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EGFR tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC) patients is inevitable. Identification of resistance mechanisms and corresponding targeting strategies can lead to more successful later-line treatment in many patients. Using spectrometry-based proteomics, we identified increased fibroblast growth factor receptor 1 (FGFR1) expression and Akt activation across erlotinib, gefitinib, and osimertinib EGFR-TKI-resistant cell line models. We show that while combined EGFR-TKI and FGFR inhibition showed some efficacy, simultaneous inhibition of FGFR and Akt or PI3K induced superior synergistic growth inhibition of FGFR1-overexpressing EGFR-TKI-resistant NSCLC cells. This effect was confirmed in vivo. Only dual FGFR and Akt inhibition completely blocked the resistance-mediating signaling pathways downstream of Akt. Further, increased FGFR1 expression was associated with significantly lower PFS in EGFR-TKI-treated NSCLC patients, and increased FGFR1 were demonstrated in a few post- vs. pre-EGFR-TKI treatment clinical biopsies. The superior therapeutic benefit of combining FGFR and Akt inhibitors provide the rationale for clinical trials of this strategy.

Background Early recurrence is a major obstacle to prolonged postoperative survival in squamous cell lung carcinoma (SqCLC). The molecular mechanisms underlying early SqCLC recurrence remain unclear, and effective prognostic biomarkers for predicting early recurrence are needed. Methods We analyzed primary tumor samples of 20 SqCLC patients using quantitative proteomics to identify differentially-expressed proteins in patients who experienced early versus late disease recurrence. The expression and prognostic significance of DDX56 was evaluated using a SqCLC tumor tissue microarray and further verified using different online databases. We performed in vitro and in vivo experiments to obtain detailed molecular insight into the functional role of DDX56 in SqCLC. Results We found that DDX56 exhibited increased expression in tumors of patients who experienced early versus late disease recurrence. Increased DDX56 expression in SqCLC tumors was subsequently confirmed as an independent prognostic factor of poor recurrence-free survival in independent SqCLC cohorts. Functionally, DDX56 promotes SqCLC cell growth and migration in vitro, and xenograft tumor progression in vivo. Mechanistically, DDX56 post-transcriptionally promotes expression of multiple Wnt signaling pathway-related genes, including CTNNB1, WNT2B, and represses a subset of miRNAs, including miR-378a-3p, a known suppressor of Wnt signaling. Detailed analysis revealed that DDX56 facilitated degradation of primary miR-378a, leading to down-regulation of mature miR-378a-3p and thus derepression of the target gene WNT2B. Conclusion We identified DDX56 as a novel independent prognostic biomarker that exerts its oncogenic effects through miRNA-mediated post-transcriptional regulation of Wnt signaling genes to promote early SqCLC recurrence. DDX56 may assist in identifying SqCLC patients at increased risk of early recurrence and who could benefit from Wnt signaling-targeted therapies.

IntroductionAcquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC.MethodsWe aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC.ResultsWe found that EGFRi resistance was mediated byup-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset ofacquired resistance to long-term EGFRi treatment invivo.AXL-expressing EGFRi-resistant cells revealed phenotypicand cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported toinhibit the autophagic flux invitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n= 1018).ConclusionOur results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-Targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-Agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

Background Epithelial to mesenchymal transition (EMT) is a well-characterized process of cell plasticity that may involve metabolic rewiring. In cancer, EMT is associated with malignant progression, tumor heterogeneity, and therapy resistance. In this study, we investigated the role of succinate dehydrogenase (SDH) as a potential key regulator of EMT. Methods Associations between SDH subunits and EMT were explored in gene expression data from breast cancer patient cohorts, followed by in-depth studies of SDH suppression as a potential mediator of EMT in cultured cells. Results We found an overall inverse association between EMT and the SDH subunit C (SDHC) when analyzing gene expression in breast tumors. This was particularly evident in carcinomas of basal-like molecular subtype compared to non-basal-like tumors, and a low SDHC expression level tended to have a prognostic impact in those patients. Studies in cultured cells revealed that EMT was induced by SDH inhibition through SDHC CRISPR/Cas9 knockdown or by the enzymatic inhibitor malonate. Conversely, overexpression of EMT-promoting transcription factors TWIST and SNAI2 caused decreased levels of SDHB and C and reduced rates of SDH-linked mitochondrial respiration. Cells overexpressing TWIST had reduced mitochondrial mass, and the organelles were thinner and more fragmented compared to controls. Conclusions Our findings suggest that downregulation of SDHC promotes EMT and that this is accompanied by structural remodeling of the mitochondrial organelles. This may confer survival benefits upon exposure to hostile microenvironment including oxidative stress and hypoxia during cancer progression. Electronic supplementary material The online version of this article (10.1186/s40170-019-0197-8) contains supplementary material, which is available to authorized users.

