Patient-Derived Cells to Guide Targeted Therapy for Advanced Lung Adenocarcinoma

Adequate preclinical model and model establishment procedure are required to accelerate translational research in lung cancer. We streamlined a protocol for establishing patient-derived cells (PDC) and identified effective targeted therapies and novel resistance mechanisms using PDCs. We generated 23 PDCs from 96 malignant effusions of 77 patients with advanced lung adenocarcinoma. Clinical and experimental factors were reviewed to identify determinants for PDC establishment. PDCs were characterized by driver mutations and in vitro sensitivity to targeted therapies. Seven PDCs were analyzed by whole-exome sequencing. PDCs were established at a success rate of 24.0%. Utilizing cytological diagnosis and tumor colony formation can improve the success rate upto 48.8%. In vitro response to a tyrosine kinase inhibitor (TKI) in PDC reflected patient treatment response and contributed to identifying effective therapies. Combination of dabrafenib and trametinib was potent against a rare BRAF K601E mutation. Afatinib was the most potent EGFR-TKI against uncommon EGFR mutations including L861Q, G719C/S768I, and D770_N771insG. Aurora kinase A (AURKA) was identified as a novel resistance mechanism to olmutinib, a mutant-selective, third-generation EGFR-TKI, and inhibition of AURKA overcame the resistance. We presented an efficient protocol for establishing PDCs. PDCs empowered precision medicine with promising translational values.ope

Targeting YAP to overcome acquired resistance to ALK inhibitors in ALK-rearranged lung cancer

Clinical benefit of ALK tyrosine kinase inhibitors (ALK-TKIs) in ALK-rearranged lung cancer has been limited by the inevitable development of acquired resistance, and bypass-molecular resistance mechanisms remain poorly understood. We investigated a novel therapeutic target through screening FDA-approved drugs in ALK-TKI-resistant models. Cerivastatin, the rate-limiting enzyme inhibitor of the mevalonate pathway, showed anti-cancer activity against ALK-TKI resistance in vitro/in vivo, accompanied by cytoplasmic retention and subsequent inactivation of transcriptional co-regulator YAP. The marked induction of YAP-targeted oncogenes (EGFR, AXL, CYR61, and TGFβR2) in resistant cells was abolished by cerivastatin. YAP silencing suppressed tumor growth in resistant cells, patient-derived xenografts, and EML4-ALK transgenic mice, whereas YAP overexpression decreased the responsiveness of parental cells to ALK inhibitor. In matched patient samples before/after ALK inhibitor treatment, nuclear accumulation of YAP was mainly detected in post-treatment samples. High expression of YAP in pretreatment samples was correlated with poor response to ALK-TKIs. Our findings highlight a crucial role of YAP in ALK-TKI resistance and provide a rationale for targeting YAP as a potential treatment option for ALK-rearranged patients with acquired resistance to ALK inhibitors.ope

Molecular landscape of osimertinib resistance in patients and patient-derived preclinical models

Introduction: Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) that is approved for the use of EGFR-mutant non-small cell lung cancer (NSCLC) patients. In this study, we investigated the acquired resistance mechanisms in NSCLC patients and patient-derived preclinical models. Methods: Formalin-fixed paraffin-embedded tumor samples and plasma samples from 55 NSCLC patients who were treated with osimertinib were collected at baseline and at progressive disease (PD). Next-generation sequencing was performed in tumor and plasma samples using a 600-gene hybrid capture panel designed by AstraZeneca. Osimertinib-resistant cell lines and patient-derived xenografts and cells were generated and whole exome sequencing and RNA sequencing were performed. In vitro experiments were performed to functionally study the acquired mutations identified. Results: A total of 55 patients and a total of 149 samples (57 tumor samples and 92 plasma samples) were analyzed, and among them 36 patients had matched pre- and post-treatment samples. EGFR C797S (14%) mutation was the most frequent EGFR-dependent mechanism identified in all available progression samples, followed by EGFR G824D (6%), V726M (3%), and V843I (3%). Matched pre- and post-treatment sample analysis revealed in-depth acquired mechanisms of resistance. EGFR C797S was still most frequent (11%) among EGFR-dependent mechanism, while among EGFR-independent mechanisms, PIK3CA, ALK, BRAF, EP300, KRAS, and RAF1 mutations were detected. Among Osimertinib-resistant cell lines and patient-derived models, we noted acquired mutations which were potentially targetable such as NRAS p.Q61K, in which resistance could be overcome with combination of osimertinib and trametinib. A patient-derived xenograft established from osimertinib-resistant patient revealed KRAS p.G12D mutation which could be overcome with combination of osimertinib, trametinib, and buparlisib. Conclusion: In this study, we explored the genetic profiles of osimertinib-resistant NSCLC patient samples using targeted deep sequencing. In vitro and in vivo models harboring osimertinib resistance revealed potential novel treatment strategies after osimertinib failure.ope

