Summary of non-synonymous mutations for EPHB6 (NM_004445 and NP_004436) found in tumors.

<p>Note: The table contains data from the databases of <a href="http://www.sanger.ac.uk/genetics/CGP/cosmic/" target="_blank">http://www.sanger.ac.uk/genetics/CGP/cosmic/</a>, <a href="http://strubiol.icr.ac.uk/extra/mokca" target="_blank">http://strubiol.icr.ac.uk/extra/mokca</a>, and the references were listed in the column of “Pubmed Id”. The NSCLC mutations identified in this study were marked as “not reported”. Two sequence homology-based tools were used to predict the potential impact of the identified non-synonymous substitutions on protein function: Sort Intolerant from Tolerant (SIFT; <a href="http://sift.bii.a-star.edu.sg/" target="_blank">http://sift.bii.a-star.edu.sg/</a>) and Polymorphism Phenotype (PolyPhen-2; <a href="http://genetics.bwh.harvard.edu/pph2/" target="_blank">http://genetics.bwh.harvard.edu/pph2/</a>). If the SIFT prediction tolerance index score was less than 0.05, the variation was considered possibly damaging. Predictions made by PolyPhen-2 were assigned as “probably damaging,” “possibly damaging” or “benign.” Deletion mutations cannot be tested by either SIFT or PolyPhen-2.</p

Proliferative activity and cell size of EPHB6 wildtype and mutant cells.

<p>A) Proliferative activity of empty vector control, EPHB6 wildtype and mutant cells were analyzed using a colorimetric MTT assay after 72 hours. Data are shown as means +/− standard deviation of three independent experiments. Differences were statistically not significant (ANOVA). B) Cell size of individual cells (n = 20) growing on plastic dishes was analyzed by live video microscopy and recorded. EPHB6 mutant cells showed a significantly reduced cell size in comparison to EPHB6 wild type and to control cells (p<0.05, t-test).</p

Migration analysis of EPHB6 expressing NSCLC cells.

<p>A) Protein expression of stably transfected A549 cell lines expressing wild type EPHB6 or the EPHB6 deletion mutant. Cells were co-transfected using an EGFP -pcDNA3.1⁺ vector for identification of selected clones. Multiple clones were pooled and further selected as bulk cultures. B) Transwell migration assays were performed with empty vector control cells, EPHB6 mutant and EPHB6 wildtype cells. Five different experiments in triplicates were analyzed. *: significant (p<0.05) differences by (EITHER ANOVA OR t-test) The provided p-value between the three different cell lines was statistically analyzed from all migrated cells by using the OneWay ANOVA-test. The analysis of the pair-wise t-test results in a significant p-value for the control cells vs. EPHB6-wt cells (p<0.015) and between the EPHB6-wt cells and the EPHB6-mut cells (p<0.005). C) <i>In vitro</i> wound healing scratch assay. Cells were scratched by a 10 µl pipette tip. The scratch areas were recorded over a periode of 17 hours. Shown are means of three different experiments, calculated as percentage from one initial point for all three cell lines. The ANOVA-test (p<0.002) indicated statistically significant differences between the three cell lines. D) Representative images of the scratch assays at the beginning and the end of the experiments.</p

Development of metastasis <i>in vivo</i>.

<p>A) Number of pulmonary metastases in evaluable NOD/SCID mice four weeks after transplantation, each with 3×10⁵ stably transfected A549 cells expressing EPHB6-wt (n = 9), EPHB6-del915-917 (n = 9) or empty vector control cells (n = 2). Dots represent individual mice and horizontal lines the median value of metastases. B) Images from representative whole lungs of NOD/SCID mice, transplanted with A549 cells expressing EPHB6-wt, EPHB6-del915-917, or empty vector control. Lung metastases are marked by black arrows. C) Images from lung sections of NOD/SCID mice, stained with hematoxylin. Metastases are marked by black arrows. Three representative examples are shown each for mice transplanted with A549 cells expressing EPHB6-wt or EPHB6-del915-917.</p

A Phase I Dose Escalation Study of the Triple Angiokinase Inhibitor Nintedanib Combined with Low-Dose Cytarabine in Elderly Patients with Acute Myeloid Leukemia

