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Application of Bliss independence to test for synergy effect of TKI combination treatment in vivo

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non–small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro. The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Supplementary Figures S1-3; Supplementary Tables S1-3

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non–small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro. The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non–small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3–DXd is an antibody–drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3–DXd across a series of EGFR inhibitor–resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3–DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3–DXd. The combination of osimertinib and HER3–DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients. Significance: EGFR inhibition leads to increased HER3 membrane expression and promotes HER3–DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3–DXd combination in EGFR-mutant lung cancer. See related commentary by Lim et al., p. 18

Purpose:Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking.Experimental Design:We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non–small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations.Results:In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors.Conclusions:This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.

Supplementary Data from Comparative Analysis and Isoform-Specific Therapeutic Vulnerabilities of KRAS Mutations in Non–Small Cell Lung Cancer

Summary of EGFR inhibitor resistant patient-derived xenografts.

Antibodies used in the study

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non–small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3–DXd is an antibody–drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3–DXd across a series of EGFR inhibitor–resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3–DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3–DXd. The combination of osimertinib and HER3–DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients.Significance:EGFR inhibition leads to increased HER3 membrane expression and promotes HER3–DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3–DXd combination in EGFR-mutant lung cancer.See related commentary by Lim et al., p. 18

Allelic Frequencies in PC-9 vs PC-9 T790M/C797S cells.

Supplementary Figures and legends

RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K, and RB pathways. Within the MAPK pathway, ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing antitumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS-mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here, we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS-mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and—equally important—to identify biomarkers for patient stratification.

Chemistry - a European journal 28(36), e202200883 (2022). doi:10.1002/chem.202200883, Published by Wiley-VCH, Weinheim

Purpose:Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking.Experimental Design:We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non–small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations.Results:In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors.Conclusions:This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.

Advanced healthcare materials 11(24), 2200989 (2022). doi:10.1002/adhm.202200989 special issue: "Special Issue:Biofabrication Applications", Published by Wiley-VCH, Weinheim

RSC Advances 10(37), 21933-21939 (2020). doi:10.1039/D0RA01607A, Published by RSC Publishing, London

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in T cell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores T cell infiltration in vivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.

Non-small-cell lung cancer (NSCLC) with Kirsten rat sarcoma (KRAS) mutations has notoriously challenged oncologists and researchers for three notable reasons: (1) the historical assumption that KRAS is “undruggable”, (2) the disease heterogeneity and (3) the shaping of the tumor microenvironment by KRAS downstream effector functions. Better insights into KRAS structural biochemistry allowed researchers to develop direct KRAS(G12C) inhibitors, which have shown early signs of clinical activity in NSCLC patients and have recently led to an FDA breakthrough designation for AMG-510. Following the approval of immune checkpoint inhibitors for PDL1-positive NSCLC, this could fuel yet another major paradigm shift in the treatment of advanced lung cancer. Here, we review advances in our understanding of the biology of direct KRAS inhibition and project future opportunities and challenges of dual KRAS and immune checkpoint inhibition. This strategy is supported by preclinical models which show that KRAS(G12C) inhibitors can turn some immunologically “cold” tumors into “hot” ones and therefore could benefit patients whose tumors harbor subtype-defining STK11/LKB1 co-mutations. Forty years after the discovery of KRAS as a transforming oncogene, we are on the verge of approval of the first KRAS-targeted drug combinations, thus therapeutically unifying Paul Ehrlich’s century-old “magic bullet” vision with Rudolf Virchow’s cancer inflammation theory.

Background Exosomes are extracellular vesicles (EVs) derived from endocytic compartments of eukaryotic cells which contain various biomolecules like mRNAs or miRNAs. Exosomes influence the biologic behaviour and progression of malignancies and are promising candidates as non-invasive diagnostic biomarkers or as targets for therapeutic interventions. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is used to assess gene expression in cancer exosomes, however, the ideal reference genes for normalization yet remain to be identified. Results In this study, we performed an unbiased analysis of high-throughput mRNA and miRNA-sequencing data from exosomes of patients with various cancer types and identify candidate reference genes and miRNAs in cancer exosomes. The expression stability of these candidate reference genes was evaluated by the coefficient of variation “CV” and the average expression stability value “M”. We subsequently validated these candidate reference genes in exosomes from an independent cohort of ovarian cancer patients and healthy control individuals by qRT-PCR. Conclusions Our study identifies OAZ1 and hsa-miR-6835-3p as the most reliable individual reference genes for mRNA and miRNA quantification, respectively. For superior accuracy, we recommend the use of a combination of reference genes - OAZ1/SERF2/MPP1 for mRNA and hsa-miR-6835-3p/hsa-miR-4468-3p for miRNA analyses.