Repotrectinib Exhibits Potent Antitumor Activity in Treatment-Naïve and Solvent-Front-Mutant ROS1-Rearranged Non-Small Cell Lung Cancer

Purpose: Although first-line crizotinib treatment leads to clinical benefit in ROS1+ lung cancer, high prevalence of crizotinib-resistant ROS1-G2032R (ROS1G2032R) mutation and progression in the central nervous system (CNS) represents a therapeutic challenge. Here, we investigated the antitumor activity of repotrectinib, a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI) in ROS1+ patient-derived preclinical models. Experimental design: Antitumor activity of repotrectinib was evaluated in ROS1+ patient-derived preclinical models including treatment-naïve and ROS1G2032R models and was further demonstrated in patients enrolled in an on-going phase I/II clinical trial (NCT03093116). Intracranial antitumor activity of repotrectinib was evaluated in a brain-metastasis mouse model. Results: Repotrectinib potently inhibited in vitro and in vivo tumor growth and ROS1 downstream signal in treatment-naïve YU1078 compared with clinically available crizotinib, ceritinib, and entrectinib. Despite comparable tumor regression between repotrectinib and lorlatinib in YU1078-derived xenograft model, repotrectinib markedly delayed the onset of tumor recurrence following drug withdrawal. Moreover, repotrectinib induced profound antitumor activity in the CNS with efficient blood-brain barrier penetrating properties. Notably, repotrectinib showed selective and potent in vitro and in vivo activity against ROS1G2032R. These findings were supported by systemic and intracranial activity of repotrectinib observed in patients enrolled in the on-going clinical trial. Conclusions: Repotrectinib is a novel next-generation ROS1-TKI with improved potency and selectivity against treatment-naïve and ROS1G2032R with efficient CNS penetration. Our findings suggest that repotrectinib can be effective both as first-line and after progression to prior ROS1-TKI.ope

Comprehensive analyses of immunodynamics and immunoreactivity in response to treatment in ALK-positive non-small-cell lung cancer

Background: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). Immune checkpoint inhibitors (ICIs) have not proved efficacy in ALK-positive non-small cell lung cancer so far. In this study, we performed a mouse clinical trial using EML4-ALK transgenic mice model to comprehensively investigate immunomodulatory effects of ALK TKI and to investigate the mechanisms of resistance to ICIs. Methods: EML4-ALK transgenic mice were randomized to three treatment arms (arm A: antiprogrammed death cell protein-1 (PD-1), arm B: ceritinib, arm C: anti-PD-1 and ceritinib), and tumor response was evaluated using MRI. Progression-free survival and overall survival were measured to compare the efficacy. Flow cytometry, multispectral imaging, whole exome sequencing and RNA sequencing were performed from tumors obtained before and after drug resistance. Results: Mouse clinical trial revealed that anti-PD-1 therapy was ineffective, and the efficacy of ceritinib and anti-PD-1 combination was not more effective than ceritinib alone in the first line. Dynamic changes in immune cells and cytokines were observed following each treatment, while changes in T lymphocytes were not prominent. A closer look at the tumor immune microenvironment before and after ceritinib resistance revealed increased regulatory T cells and programmed death-ligand 1 (PD-L1)-expressing cells both in the tumor and the stroma. Despite the increase of PD-L1 expression, these findings were not accompanied by increased effector T cells which mediate antitumor immune responses. Conclusions: ALK-positive tumors progressing on ceritinib is not immunogenic enough to respond to immune checkpoint inhibitors.ope

