Lastly, the introduction of mutant p53-R273H induced the epithelial-to-mesenchymal transition and exerted resistance to EGFR-TKIs in cells with activating EGFR mutations. These findings indicate that p53 mutations can be associated with primary or acquired resistance to EGFR-TKIs. Thus, the status or mutations of p53 may be considered as routes to improving the therapeutic effects of EGFR-TKIs in NSCLC.

Discovery of a novel CDK7 inhibitor YPN-005 in small cell lung cancer.

In contrast to non-small cell lung cancer, there has been no significant progress in the development of therapies for the small cell lung cancer (SCLC) in recent decades. Although various targeted agents, including immunotherapies, have recently been developed for testing in clinical trials, novel therapeutic agents are still needed for SCLC. We developed a potent inhibitor of cyclin-dependent kinase 7 (CDK7), designated YPN-005, and sought to determine whether it showed any anticancer effects in SCLC cells, cisplatin or etoposide-resistant cells, or organoids derived from SCLC patients. In a panel of kinases assay, YPN-005 was highly selective for CDK7 and showed antiproliferative effects in SCLC and cells with acquired resistance to conventional anticancer drugs. Similar to other CDK7 inhibitors, YPN-005 treatment significantly decreased the phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Consistent with the in vitro results, YPN-005 treatment showed a significant inhibition of tumor growth through the suppression of RNA polymerase II phosphorylation. Finally, YPN-005 showed potent anticancer effects in organoids derived from SCLC patients compared to another CDK7 inhibitor, THZ1. Therapeutic targeting of CDK7 in SCLC might be suitable for clinical investigation, and YPN-005 may be a promising therapeutic option for primary SCLC and SCLC with acquired resistance to conventional therapy.

Exosomal miR-1260b derived from non-small cell lung cancer promotes tumor metastasis through the inhibition of HIPK2.

Tumor-derived exosomes (TEXs) contain enriched miRNAs, and exosomal miRNAs can affect tumor growth, including cell proliferation, metastasis, and drug resistance through cell-to-cell communication. We investigated the role of exosomal miR-1260b derived from non-small cell lung cancer (NSCLC) in tumor progression. Exosomal miR-1260b induced angiogenesis by targeting homeodomain-interacting protein kinase-2 (HIPK2) in human umbilical vein endothelial cells (HUVECs). Furthermore, exosomal miR-1260b or suppression of HIPK2 led to enhanced cellular mobility and cisplatin resistance in NSCLC cells. In patients with NSCLC, the level of HIPK2 was significantly lower in tumor tissues than in normal lung tissues, while that of miR-1260b was higher in tumor tissues. HIPK2 and miR-1260b expression showed an inverse correlation, and this correlation was strong in distant metastasis. Finally, the expression level of exosomal miR-1260b in plasma was higher in patients with NSCLC than in healthy individuals, and higher levels of exosomal miR-1260b were associated with high-grade disease, metastasis, and poor survival. In conclusion, exosomal miR-1260b can promote angiogenesis in HUVECs and metastasis of NSCLC by regulating HIPK2 and may serve as a prognostic marker for lung cancers.

Establishment and Long-Term Expansion of Small Cell Lung Cancer Patient-Derived Tumor Organoids.

Differential chemo-sensitivity of cancer cells, which is attributed to the cellular heterogeneity and phenotypic variation of cancer cells, is considered to be the main reason for tumor recurrence after chemotherapy. Here, we generated small cell lung cancer patient-derived tumor organoids and subjected them to long-term expansion with the addition of WNT3A or R-spondin1. We confirmed that the organoids have similar genetic profiles, molecular characteristics, and morphological architectures to the corresponding patient tumor tissue during and after long-term expansion. Interestingly, the cellular heterogeneity of organoids is reflected in their differential response to cisplatin or etoposide. We propose to utilize the organoids as small cell lung cancer patient avatar models that would be ideal for investigating the mechanisms underlying tumor recurrence after chemotherapy, and would ultimately help to develop personalized medicine.

Efficacy of the CDK7 Inhibitor on EMT-Associated Resistance to 3rd Generation EGFR-TKIs in Non-Small Cell Lung Cancer Cell Lines.

Epithelial to mesenchymal transition (EMT) is associated with resistance during EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. Here, we investigated whether EMT is associated with acquired resistance to 3rd generation EGFR-TKIs, and we explored the effects of cyclin-dependent kinase 7 (CDK7) inhibitors on EMT-mediated EGFR-TKIs resistance in non-small cell lung cancer (NSCLC). We established 3rd generation EGFR-TKI resistant cell lines (H1975/WR and H1975/OR) via repeated exposure to WZ4002 and osimertinib. The two resistant cell lines showed phenotypic changes to a spindle-cell shape, had a reduction of epithelial marker proteins, an induction of vimentin expression, and enhanced cellular mobility. The EMT-related resistant cells had higher sensitivity to THZ1 than the parental cells, although THZ1 treatment did not inhibit EGFR activity. This phenomenon was also observed in TGF-ß1 induced EMT cell lines. THZ1 treatment induced G2/M cell cycle arrest and apoptosis in all of the cell lines. In addition, THZ1 treatment led to drug-tolerant, EMT-related resistant cells, and these THZ1-tolerant cells partially recovered their sensitivity to 3rd generation EGFR-TKIs. Taken together, EMT was associated with acquired resistance to 3rd generation EGFR-TKIs, and CDK7 inhibitors could potentially be used as a therapeutic strategy to overcome EMT associated EGFR-TKI resistance in NSCLC.

Efficacy of natural killer cell activity as a biomarker for predicting immunotherapy response in non-small cell lung cancer.

BACKGROUND: The establishment of biomarkers that can be used to predict the response to immunotherapy for malignancy is extremely important. In particular, noninvasive analysis of immune cells from peripheral blood before treatment has gained increased attention, and natural killer (NK) cell activity has been shown to be related to treatment response. Here, we aimed to confirm the relationship between the response to immunotherapy and NK cell activity. METHODS: In this prospective observational study, patients with advanced NSCLC who were scheduled for immunotherapy from October 2018 to December 2019 were enrolled. Baseline NK cell activity was compared according to the best clinical response to immunotherapy. RESULTS: A total of 54 patients with advanced NSCLC were enrolled, and 34 patients were analyzed. The baseline NK cell activity was significantly higher in the non-PD group than in the PD group (P = 0.002). At the cutoff level of ≥1200 pg/mL, baseline NK cell activity yielded a sensitivity of 80% and a specificity of 68.4% in predicting the response to immunotherapy (AUC = 0.8, P < 0.003). The median progression-free survival (PFS) was significantly better in the high NK group (P = 0.003), and correlation between baseline NK cell activity and PFS was also confirmed (r = 0.517, P = 0.002). CONCLUSIONS: Baseline NK cell activity was related to the response to immunotherapy and the PFS. We suggest that NK cell activity from peripheral blood before immunotherapy is a noninvasive, simple, and novel way to predicting the treatment response in patients with NSCLC. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: The response to immunotherapy was significantly better in patients with high baseline NK cell activity, and there was a significant correlation between baseline NK cell activity and PFS. What this study adds This study demonstrates the efficacy of baseline NK cell activity from peripheral blood as a biomarker for predicting immunotherapy response.