Background Exosomes are extracellular vesicles (EVs) derived from endocytic compartments of eukaryotic cells which contain various biomolecules like mRNAs or miRNAs. Exosomes influence the biologic behaviour and progression of malignancies and are promising candidates as non-invasive diagnostic biomarkers or as targets for therapeutic interventions. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is used to assess gene expression in cancer exosomes, however, the ideal reference genes for normalization yet remain to be identified. Results In this study, we performed an unbiased analysis of high-throughput mRNA and miRNA-sequencing data from exosomes of patients with various cancer types and identify candidate reference genes and miRNAs in cancer exosomes. The expression stability of these candidate reference genes was evaluated by the coefficient of variation “CV” and the average expression stability value “M”. We subsequently validated these candidate reference genes in exosomes from an independent cohort of ovarian cancer patients and healthy control individuals by qRT-PCR. Conclusions Our study identifies OAZ1 and hsa-miR-6835-3p as the most reliable individual reference genes for mRNA and miRNA quantification, respectively. For superior accuracy, we recommend the use of a combination of reference genes - OAZ1/SERF2/MPP1 for mRNA and hsa-miR-6835-3p/hsa-miR-4468-3p for miRNA analyses.

RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K and RB pathways. Within the MAPK pathway ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing anti-tumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and - equally important - to identify biomarkers for patient stratification.

Background In cancer cells, fusion genes can produce linear and chimeric fusion-circular RNAs (f-circRNAs), which are functional in gene expression regulation and implicated in malignant transformation, cancer progression, and therapeutic resistance. For specific cancers, proteins encoded by fusion transcripts have been identified as innovative therapeutic targets (e.g., EML4-ALK). Even though RNA sequencing (RNA-Seq) technologies combined with existing bioinformatics approaches have enabled researchers to systematically identify fusion transcripts, specifically detecting f-circRNAs in cells remains challenging owing to their general sparsity and low abundance in cancer cells but also owing to imperfect computational methods. Results We developed the Python-based workflow “Fcirc” to identify fusion linear and f-circRNAs from RNA-Seq data with high specificity. We applied Fcirc to 3 different types of RNA-Seq data scenarios: (i) actual synthetic spike-in RNA-Seq data, (ii) simulated RNA-Seq data, and (iii) actual cancer cell–derived RNA-Seq data. Fcirc showed significant advantages over existing methods regarding both detection accuracy (i.e., precision, recall, F-measure) and computing performance (i.e., lower runtimes). Conclusion Fcirc is a powerful and comprehensive Python-based pipeline to identify linear and circular RNA transcripts from known fusion events in RNA-Seq datasets with higher accuracy and shorter computing times compared with previously published algorithms. Fcirc empowers the research community to study the biology of fusion RNAs in cancer more effectively.

Patients (pts) with locally advanced (LAPC) or metastatic pancreatic ductal adenocarcinoma (mPDAC) have a dismal prognosis. Recently, new combination chemotherapies such as FOLFIRINOX and nab-paclitaxel/gemcitabine have demonstrated superiority over gemcitabine monotherapy. However, a substantial proportion of pts cannot tolerate these intensive front-line protocols. Moreover, the long-term superiority of multiagent protocols over less intensive strategies remains to be shown. To provide a benchmark for future studies, we analyzed the outcome of patients with LAPC or mPDAC treated at the West German Cancer Center before the FOLFIRINOX/nab-paclitaxel + gemcitabine era. This retrospective analysis included 201 consecutive pts with LAPC and mPDAC treated between 2007 and 2011. Efficacy parameters were correlated with type of chemotherapy, number of treatment lines and clinicopathological parameters. Gemcitabine monotherapy was given as first-line therapy in 51.1%, whereas 48.9% received combination chemotherapies such as gemcitabine/oxaliplatin or FOLFOX. Patients received a median of two lines of treatment, with 54.8% receiving second-line and 37.9% receiving third- and further-line therapies. There was no significant difference between gemcitabine monotherapy and combination therapies. Despite moderate activity of first-line treatment, median overall survival for LAPC was 11.3 months and 8.7 months for mPDAC. Multivariate analysis identified age and number of treatment lines as prognostic markers. The long-term outcome of unselected pts with LAPC and mPDAC treated before the introduction of aggressive multiagent chemotherapy protocols compares favorably with the results of contemporary benchmark trials. This suggests a multifactorial benefit from interdisciplinary care provided over sequential treatment lines at high volume expert centers.