Modeling Clinical Responses to Targeted Therapies by Patient-Derived Organoids of Advanced Lung Adenocarcinoma

Purpose: Patient-derived organoids (PDO) of lung cancer has been recently introduced, reflecting the genomic landscape of lung cancer. However, clinical relevance of advanced lung adenocarcinoma organoids remains unknown. Here, we examined the ability of PDOs to predict clinical responses to targeted therapies in individual patients and to identify effective anticancer therapies for novel molecular targets. Experimental design: Eighty-four organoids were established from patients with advanced lung adenocarcinoma. Formalin-fixed, paraffin-embedded tumor specimens from corresponding patients were analyzed by whole-exome sequencing (n = 12). Organoids were analyzed by whole-exome sequencing (n = 61) and RNA sequencing (n = 55). Responses to mono or combination targeted therapies were examined in organoids and organoid-derived xenografts. Results: PDOs largely retained somatic alterations including driver mutations of matching patient tumors. PDOs were able to recapitulate progression-free survival and objective responses of patients with non-small cell lung cancer receiving clinically approved tyrosine kinase inhibitors. PDOs recapitulated activity of therapeutic strategies under clinical investigation. YUO-071 harboring an EGFR exon 19 deletion and a BRAF G464A mutation and the matching patient responded to dabrafenib/trametinib combination therapy. YUO-004 and YUO-050 harboring an EGFR L747P mutation was sensitive to afatinib, consistent with the response in the matching patient of YUO-050. Furthermore, we utilized organoids to identify effective therapies for novel molecular targets by demonstrating the efficacy of poziotinib against ERBB2 exon 20 insertions and pralsetinib against RET fusions. Conclusions: We demonstrated translational relevance of PDOs in advanced lung adenocarcinoma. PDOs are an important diagnostic tool, which can assist clinical decision making and accelerate development of therapeutic strategies.ope

Antitumor Activity and Acquired Resistance Mechanism of Dovitinib (TKI258) in RET-Rearranged Lung Adenocarcinoma

RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, in RET-rearranged LADC has not been reported. The aims of the study are to explore antitumor effects and mechanisms of acquired resistance of dovitinib in RET-rearranged LADC. Using structural modeling and in vitro analysis, we demonstrated that dovitinib induced cell-cycle arrest at G0-G1 phase and apoptosis by selective inhibition of RET kinase activity and ERK1/2 signaling in RET-rearranged LC-2/ad cells. Strong antitumor effect of dovitinib was observed in an LC-2/ad tumor xenograft model. To identify the acquired resistance mechanisms to dovitinib, LC-2/ad cells were exposed to increasing concentrations of dovitinib to generate LC-2/ad DR cells. Gene-set enrichment analysis of gene expression and phosphor-kinase revealed that Src, a central gene in focal adhesion, was activated in LC-2/ad DR cells. Saracatinib, an src kinase inhibitor, suppressed ERK1/2 phosphorylation and growth of LC-2/ad DR cells. Taken together, these findings suggest that dovitinib can be a potential therapeutic option for RET-rearranged LADC, in which acquired resistance to dovitinib can be overcome by targeting Src.ope