Background: Erlotinib, an EGFR selective reversible inhibitor, has dramatically changed the treatment of non-small cell lung cancer (NSCLC) as approximately 70% of patients show significant tumor regression upon treatment. However, all patients eventually relapse due to development of acquired resistance, which in 43-50% of cases is caused by a secondary mutation (T790M) in EGFR, and in 5-15% of cases is caused by MET amplification. However, a majority of resistance cases are still unexplained. Consequently, our aim was to identify novel resistance mechanisms – and potentially new drug targets - in erlotinib-resistant subclones of the NSCLC cell line HCC827. Materials and methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively), and prepared cDNA libraries of purified RNA from biological duplicates using TruSeq® Stranded Total RNA Ribo-Zero™ Gold (Illumina) prior to sequencing on an Illumina HiSeq platform (100bp paired end). The resistant subclones were examined both in presence and absence of erlotinib. The data was analyzed by an in-house developed pipeline including quality control by Trim Galore v0.3.3, mapping of reads to HG19 by TopHat2 v.2.0.10 and differential expression analysis by CuffDiff from the Cufflinks package v.2.2.1. Results: Significant differences in viability between the resistant clones and parental HCC827 were observed when incubated with erlotinib. Importantly, the resistant clones did not acquire the T790M mutation or other EGFR/KRAS mutations, indicating that other mechanisms are responsible for the resistance in these cells. We identified 303-581 transcripts being >2-fold upregulated (p < 0.001), and 460-679 transcripts being >2-fold downregulated (p < 0.001) in the three resistant clones treated with erlotinib compared to the parental cell line, HCC827. Among others, we found networks such as growth and proliferation, migration and oxidative stress to be upregulated by Ingenuity Pathway Analysis (IPA), whereas adherens junction signaling was downregulated. More specifically, transcripts encoding potential resistance drivers such as AXL kinase and Fibroblast Growth Factor Receptor 1 (FGFR1) were found consistently upregulated together with a number of central metabolic enzymes, which may also be important for resistance. Vimentin and CD44 encoding transcripts involved with epithelial-to-mesenchymal transition were consistently upregulated and CDH1 encoding E-cadherin was downregulated, indicating that EMT may be correlated with resistance to erlotinib. EGFR was either not regulated or downregulated in the three clones. MET was not regulated, whereas p53 expression was consistently downregulated. Conclusions: We established 3 erlotinib-resistant NSCLC subclones, which did not harbor any of the common resistance mutations, thus allowing for the identification of novel resistance mechanisms in these subclones. Cancer-related networks such as proliferation and migration were upregulated in the resistant clones, supporting the validity of the model. While EGFR was either not regulated or down-regulated, other potential resistance drivers such as AXL kinase and FGFR1, among others, were found consistently upregulated in the three resistant clones, which may indicate a cooperative bypass signaling mechanism to obtain resistance to erlotinib. Citation Format: Kirstine Jacobsen, Nicolas Alcaraz, Rikke Raaen Lund, Henrik Jørn Ditzel. Resistance mechanisms to erlotinib in the non-small cell lung cancer cell line, HCC827 examined by RNA-seq. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-59.

We have previously shown that the phenothiazine, thioridazine, acts in synergy with the beta-lactam antibiotic, dicloxacillin, to kill methicillin-resistant Staphylococcus aureus. In this study, we investigated whether synergy by combining these two drugs could also be observed in vancomycin intermediate susceptible S. aureus (VISA) and methicillin-resistant Staphylococcus epidermidis (MRSE). Synergy was observed in three of four tested VISA strains, suggesting that the thickening of cell wall does not interfere with the effects of thioridazine. In S. epidermidis, no synergy was observed in all tested strains, suggesting that synergy by combining thioridazine and dicloxacillin is isolated to S. aureus species.

Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms., EGFR-mutant non-small cell lung cancer are often resistant to EGFR tyrosine kinase inhibitor treatment. In this study, the authors show that resistant tumors display high Akt activation and that a combined treatment with AKT inhibitors causes synergistic tumour growth inhibition in vitro and in vivo.

The lac promoter is one of the most commonly used promoters for expression control of recombinant genes in E. coli. In the absence of galactosides, the lac promoter is repressed by its repressor protein LacI. Since the lac promoter is regulated by a repressor, overexpression of LacI is necessary for regulation when the promoter is introduced on a high-copy plasmid. For that purpose, a modified variant of LacI, a LVA-tagged LacI, was submitted to the Registry of Standard Biological Parts and has been used for more than 500 constructs since then. We have found, however, that natural LacI is superior to the LVA-tagged LacI as controller of expression.

Background In an attempt to explore cynomolgus monkeys as an animal model for Alzheimer’s disease, the present study focused on the Alzheimer’s biomarkers beta amyloid 1–42 (Ab42) in serum, and total tau (t-tau) and phosphorylated tau (p-tau) levels in cerebrospinal fluid. Methods We measured biomarker levels in Young and Aged cynomolgus monkeys and correlated these with performance on three delayed response tasks. Results The Ab42 concentration of the Aged monkeys was significantly lower than in the Young subjects, while the t-tau and p-tau did not significantly differ between the groups. The Young subjects performed significantly better than the Aged individuals on the memory tests. Only Ab42 levels were significantly correlated with performance in the three delayed response tasks. Conclusions Circulating Ab42 levels were lower in Aged monkeys and were correlated with inferior performance on delayed response tasks in Aged animals; therefore, both measures may be useful in establishing cynomolgus monkeys as models for studies of AD.

The neuroleptic antipsychotic derivate thioridazine has been shown to increase the susceptibility of a methicillin-resistant Staphylococcus aureus (MRSA) isolate towards dicloxacillin. The aim of this study was to investigate the combinatorial effect of the two drugs on a broad selection of staphylococcal strains by analyzing a large collection of MRSA strains carrying different types of SCCmec, as well as MSSA strains. Transcription and translation of the resistance marker PBP2a encoded by mecA within the SCCmec cassette were analyzed by primer extension and western blotting. We observed increased susceptibility to dicloxacillin in the presence of thioridazine in all tested MRSA isolates. In contrast to previously published results, the synergistic effect was also applicable to methicillin-susceptible S. aureus (MSSA). We conclude that the combination of dicloxacillin and thioridazine potentiates the killing effect against S. aureus in a broad selection of clinical isolates. Additionally, the study indicates that the killing effect by the combinatorial treatment is independent of PBP2a-mediated resistance mechanisms.

Background: EGFR inhibitors, erlotinib and gefitinib, cause tumor shrinkage in approximately 70% of non-small cell lung cancer (NSCLC) patients with activating mutations in EGFR, but may also be efficient in a subset of patients with EGFR wildtype, as indicated by a retrospective analysis of the BR.21 trial. However, there is currently a lack of biomarkers predictive for outcome of EGFR inhibitor-treated NSCLC patients with EGFR wildtype. The identification of such biomarkers was the aim of this study. Materials and methods: Formalin-fixed paraffin-embedded tissue blocks from 50 NSCLC patients with EGFR wildtype, who received treatment with erlotinib/gefitinib were included and stratified according to progression-free-survival (PFS). Twelve patients experienced no progression for >6 months (Good Response Group), while 38 patients experienced progression in Results: The patient cohort represented 20 men and 30 women. Two were non-smokers, 45 were smokers/former smokers and three were unknown. 42 exhibited adenocarcinoma histology and eight exhibited bronchioalveolar carcinoma histology. The majority of patients were treated with erlotinib (n = 47), while three received gefitinib. Mean PFS of patients in the Good Response Group was 455 days, while patients in the Poor Response Group had a mean PFS of 66 days, indicating that there are indeed patients with EGFR wildtype who benefit from EGFR inhibitors. From the quantitative LC-MS/MS analysis, we identified and quantified 3152 high-confident proteins across the 50 patients with ≥2 unique peptides. Softwares R and GProX were used for statistical analysis. Data was quantile normalized and revealed 30 proteins which were found to be differentially expressed between the two groups with a p-value Conclusions: From the PFS analysis of our patient cohort, it is clear that patients exhibiting EGFR wildtype may also benefit from treatment with EGFR inhibitors. Using unbiased mass spectrometry, we identified 30 differentially expressed proteins with the potential to predict outcome of EGFR inhibitor-treated NSCLC patients with EGFR wildtype. Citation Format: Kirstine Jacobsen, Brian Hood, Karen Ege Olsen, Thomas Conrads, Henrik Ditzel. Protein biomarkers predictive of outcome in EGFR inhibitor-treated non-small cell lung cancer patients with EGFR wildtype. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C11.