In the course of gaining new insights into the secondary metabolite profile of various Stachybotrys strains, in particular concerning triprenyl phenol-like compounds, so far, unknown metabolites with analogous structural features were discovered. Three novel meroterpenoids containing a chromene ring moiety, namely stachybotrychromenes A–C, were isolated from solid culture of the filamentous fungus Stachybotrys chartarum DSMZ 12880 (chemotype S). Their structures were elucidated by means of comprehensive spectroscopic analysis (1D and 2D NMR, ESI-HRMS, and CD) as well as by comparison with spectroscopic data of structural analogues described in literature. Stachybotrychromenes A and B exhibited moderate cytotoxic effects on HepG2 cells after 24 h with corresponding IC50 values of 73.7 and 28.2 μM, respectively. Stachybotrychromene C showed no significant cytotoxic activity up to 100 μM. Moreover, it is noteworthy that stachybotrychromenes A–C are produced not only by S. chartarum chemotype S but also S. chartarum chemotype A and Stachybotrys chlorohalonata. Electronic supplementary material The online version of this article (10.1007/s12550-018-0312-7) contains supplementary material, which is available to authorized users.

Background: Lung cancer accounts for one in five cancer deaths worldwide and mutations in the gene encoding for the Kirsten rat sarcoma (KRAS) oncoprotein define the largest molecular subset of non-small cell lung cancer (NSCLC). These tumors are characterized by activated MAPK signaling, however, no targeted inhibitors of mutant KRAS or of downstream signaling molecules have yet been approved for routine clinical use. Objective: The primary objective of this review is to critically summarize the current developmental state of MEK and ERK inhibitors in pre-clinical models and in human clinical trials for KRAS mutant lung cancer particularly in light of the newly emerging concept of immune checkpoint blockade. Method: We performed a Pubmed-based literature search and considered publications from the fields of basic and translational biomedicinal and biochemistry research, as well as from past and ongoing human clinical trials (www.clinicaltrials.gov). Results and Conclusions: MAPK pathway targeting agents are efficacious in pre-clinical models but their benefit is limited for patients with KRAS mutant NSCLC due to the lack of predictive factors, toxicity and the adaptive dynamic kinome reprogramming within the tumor. Overall, MEK inhibitors have advanced further in clinical development compared to ERK inhibitors. New treatment strategies as e.g. immune checkpoint blockade are currently revolutionizing the treatment paradigms and future clinical trials need to show if they replace MAPK targeting strategies or are used as add-on.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have changed the treatment landscape for EGFR-mutant non-small cell lung cancers (NSCLC); however, most patients develop resistance over time. HER3 is a unique pseudokinase member of the ERBB family which functions through dimerization with other ERBB family members (EGFR and HER2) and has been difficult to target with conventional kinase inhibitor strategies. HER3 is frequently over expressed in EGFR-mutant NSCLC. U3-1402 is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody (patritumab) linked to a topoisomerase I inhibitor (DX-8951 derivative, or DXd). U3-1402 is currently in clinical development as a single agent in metastatic EGFR-mutant NSCLC (NCT03260491). We aimed to develop a preclinical strategy to enhance the efficacy of U3-1402. Pre-treatment with EGFR TKIs (gefitinib or osimertinib) increased HER3 membrane levels in six different EGFR-mutant cell lines by increasing both the amount of HER3 positive cells and the intensity of HER3 expression. Furthermore, using a proximity-ligation assay (PLA), we noted an increase in membrane EGFR:HER3 interaction following EGFR inhibitor treatment. HER3 protein levels and EGFR:HER3 interaction were similarly increased in four EGFR-mutant patient-derived xenograft (PDX) models following osimertinib treatment. HER3 expression peaked between 24 and 48 hours, and the increase in HER3 was regulated by both increased gene expression and in the level of protein stability. We further evaluated the biological consequences of increased HER3 expression on U3-1402 intake and activity. We used pH-sensitive pHrodo Red conjugate to label U3-1402 to monitor the intake of the ADC using Incucyte live cell imaging. In three EGFR-mutant cell lines, there was a 3- to 5-fold higher intake of U3-1402 over time in cell lines pre-treated with osimertinib, compared to cell lines without pre-treatment. Osimertinib pre-treatment also increased the uptake of U3-1402 in HER3-low expressing EGFR-mutant models. In contrast, osimertinib pre-treatment did not increase the uptake of a control IgG-ADC. In long-term in vitro cell growth assays, the combination of osimertinib and U3-1402 was superior to single agent U3-1402 in four EGFR-mutant cell lines. Preliminary in vivo studies demonstrated no significant toxicities as measured by mouse body weights and complete blood counts. In vivo experiments evaluating the antitumor efficacy of the combination of osimertinib and U3-1402 are currently underway. Our studies reveal that EGFR inhibitor treatment increased membrane expression of HER3 which was associated with enhanced internalization of U3-1402 in EGFR-mutant NSCLC. The combination of osimertinib and U3-1402 may be an effective treatment approach and should be evaluated in future clinical trials in patients with EGFR-mutant NSCLC. Citation Format: Heidi M. Haikala, Jens Köhler, Timothy Lopez, Pinar Eser, Man Xu, Channing Yu, Yoshinobu Shiose, Yang Qiu, Prafulla Gokhale, Pasi A. Jänne. EGFR inhibition enhances the cellular uptake and antitumor activity of the novel HER3 antibody drug conjugate U3-1402 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5192.

