MDSC subtypes and CD39 expression on CD8+ T cells predict the efficacy of anti-PD-1 immunotherapy in patients with advanced NSCLC.

The major suppressive immune cells in tumor sites are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and Treg cells, and the major roles of these suppressive immune cells include hindering T-cell activities and supporting tumor progression and survival. In this study, we analyzed the pattern of circulating MDSC subtypes in patients with non-small cell lung cancer (NSCLC) whether those suppressive immune cells hinder T-cell activities leading to poor clinical outcomes. First, we verified PMN-MDSCs, monocytic-MDSCs (M-MDSCs), and Treg cells increased according to the stages of NSCLC, and MDSCs effectively suppressed T-cell activities and induced T-cell exhaustion. The analysis of NSCLC patients treated with anti-PD-1 immunotherapy demonstrated that low PMN-MDSCs, M-MDSCs, and CD39+ CD8+ T cells as an individual and all together were associated with longer progression free survival and overall survival, suggesting PMN-MDSCs, M-MDSCs, and CD39+ CD8+ T cells frequencies in peripheral blood might be useful as potential predictive and prognostic biomarkers.

Entrectinib resistance mechanisms in ROS1-rearranged non-small cell lung cancer.

Entrectinib is a pan-tyrosine-kinase inhibitor that targets oncogenic rearrangements in NTRK, ROS1 and ALK. The combined results of two clinical trials demonstrated the efficacy of entrectinib in ROS1-rearranged NSCLC. Because the development of drug resistance is inevitable, it would be helpful to determine the mechanisms of entrectinib resistance in a ROS1-rearranged tumor model so that future therapeutic strategies can be developed. Here, we characterized the molecular basis of resistance in entrectinib-resistant ROS1-rearranged HCC78 cells (HCC78ER cells). These cells were analyzed by next-generation sequencing and genetic profiling, which revealed the acquisition of KRAS G12C and the amplification of KRAS and FGF3. However, there were no secondary mutations in the ROS1 kinase domain. We also found that sustained ERK activation was involved in entrectinib resistance, and that combined treatment with selumetinib resensitized HCC78ER cells to entrectinib in cell viability and colony formation assays. Our data suggest that activation of the RAS signaling pathway can cause entrectinib resistance in ROS1-rearranged NSCLC, and is unlikely to be overcome by sequential single agent ROS1-targeting strategies against such tumors. Instead, co-targeting ROS1 and MEK may be an effective strategy for overcoming entrectinib resistance in ROS1-rearranged NSCLC.

AZD9291 overcomes T790 M-mediated resistance through degradation of EGFR(L858R/T790M) in non-small cell lung cancer cells.

The discovery of activating mutations of epidermal growth factor receptor (EGFR) has resulted in the development of more effective treatments for non-small cell lung cancer (NSCLC). Although first-generation EGFR tyrosine kinase inhibitors (EGFR TKIs) provide significant clinical benefit, acquired resistance often occurs, most commonly (>50 %) via a T790 M resistance mutation. Although AZD9291 is selective for both T790 M and activating EGFR mutations over wild-type EGFR, it is highly active when T790 M is present, especially EGFR(L858R/T790M), and modestly active when T790 M is absent. The aim of this study was to elucidate the underlying mechanism of the high sensitivity of NSCLC cells harboring EGFR(L858R/T790M) to AZD9291. In H1975 cells harboring EGFR(L858R/T790M), AZD9291 potently inhibited cellular growth and EGFR signaling pathways together with depletion of mutant EGFR protein. AZD9291-induced depletion of EGFR(L858R/T790M) protein was abrogated through inhibition of the proteasome with MG132. However, AZD9291 had no effect on protein levels of EGFR(WT) and EGFR(L858R). In addition, AZD9291 induced apoptosis and caused expression changes in cell cycle-related genes. Moreover, oral administration of AZD9291 as a single agent induced tumor regression in vivo in a H1975 tumor xenograft model and reduced EGFR(L858R/T790M) protein levels in xenograft tumors. Taken together, our results provide a potential mechanism for the sensitivity of EGFR(L858R/T790M) cells to AZD9291 and suggest that AZD9291 may be effective in cases of T790 M-positive EGFR resistance.

Vascular calcifying progenitor cells possess bidirectional differentiation potentials.

Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1(+) (Sca-1(+)/PDGFRα(+) and Sca-1(+)/PDGFRα(-)) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1(-) (Sca-1(-)/PDGFRα(+) and Sca-1(-)/PDGFRα(-)) progenitor cells. Among Sca-1(+) progenitor populations, Sca-1(+)/PDGFRα(-) cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1(+)/PDGFRα(+) cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1(+)/PDGFRα(-) cells preferentially differentiated into osteoclast-like cells. Sca-1(+) progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1(+) calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1(+) progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1(+)/PDGFRα(-) cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1(+)/PDGFRα(-) cells into Apoe(-/-) mice increased the severity of calcified atherosclerotic plaques. However, Sca-1(+)/PDGFRα(-) cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1(+)/PDGFRα(-) cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.