(A) study on the teaching method of ballet calss for its efficiency

무용은 학교에서 이루어지는 교육의 일 분야로서 그 교육목표는 일반 교육의 목표인 전인적 인간 양성을 지향하는 것과 같다. 즉 무용교육을 통해 지적, 정서적, 정신적, 사회적발달 뿐만아니라 미적 감각을 높여 생활의 활력소를 제공한다는데 그 의의를 찾을 수 있다. 오늘날 학교 교육은 고도로 산업화, 기계화, 과학화, 자동화되어지는 시대에 부응하는 전문인 양성 교육에 역점을 두고 있다. 전문교육이란 각 분야에서 전문적 지식을 갖고 그 방법을 터득하는 전문 지식과 그것을 학생들에게 전달할 수 있는 적절한 교수 기능을 포함하는 것으로 장래의 직업까지 연결된다. 이러한 전문 교육은 무용교육에 있어서도 "무용을 통한 교육"으로 오늘날의 사회속에서 그 필요성이 절대적으로 요구되어 지고 있다. 그중 오늘날 가장 과학적이고 능률적으로 고안된 움직임을 갖고 있다는 발레는 각 나라마다 그 특색에 맞는 교육과정과 교육체제를 마련하여 보다 체계적이고 효율적인 방법아래 기술의 습득과 향상을 꾀하고 있다. 그러나 우리나라 발레교육은 무용수들의 신체적, 기술적 조건에 있어서는 많은 향상을 하고 있지만 그 훈련방법에 있어서 체계가 소홀하고 미흡한 상태이다. 그 이유는 대부분의 교사들이 그들의 경험과 단순한 답습에 의존하여 확실한 과학적인 근거를 제시하지 못하고 있는 실정이기 때문이다. 이러한 현시점에서 우리나라 발레 교육의 향상을 위해 몇가지 문제점을 제시하면 첫째, 교사의 자질에 대한 문제가 시급하다는 점 둘째, 체계적인 교수 방법의 연구와 개발의 미비로 적절한 교수법이 정립되지 못하고 있다는 점 셋째, 신체의 기능과 역할범위에 따른 과학적교수의 접근을 소홀히 하는 경향이 있다는 점등이다. 따라서 본 논문은 이러한 문제점들의 개선을 위한 방안이자 또 발레수업의 효율화를 도모하기 위한 목적으로 무용교사의 자질, 역사적으로 위대한 무용가들의 교육체제 및 내용, 효율적 발레 교수방법의 개발을 이론적, 기능학적 접근방법을 통해 고찰해 본 결과 다음과 같다. 첫째, 훌륭한 무용교사란 진정한 교육애를 통한 교육 경험에 의한 교육이 이루어져야 한다. 둘째, 블라시스, 체께띠, 바가노바의 교수이론을 살펴본 바, 이론과 실기를 겸비한 교사만이 훌륭한 무용수를 지도 할 수 있다는 것이다. 셋째, 발레 수업의 지도형태중 기술적, 과학적, 예술적, 창조적 접근 방법이 기술 개발을 위해서는 어는 하나를 택하기 보다는 지도시 4가지 교수방법이 적절히 조화롭게 이루어져야 가장 효과적인 교육이 이루어진다. 넷째, 신체를 자세, 상부, 골반, 하지, 턴 아웃으로 분류하여 그 기능을 살펴본 바, 그 사용법을 터득해야만 동작을 지도함에 있어 보다 효율적이고, 신체의 상해를 예방하게 된다는 것이다.;As a dancing is one part of education taking place in a school, its educational purpose is as same as the purpose of general education in intending to cultivate the man of all-round personality. So to speak, we can find the meaning of the dancing deucation in the fact that it not only leads to the intellectual, emotional, mental and social developmet but also provides people with vitality of life by enhancing the esthetic sense. Today the school education stresses its importance on fostering the professionals to live up to the highly industrialized, mechanized, scientific and automatic age. When it is called the professional education, it should have professional knowledge how to get it and should include proper teaching method to instruct the students for the transfer of professional knowledge, Thereby the professional education can be easily connected to the vocation. Under the professional education tendency, the need of dancing education has been desperately required. Among dancing education, the ballet conducted by the most scientific and effective movement has been adjusted suitably to particular educational curriculum and its systems of each country under the well-organized and efficient methods. In the meantime regarding the ballet deucation of our country while the physical and technical conditions has been much improved, the organization of training program is still carless and inferior. The reason is that most teachers relying on their experience and brief performance, they don't prove the scientific and definite grounds. Accordingly, I would like to suggest some problems for the improvement of ballet education First, the emergent problem of teachers standard. Secondly, non-establishment of correct teaching method due to lack of systematic teaching method and its development. Thirdly, carelessness of scientific teaching approach based on the physical function and its roles. Accordingly, this thesis intends to not improve these problems but also push for efficiency of ballet class. Under this intention, the standard of dancing teacher, education curriculun and its contents of historic dancers, the developement of effective ballet teaching method have been researched by theoretical and functional appriach. Regarding the information obtained from the research, I propose the following measure. First, Regarding the teaching method, the four technological, scientific, art, creative methods, for technique development should be harmonized for the great effect of ballet class, not to select only one method. Secondly, The phtsical function of posture, the upper of body, pelvis, the lower limbs, turnout should be acknowledged to lead the class effectively, as well as protect any injury in advance.목차 = ⅲ 논문개요 = ⅶ Ⅰ. 서론 = 1 A. 연구의 필요성 및 목적 = 1 B. 연구의 방법 및 내용 = 3 Ⅱ. 발레의 발달 배경 및 교사의 자질 = 4 A. 발레의 발달 배경 = 4 B. 발레교사의 자질 = 12 Ⅲ. 발레지도의 기본 교수이론 = 16 A. 카를로 블라시스 (Carlo Blasis, 1797~1878) = 16 B. 앤리꼬 체께띠 (Enrico Cecchetti 1850~1928) = 22 C. 아그리삐나 바가노바 (Agrippina Vaganova, 1879~1951) = 26 Ⅳ. 발레지도의 효율적 접근 방법 = 31 A. 이론적 접근 = 32 1. 기술적 방법 = 32 2. 과학적 방법 = 33 3. 예술적 방법 = 36 4. 창조적 방법 = 38 B. 기능학적 접근 = 40 1. 자세 = 40 2. 상부 = 46 3. 골반 = 49 4. 하지 = 54 5. 턴-아웃 (Turn-Out) = 58 Ⅴ. 결론 = 62 Ⅵ. 참고문헌 = 65 Abstract = 6