Background: KRAS mutations are the most frequent oncogenic aberration in lung adenocarcinoma. Due to differences in protein structure and GTPase activity, KRAS mutant isoforms shape tumor biology and therefore may influence the treatment response in non-small-cell lung cancer. This heterogeneity challenges the development of effective targeted therapies for KRAS-driven lung cancer. Methods: Here, we systematically investigated MEK/ERK inhibitors sensitivity for different KRAS mutant isoforms. Then we developed an integrative pharmacogenomics analysis to identify potential targets in lung cancer with KRAS(G12C) mutation, the most frequent aberration in patients with primary or metastatic KRAS mutant non-small cell lung cancer. We further validated our prediction by siRNA-mediated gene knockdown and TOPFlash reporter assay. Findings: Our computational analysis identifies casein kinase 2A1 (CSNK2A1) as a mediator of MEK inhibitor resistance in KRAS(G12C) mutant cells which is not observed in cells with non-KRAS(G12C) mutations and in those harboring other oncogenic drivers as e.g. activating mutations in EGFR, BRAF, or NRAS. Knockdown of CSNK2A1 reduces proliferation, inhibits Wnt/I²-catenin signalling and increases the anti-proliferative effect of selumetinib in KRAS(G12C) mutant lung cancer cells. Interpretation: Our study suggested that accurate patients stratification will be necessary in order to observe significant benefit upon CK2 inhibition - alone or in combination - in a subset of patients with a favorable intratumoral genetic context. We provide a promising approach towards developing precision treatments for various subtypes of KRAS mutant lung cancer. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (31571363, 31771469, and 81573023 to HW) the National key research and development program of China (2017YFC0908500 to HW), the Lung Cancer Research Foundation (to CA) and a Mildred-Scheel postdoctoral fellowship from the German Cancer Aid Foundation (70111755 to JK). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: Not required.

Anisometric microgels are prepared via thermal crosslinking using an in-mold polymerization technique. Star-shaped poly(ethylene oxide-stat-propylene oxide) polymers, end-modified with amine and epoxy groups, form hydrogels, of which the mechanical properties and gelation rate can be adjusted by the temperature, duration of heating, and polymer concentration. Depending on the microgel stiffness, the rod-shaped microgels self-assemble into ordered or disordered structures.