<div><p>Nintedanib (BIBF 1120), a potent multikinase inhibitor of VEGFR-1/-2/-3, FGFR-1/-2/-3 and PDGFR-α/-β, exerts growth inhibitory and pro-apoptotic effects in myeloid leukemic cells, especially when used in combination with cytarabine. This phase I study evaluated nintedanib in combination with low-dose cytarabine (LDAC) in elderly patients with untreated or relapsed/refractory acute myeloid leukemia (AML) ineligible for intensive chemotherapy in a 3+3 design. Nintedanib (dose levels 100, 150, and 200 mg orally twice daily) and LDAC (20 mg subcutaneous injection twice daily for 10 days) were administered in 28-day cycles. Dose-limiting toxicity (DLT) was defined as non-hematological severe adverse reaction CTC grade ≥ 4 with possible or definite relationship to nintedanib. Between April 2012 and October 2013, 13 patients (median age 73 [range: 62–86] years) were enrolled. One patient did not receive study medication and was replaced. Nine (69%) patients had relapsed or refractory disease and 6 (46%) patients had unfavorable cytogenetics. The most frequently reported treatment-related adverse events (AE) were gastrointestinal events. Twelve SAEs irrespective of relatedness were reported. Two SUSARs were observed, one fatal hypercalcemia and one fatal gastrointestinal infection. Two patients (17%) with relapsed AML achieved a complete remission (one CR, one CRi) and bone marrow blast reductions without fulfilling PR criteria were observed in 3 patients (25%). One-year overall survival was 33%. Nintedanib combined with LDAC shows an adequate safety profile and survival data are promising in a difficult-to-treat patient population. Continuation of this trial with a phase II recommended dose of 2 x 200 mg nintedanib in a randomized, placebo-controlled phase II study is planned. The trial is registered to EudraCT as 2011-001086-41.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01488344" target="_blank">NCT01488344</a></p></div

MTSS1 expression is increased by ATRA treatment in t(15;17) AML.

<p>MTSS1 mRNA levels were measured in human AML cells (U937) transduced with either empty vector control (PMT Control) or an inducible PML-RARα (PR9) after 24 hours of activation with zinc (Zn) by qRT-PCR (n = 3; <b>A</b>). Similarly, MTSS1 mRNA levels were measured after treatment with all-trans retinoic acid (0.5μM ATRA) or 0.5μM DMSO control after 48 and 72 hours in human AML cells carrying the PML-RARα translocation (NB4) (n = 3; <b>B</b>).</p

FLT3-ITD mediated suppression of MTSS1 is pharmacologically reverted by PKC-412.

<p><i>Mtss1</i> mRNA levels were measured in murine 32D cells transduced with either empty vector control (EV) or a FLT3-ITD overexpression vector by qRT-PCR (FLT3-ITD; n = 3; <b>A</b>). Similarly, FLT3-ITD transduced murine 32D cells and human FLT3-ITD⁺ AML cells (MV4;11) were treated with the pharmacological FLT3-ITD inhibitor PKC-412 and DMSO control and <i>MTSS1</i> mRNA levels were measured after 24 hours (n = 3; <b>B</b> and <b>C</b>).</p

Frequency of patients with nintedanib-related adverse events across all dose levels.

<p>Frequency of patients with nintedanib-related adverse events across all dose levels.</p

Kaplan-Meier curve of overall survival.

<p>Median OS of treated patients was 234 days, and one-year OS (95% CI) was 33.3% (10.3–58.8%).</p

MTSS1 is positively regulated by AML1-ETO in human AML cells.

<p>The gene expression of MTSS1 in human AML cells (Kasumi-1 without transduction (no Trans), scramble siRNA (ctr RNAi) or siRNA targeting the AML1-ETO translocation is shown (GEO accession numbers GSE15646; <b>A</b>). Similarly, the gene expression of MTSS1, DNMT3B and 3A are measured in a gene expression analysis of human cord-blood derived CD34⁺ cells and patient-derived AML samples with the AML-1 ETO translocation (GEO accession numbers GSE8023; <b>B</b> and <b>C</b>). Human AML cell lines carrying either a FLT3-ITD mutation (MV4-11), t(15;17) (NB4), t(8;21) (Kasumi-1) or not defined (U937) were transduced with a MTSS1 firefly / renilla construct and the MTSS1 promoter activity as a normalized relative ratio was measured (n = 3;<b>D</b>).</p