Sex ratio of Acartia omorii with short period sampling in Jangmok Bay, Keoge

2001년 1월부터 2004년 6월까지 158차례의 단주기적 관측을 통하여 요각류인 Acartia omorii 의 성비(sex ratio) 변화 양상을 관찰하였다. 동물플랑크톤은 망목 300인 NORPAC 네트를 사용하여 수직채집하였고, Patch 영향을 최소화하기 위해 3회 반복 실시 하였다.2

ERK-dependent IL-6 autocrine signaling mediates adaptive resistance to pan-PI3K inhibitor BKM120 in head and neck squamous cell carcinoma

Hyperactivation of phosphatidylinositol 3-kinase (PI3K) pathway occurs frequently in head and neck squamous cell carcinoma (HNSCC). However, clinical outcomes of targeting the PI3K pathway have been underwhelming. In present study, we investigated the resistant mechanisms and potential combination therapeutic strategy to overcome adaptive resistance to PI3K inhibitor in HNSCC. Treatment of NVP-BKM120, a pan-PI3K inhibitor, led to upregulation of interleukin-6 (IL-6) and subsequent activation of either extracellular signal-regulated kinase (ERK) or signal transducers and activators of transcription 3 (STAT3), causing modest antitumor effects on the growth of HNSCC cells. Blockade of autocrine IL-6 signaling with siRNA or neutralizing antibody for IL-6 receptor (IL-6R) completely abolished NVP-BKM120-induced activation of ERK and STAT3 as well as expression of c-Myc oncogene, which resulted in enhanced sensitivity to NVP-BKM120. Moreover, when compared with a pharmacologic inhibitor or silencing of STAT3, trametinib, a MEK inhibitor, in combination with NVP-BKM120 yielded more potent anti-proliferative effects by inhibiting S phase transition, arresting cells at G0/G1 phase, and downregulating IL-6 and c-Myc expression. Furthermore, as compared with either agent alone, combination of NVP-BKM120 with trametinib or tocilizumab, a humanized anti-IL-6R antibody, significantly suppressed tumor growth in NVP-BKM120-resistant patient-derived tumor xenograft (PDTX) models, which was also confirmed in PDTX-derived cell lines. Collectively, these results suggested that IL-6/ERK signaling is closely involved in adaptive resistance of NVP-BKM120 in HNSCC cells, providing a rationale for a novel combination therapy to overcome resistance to PI3K inhibitors.restrictio