Background: Erlotinib (Tarceva®, Roche) has significantly changed the treatment of non-small cell lung cancer (NSCLC) as 70% of patients show significant tumor regression when treated. However, all patients relapse due to development of acquired resistance, which in 43-50% of cases are caused by a secondary mutation (T790M) in EGFR. Importantly, a majority of resistance cases are still unexplained. Our aim is to identify novel resistance mechanisms in erlotinib-resistant subclones of the NSCLC cell line HCC827. Materials & Methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively), and analyzed these by quantitative proteomics. The resistant subclones were examined both in absence and presence of erlotinib, and in biological triplicates on a Q-Exactive mass spectrometer. Results: Importantly, the resistant clones did not acquire the T790M or other EGFR or KRAS mutations, potentiating the identification of novel resistance mechanisms. We identified 2875 cytoplasmic proteins present in all 4 cell lines. Of these 87, 56 and 23 are upregulated >1.5 fold; and 117, 72 and 32 are downregulated >1.5 fold, respectively, in the 3 resistant clones compared to the parental cell line. By network analysis, we found cell survival, proliferation and migration to be induced, and apoptosis and adhesion to be repressed across the 3 resistant clones vs the parental cell line. The resistant cells generally lost phosphorylation of EGFR, MET, FGFR and Src, but surprisingly not of AKT and FOXO1/3a, indicating that AKT is the main signaling hub for survival. Also Erk1/2 phsphorylation is pertained although at decreased levels. Conclusions: In conclusion, cancer-related networks such as proliferation and apoptosis were found to be regulated, supporting the validity of the model. Phosphorylation of EGFR was lost in resistant cells. Activation of AKT was pertained in the resistant cells, indicating a possible mechanism of erlotinib resistance.

Few drugs are available against methicillin-resistant S. aureus (MRSA), and decreased susceptibility among staphylococci to newly introduced agents such as linezolid, daptomycin, and tigecycline has been observed [1-3]. Consequently, new treatment strategies are urgently needed. Thioridazine is a phenothiazine derivate, which is known to restore in vitro susceptibility of MRSA strains to β-lactam antibiotics (e.g. oxacillin) [4]. Previously, we have demonstrated that thioridazine prevents the oxacillin-induced transcription of the resistance determinant mecA in a hospital-associated MRSA isolate leading to a decreased level of penicillin-binding protein 2a (PBP2a) [5]. In the community-associated MRSA strain USA300, however, thioridazine does not affect the mecA mRNA level, thus a decreased PBP2a level is unlikely to constitute the basis of resensitization (Bonde M, Jacobsen K, unpublished data). In order to examine the underlying mechanism we are conducting a global transcriptional analysis of the response of strain USA300 to thioridazine treatment with and without oxacillin. We show that thioridazine by itself elicits extensive transcriptional changes, including pathways involved in amino acid synthesis and transport, cell wall maintenance, and energy metabolism. Identification of the critical pathways involved in restoration of β-lactam sensitivity will aid the optimization of phenothiazines for future combinatorial treatment of infections caused by MRSA strains. 1. Ross JE et al. J Chemother 2011, 23: 71-62. Sakoulas G et al. J Clin Microbiol 2008, 46: 220-43. Fritsche TR et al. Diagn Microbiol Infect Dis 2005, 52: 195-2014. Kristiansen MM et al. Int J Antimicrob Agents 2003, 22:250-35. Klitgaard JK et al. J Antimicrob Chemother 2008, 62: 1215-21