Castleman disease (CD) is a rare lymphoproliferative disorder of unknown etiology and one of the uncommon causes of nonneoplastic lymphadenopathy. Earlier synonyms of CD included angiofollicular lymph node hyperplasia, giant lymph node hyperplasia, lymphoid hamartoma, benign lymphoma and follicular lymphoreticuloma. This disease was first reported by Castleman et al. in 1954 [1] and later defined by Castleman et al. in 1956 in a series of 13 patients with unicentric hyaline vascular CD presenting with large thymoma-like masses in the anterior mediastinum [2]. The condition was later found to be occasionally associated with hypochromic anemia, hypergammaglobulinemia and bone marrow plasmacytosis [3, 4]. By the late 1960s and the early 1970s, the histological features of the different forms of the disease and the classification in current use had been established [5, 6]. The disease features three main histopathological presentations: hyaline vascular, plasma cell-rich, and mixed variants [6, 7]. The exact incidence of CD is unknown. Association of certain forms of the disease with the human immunodeficiency virus (HIV) and human herpes virus-8 (HHV-8) infections has been long recognized. In 1985, Lachant et al. reported on 2 patients with the acquired immunodeficiency syndrome (AIDS) who developed multicentric CD followed by Kaposi sarcoma [8]. In 1995, Soulier et al. reported frequent coincidence of HHV-8 in 31 patients with multicentric CD [9].

The inducement of compressive residual stress by means of deep rolling (also known as ball or roller burnishing) has been widely investigated over the last decades; however, the influence of cyclic loading on stress relaxation of rolled components has not received the same attention. Therefore, the principal aim of this work is to study the behaviour of macro- and microresidual stresses (the latter assessed in terms of the full width at half maximum, FWHM) on the surface of AISI 1060 steel specimens under rotating bending tests. For this purpose, samples with three distinct heat treatments (subcritical annealing, full annealing and hardening by quenching and tempering) were deep rolled at various combinations of rolling pressure and number of passes. The results indicate that the tensile residual stress obtained after turning shifted to compressive stress after deep rolling. The FWHM decreased after deep rolling at the lowest pressure, thus suggesting that deep rolling promoted a better distribution of the dislocations induced by previous turning; however, the further elevation of rolling pressure and number of passes caused the elevation of the FWHM. The macro- and microresidual stress values were not drastically altered after rotating bending testing with an applied stress corresponding to 50 % of the yield strength. Nevertheless, residual stress relaxation was observed with an applied stress of 80 % of the yield strength, though the FWHM did not provide evidence of microresidual stress relaxation. Finally, the analysis of the fractured cross-sections of hardened specimens indicated that the location of the crack initiation site deepens with the elevation of rolling pressure.

This article presents a technical concept and machining possibilities for milling of micro structures with a fast tool servo (FTS). The tool is capable of tool tip displacements with a variable frequency up to 4000Hz and an amplitude up to 30μm. The control system of the actuator is linked to the angular spindle position, allowing to freely manipulate the excitation signal. Combining this tool system with a single tooth face milling head, micro structures on flat surfaces can be generated on conventional milling centers. Selected micro structures are machined and evaluated in terms of precision and surface quality.Video abstract

Natural healing is based on highly orchestrated processes, in which the extracellular matrix plays a key role. To resemble the native cell environment, we introduce an artificial extracellular matrix (aECM) with the capability to template hierarchical and anisotropic structures in situ, allowing a minimally-invasive application via injection. Synthetic, magnetically responsive, rod-shaped microgels are locally aligned and fixed by a biocompatible surrounding hydrogel, creating a hybrid anisotropic hydrogel (Anisogel), of which the physical, mechanical, and chemical properties can be tailored. The microgels are rendered cell-adhesive with GRGDS and incorporated either inside a cell-adhesive fibrin or bioinert poly(ethylene glycol) hydrogel to strongly interact with fibroblasts. GRGDS-modified microgels inside a fibrin-based Anisogel enhance fibroblast alignment and lead to a reduction in fibronectin production, indicating successful replacement of structural proteins. In addition, YAP-translocation to the nucleus increases with the concentration of microgels, indicating cellular sensing of the overall anisotropic mechanical properties of the Anisogel. For bioinert surrounding PEG hydrogels, GRGDS-microgels are required to support cell proliferation and fibronectin production. In contrast to fibroblasts, primary nerve growth is not significantly affected by the biomodification of the microgels. In conclusion, this approach opens new opportunities towards advanced and complex aECMs for tissue regeneration.

The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD¹⁵⁴Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers. The tumor-suppressive function of wild-type KRAS depends on its dimerization capacity with mutant KRAS.

