Kirstine Jacobsen

A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB

The multicopy sRNA LhrC of the intracellular pathogen Listeria monocytogenes has been shown to be induced under infection-relevant conditions, but its physiological role and mechanism of action is not understood. In an attempt to pinpoint the exact terms of LhrC expression, cell envelope stress could be defined as a specific inducer of LhrC. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. lapB mRNA, encoding a cell wall associated protein that was recently identified as an important virulence factor, was disclosed to be directly bound by LhrC leading to an impediment of its translation. Although LhrC binds to Hfq, it does not require the RNA chaperone for stability or lapB mRNA interaction. The mechanism of LhrC-lapB mRNA binding was shown to involve three redundant CU-rich sites and a structural rearrangement in the sRNA. This study represents an extensive depiction of a so far uncharacterized multicopy sRNA and reveals interesting new aspects concerning its regulation, virulence association and mechanism of target binding.

Thioridazine potentiates the effect of a beta-lactam antibiotic against Staphylococcus aureus independently of mecA expression

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.

Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer

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.

Abstract C11: Protein biomarkers predictive of outcome in EGFR inhibitor-treated non-small cell lung cancer patients with EGFR wildtype

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.

Abstract A2-59: Resistance mechanisms to erlotinib in the non-small cell lung cancer cell line, HCC827 examined by RNA-seq

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 2-fold downregulated (p 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 Jorn 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.