BYL719, a selective inhibitor of phosphoinositide 3-Kinase α, enhances the effect of selumetinib (AZD6244, ARRY-142886) in KRAS-mutant non-small cell lung cancer.

PURPOSE: KRAS is frequently mutated in non-small cell lung cancers (NSCLC), resulting in activation of the MEK/ERK pathway. Because there are currently no drugs that target oncogenic KRAS, MEK inhibitors have been tested clinically as a possible treatment option for patients with NSCLC. However, KRAS-mutant cancers exhibit resistance to MEK inhibitors. Therefore, a combinational strategy is necessary for effective therapy. To address this, we investigated the therapeutic effects of combining selumetinib, a MEK1/2 inhibitor, with BYL719, a PI3Kα inhibitor. METHODS: We evaluated the effects of selumetinib and BYL719 in vitro and in vivo in NSCLC cell lines. RESULTS: The combination of BYL719 and selumetinib resulted in synergistic cytotoxic activity compared with the single agents alone in KRAS-mutant NSCLC cells. At the molecular level, we found that AKT activation strongly influenced the sensitivity of KRAS-mutant NSCLC cells to selumetinib. Selumetinib upregulated phospho-AKT and phosphorylated BAD at ser136, which is responsible for intrinsic drug resistance in KRAS-mutant NSCLC cells. In contrast, inhibition of the PI3K/AKT pathway by BYL719 hindered selumetinib-induced BAD phosphorylation and increased the antitumor efficacy of selumetinib. Furthermore, selumetinib and BYL719 combination therapy showed synergy in the suppression of A549 xenograft tumor growth. On analysis of the pharmacodynamics, selumetinib and BYL719 together resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. CONCLUSION: Taken together, these data suggest that combination treatment with selumetinib and BYL719 is a promising therapeutic approach to overcoming resistance to MEK inhibitors.

Structural Equation Modeling of Person-Centered Nursing in Hospital Nurses.

Background: This study aimed to develop and test a model of person-centered nursing (PCN) for hospital nurses using Structural Equation Modeling (SEM). Methods: This was a cross-sectional descriptive study. The subjects of this study were 340 clinical nurses in South Korea. A survey was conducted using an online questionnaire. Data were analyzed using SPSS 18.0 and AMOS 21.0. Results: Results of the validity test of the hypothesized model showed that goodness-of-fit indices satisfied the criteria (χ2 = 573.767 (df = 257, p < 0.001), TLI = 0.92, CFI = 0.93, SRMR = 0.02, RMSEA = 0.06), and 10 of the 15 paths established in the hypothesized model were statistically supported. The model's explanatory power was 82%, which showed that empathy directly and indirectly affected PCN, and that nursing competency directly affected clinical nurse performance of PCN. In addition, nursing competency, interpersonal relationship ability, and moral sensitivity indirectly influenced PCN through empathy. Conclusions: In order to promote PCN for clinical nurses, it is desirable to develop in-hospital education programs that can improve nursing competency, interpersonal relationship ability, and moral sensitivities, emphasizing elements of empathy. Empathy was an important mediating factor that influenced the relationships between PCN and related variables.

Extracellular Vesicles Generated Using Bioreactors and their Therapeutic Effect on the Acute Kidney Injury Model.

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, having beneficial effects for various types of regenerative processes. Although EVs have shown promising effects as therapeutic agents, these effects are difficult to research due to the limitations of EV production. In this study, an EV production method based on a flat-plate bioreactor is introduced. The bioreactor produces approximately seven times more mesenchymal stem cell-derived EVs than static culture conditions. The mechanism underlying the increased production of EVs in a flat-plate bioreactor and its application to acute kidney injury is investigated. This study describes the mechanism of EV production by demonstrating the link between EV biogenesis and increased calcium ion concentration under flow conditions. EVs secreted by cells cultured in the bioreactor have therapeutic efficacy in terms of improving kidney damage, resulting in tissue regeneration in a cisplatin-induced acute kidney injury model. This method will help overcome the limitations of EV production, and the analysis of the application of EVs will increase their reliability as well as the understanding of the use of bioreactor-derived EVs as therapeutic agents.

Mutational status of TP53 defines the efficacy of Wee1 inhibitor AZD1775 in KRAS-mutant non-small cell lung cancer.