This work addresses the influence of distinct microstructures and deep rolling parameters on the behaviour of AISI 1060 steel. For this purpose, the work material was initially subjected to subcritical and full annealing as well as to hardening through quenching and tempering. The specimens were subsequently deep rolled under different rolling pressures and numbers of passes. The findings indicate that plastic deformation increases with rolling pressure and number of passes due to more intense cold working and that under identical deep rolling conditions the fully annealed material presents more severe deformation than the subcritically annealed samples. Moreover, the ability of deep rolling to increase surface hardness decreases with the elevation of the hardness of the original material. The values of the yield and ultimate tensile strength were affected in different manners by deep rolling depending on work material condition and the tensile residual stresses observed after turning were converted into compressive values by deep rolling. Finally, the elevation of rolling pressure and number of passes presented distinct effects on the microhardness distribution beneath the surface depending on the work material condition.

The use of cemented tungsten carbide inserts in hard machining requires proper coatings and edge micro-geometries. A suitable choice of these factors enables the reduction of cutting forces, the increase of tool life and the improvement of workpiece surface quality. However, the effects of the edge preparation method and coating process on the substrate properties influence the performance of the insert during cutting. In this context, the influence of two different edge preparation methods (plunge-face grinding and brushing) on the performance of TiAlN-coated cemented tungsten carbide inserts during hard turning is investigated. In general, higher values of surface roughness (Rz ≅ 0.3 μm) and edge chipping (Rk ≅ 4.5 μm) were observed for brushed inserts in comparison to the ground tools (Rz ≅ 0.2 μm, Rk ≅ 3.7 μm). Moreover, higher compressive residual stresses are induced by brushing in comparison to grinding (≅ 500 MPa against ≅ 400 MPa). In turning tests, cutting forces for the ground inserts are approximately 10% higher than for the brushed inserts. However, higher wear values are better related to lower compressive residual stresses in the substrate than to higher cutting forces.

In various tumours PET/CT with [(18)F]FDG is widely accepted as the diagnostic standard of care. The purpose of this study was to compare a dedicated [(18)F]FDG PET/MRI protocol with [(18)F]FDG PET/CT for TNM staging in a cohort of oncological patients.A dedicated [(18)F]FDG PET/MRI protocol was performed in 73 consecutive patients (mean age of 59 years, range 21 - 85 years) with different histologically confirmed solid primary malignant tumours after a routine clinical FDG PET/CT scan (60 min after injection of 295 ± 45 MBq [(18)F]FDG). TNM staging according to the 7th edition of the AJCC Cancer Staging Manual was performed by two readers in separate sessions for PET/CT and PET/MRI images. Assessment of the primary tumour and nodal and distant metastases with FDG PET/CT and FDG PET/MRI was based on qualitative and quantitative analyses. Histopathology, and radiological and clinical follow-up served as the standards of reference. A McNemar test was performed to evaluate the differences in diagnostic performance between the imaging procedures.From FDG PET/CT and FDG PET/MRI T stage was correctly determined in 22 (82 %) and 20 (74 %) of 27 patients, N stage in 55 (82 %) and 56 (84 %) of 67 patients, and M stage in 32 (76 %) and 35 (83 %) of 42 patients, respectively. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy for lymph node metastases were 65 %, 94 %, 79 %, 89 % and 87 % for PET/CT, and 63 %, 94 %, 80 %, 87 % and 85 % for PET/MRI. The respective values for the detection of distant metastases were 50 %, 82 %, 40 %, 88 % and 76 % for PET/CT, and 50 %, 91 %, 57 %, 89 % and 83 % for PET/MRI. Differences between the two imaging modalities were not statistically significant (P 0.05).According to our results, FDG PET/CT and FDG PET/MRI are of equal diagnostic accuracy for TNM staging in patients with solid tumours.

Therapeutic decisions in non-small cell lung cancer (NSCLC) patients depend on the tumor stage. PET/CT with (18)F-FDG is widely accepted as the diagnostic standard of care. The purpose of this study was to compare a dedicated pulmonary (18)F-FDG PET/MR imaging protocol with (18)F-FDG PET/CT for primary and locoregional lymph node staging in NSCLC patients using histopathology as the reference.Twenty-two patients (12 men, 10 women; mean age ± SD, 65.1 ± 9.1 y) with histopathologically confirmed NSCLC underwent (18)F-FDG PET/CT, followed by (18)F-FDG PET/MR imaging, including a dedicated pulmonary MR imaging protocol. T and N staging according to the seventh edition of the American Joint Committee on Cancer staging manual was performed by 2 readers in separate sessions for (18)F-FDG PET/CT and PET/MR imaging, respectively. Results from histopathology were used as the standard of reference. The mean and maximum standardized uptake value (SUV(mean) and SUV(max), respectively) and maximum diameter of the primary tumor was measured and compared in (18)F-FDG PET/CT and PET/MR imaging.PET/MR imaging and (18)F-FDG PET/CT agreed on T stages in 16 of 16 of patients (100%). All patients were correctly staged by (18)F-FDG PET/CT and PET/MR (100%), compared with histopathology. There was no statistically significant difference between (18)F-FDG PET/CT and (18)F-FDG PET/MR imaging for lymph node metastases detection (P = 0.48). For definition of thoracic N stages, PET/MR imaging and (18)F-FDG PET/CT were concordant in 20 of 22 patients (91%). PET/MR imaging determined the N stage correctly in 20 of 22 patients (91%). (18)F-FDG PET/CT determined the N stage correctly in 18 of 22 patients (82%). The mean differences for SUV(mean) and SUV(max) of NSCLC in (18)F-FDG PET/MR imaging and (18)F-FDG PET/CT were 0.21 and -5.06. These differences were not statistically significant (P0.05). The SUV(mean) and SUV(max) measurements derived from (18)F-FDG PET/CT and (18)F-FDG PET/MR imaging exhibited a high correlation (R = 0.74 and 0.86, respectively; P0.0001). Size measurements showed an excellent correlation between (18)F-FDG PET/MR imaging and (18)F-FDG PET/CT (R = 0.99; P0.0001). The lower and upper limits of agreement between (18)F-FDG PET/CT and (18)F-FDG PET/MR imaging using Bland-Altman analysis were -2.34 to 3.89 for SUV(mean), -7.42 to 4.40 for SUV(max), and -0.59 to 0.83 for the tumor size, respectively.(18)F-FDG PET/MR imaging using a dedicated pulmonary MR imaging protocol, compared with (18)F-FDG PET/CT, does not provide advantages in thoracic staging in NSCLC patients.

Plunge-face grinding is commonly used to finish PCBN cutting inserts. In order to reach an adequate process design, an investigation of the influence of the grinding parameters on the quality of high content PCBN inserts is carried out in this work. For this, the inserts are ground with different grinding wheels (including a variation of grain size and bonding), dressing feed rates, feed and cutting speeds and the edge chipping and flank face roughness are measured. It was found that a reduction of the abrasive grain size as well as an increase of the dressing feed rate lead to an improvement of the insert edge and surface quality. Moreover, a variation of the cutting and feed speeds has only a small influence on the PCBN insert quality.

The aim of this work is to study the influence of selected deep rolling parameters (rolling pressure and number of passes) on the surface integrity of fully annealed AISI 1060 high carbon steel. In addition to the mechanical properties, a comprehensive investigation on surface integrity is carried out. The findings indicate that despite the increase in surface hardness and ultimate tensile stress, deep rolling can negatively affect the yield strength. The amplitude and functional roughness parameters show a considerable reduction after deep rolling, however, increasing rolling pressure and number of passes leads to poorer surface finish. Finally, the tensile residual stress generated by turning shifts to compressive values after deep rolling and the microhardness and microstructure analyses indicate that the depth of the layer affected by deep rolling depends on both the rolling pressure and number of passes. DFG/CRC/653 CAPES Foundation, Ministry of Education of Brazil

PCBN cutting inserts have been more often used in order to attend to the demands of an economically viable process and to lead to a proper workpiece surface quality. A proper application of this cutting material requires its adequate processing. Plunge-face grinding is used for finishing the inserts after sintering. To choose a suitable grinding tool and process parameters, the properties of the ground cutting inserts must be taken into account. Therefore, the influence of PCBN grain size and composition on the insert cutting edge and surface quality has to be investigated. This work aims to give an overview of material removal mechanisms, process forces and abrasive grain wear during grinding different PCBN inserts. It was found that the insert quality depends mainly on the material removal mechanism, which in the studied case is defined by the PCBN grain size.