KRAS is frequently mutated in non-small cell lung cancer (NSCLC). However, direct targeting of KRAS has proven to be challenging, and inhibition of KRAS effectors has resulted in limited clinical efficacy. Wee1 kinase is an important regulator of the G2 checkpoint and is overexpressed in various cancers. Inhibition of Wee1 exerts anticancer effects as a monotherapy or in combination with DNA-damaging agents when cancer cells harbor TP53 mutations. However, its role in KRAS-mutant NSCLC, especially as a single agent, has not been explored. Here, we investigate the anticancer potential of Wee1 inhibitor AZD1775 as a monotherapy and uncover a possible cellular context underlying sensitivity to AZD1775. Our data show that treatment with AZD1775 significantly inhibited cell survival, growth, and proliferation of TP53-mutant (TP53MUT) compared to TP53 wild-type (TP53WT) in KRAS-mutant (KRASMUT) NSCLC cells. In KRASMUT/TP53MUT cells, AZD1775 treatment led to DNA damage, a decrease of survival signaling, and cell death by apoptosis. Interestingly, cell death through apoptosis was found to be heavily dependent on specific cellular genetic context, rather than inhibition of Wee1 kinase activity alone. In addition, AZD1775 treatment was well tolerated and displayed single-agent efficacy in a mouse xenograft model. This study provides rationale for inhibiting Wee1 using AZD1775 as a potential anticancer therapy against the TP53MUT subgroup of KRASMUT NSCLC.

The CDK4/6 inhibitor LY2835219 has potent activity in combination with mTOR inhibitor in head and neck squamous cell carcinoma.

Deletion of CDKN2A (p16) or amplification of CCND1 (cyclin D1) occurs commonly in head and neck squamous cell carcinoma (HNSCC) and induces sustained cyclin-dependent kinase (CDK) 4/6 activation. Here, we report the antiproliferative activity of LY2835219, a selective CDK4/6 inhibitor through inhibition of CDK4/6-dependent Ser780 phosphorylation in retinoblastoma (RB) and induction of cell cycle arrest in HNSCC cells. In addition, we demonstrated the antitumor effects of HNSCC xenografts to LY2835219 in vivo. Given the limited effect in HNSCC as a single-agent treatment with LY2835219, a combinational strategy is required to enhance antitumor activity. At the molecular level, we found that LY2835219 inhibited activation of AKT and ERK, but not mTOR. The combination of LY2835219 with mTOR inhibitor was found to be more effective than either drug alone in vitro and in vivo. Taken together, our findings suggest that a combinational treatment with LY2835219 and mTOR inhibitor is a promising therapeutic approach for HNSCC.

A Phase Ib/II Study of Afatinib in Combination with Nimotuzumab in Non-Small Cell Lung Cancer Patients with Acquired Resistance to Gefitinib or Erlotinib.

PURPOSE: In this phase Ib/II study, we aimed to assess the safety and efficacy of afatinib plus nimotuzumab (N) in advanced non-small cell lung cancer (NSCLC) patients with acquired resistance to gefitinib or erlotinib. EXPERIMENTAL DESIGN: In phase Ib stage, patients received afatinib (40 mg or 30 mg once daily) plus nimotuzumab (100 mg or 200 mg once weekly) for 28-day cycles to determine the recommended phase II dose (RPIID). The safety and efficacy of RPIID dose was evaluated in phase II stage. RESULTS: In total, 50 patients were enrolled (13 to phase Ib and 37 to phase II). In the first dose-finding cohort (afatinib 40 mg plus nimotuzumab 100 mg), one patient experienced dose-limiting toxicity (DLT) of grade 3 diarrhea and in the subsequent cohort (afatinib 40 mg plus nimotuzumab 200 mg), two DLTs (grade 3 diarrhea and grade 3 neutropenia) occurred in 2 of 6 patients. Accordingly, RPIID was determined as afatinib 40 mg plus nimotuzumab 100 mg. In 44 patients treated with RPIID, 7 (16%) patients had grade 3 toxicities; skin rash (7%), diarrhea (5%), acne (2%), and fatigue (2%). The overall response rate was 23% and the median duration of response was 4.3 months (range, 0.7-16.2 months). The median progression-free survival and overall survival were 4.0 months [95% confidence interval (CI), 2.3-5.7 months] and 11.7 months (95% CI, 9.4-14.0 months), respectively. CONCLUSIONS: Combination treatment of afatinib and nimotuzumab demonstrated an acceptable safety profile and encouraging antitumor activity in advanced NSCLC patients with acquired resistance to gefitinib or erlotinib. Larger phase III trial is warranted to confirm its efficacy and safety. Clin Cancer Res; 22(9); 2139-45. ©2015 AACR.