👤 Stefan Terjung

ROS1 fusions are well treatable aberrations in NSCLC. Besides solvent-front mutations (SFM) in resistance to targeted therapy, small-scale ROS1 mutations are largely unknown. We exploratively analyzed the clinical and molecular characteristics of small-scale ROS1 mutations in NSCLC patients without activating ROS1 fusions or SFMs. Next-generation sequencing was performed on tissue samples from NSCLC patients within the Network Genomic Medicine. Patients with ROS1 fusions and SFMs were excluded. We analyzed clinical characteristics of patients harboring small-scale ROS1-mutations, ROS1- and co-occurring mutations, and their response to systemic therapy. Of 10,396 patients analyzed, 101 (1.0%) patients harbored small-scale ROS1 mutations. Most patients were male (73.3%) and smokers (96.6%). Nearly half of the patients presented with squamous-cell carcinoma (SqCC, 40.4%). Most mutations were transversions (50.5%), and 66% were in the kinase domain. Besides TP53 mutations (65.3%), KRAS (22.8%), EGFR (5.9%), PIK3CA (9.9%) and FGFR1-4 mutations (8.9%) co-occurred. In 10 (9.9%) patients, ROS1 mutation was the only aberration detected. Median overall survival (mOS) differed significantly in patients with or without KRAS co-mutations (9.7 vs 21.5 months, p = 0.02) and in patients treated with or without immune-checkpoint blockade (ICB) during treatment (21.5 vs 4.4 months, p = 0.003). The cohort's clinical characteristics contrasted ROS1-fused cohorts. Co-occurrence of KRAS mutations led to shortened survival and patients benefited from ICB. Our data does not support the idea of ROS1 small-scale mutations as strong oncogenic drivers in NSCLC, but rather as relevant bystanders altering the efficacy of treatment approaches. Show less

We have compared the distribution of endogenous heterochromatin protein 1 (HP1) proteins (alpha, beta and gamma) in different epithelial lines, pluripotent stem cells and embryonic fibroblasts. In parallel, we have interrogated assembly and dynamics of newly expressed HP1-GFP proteins in cells lacking both HP1alpha and HP1beta alleles, blocked at the G1-S boundary, or cultured in the presence of HDAC and HAT inhibitors. The results reveal a range of cell type and differentiation state-specific patterns that do not correlate with 'fast' or 'slow' subunit exchange in heterochromatin. Furthermore, our observations show that targeting of HP1gamma to heterochromatic sites depends on HP1alpha and H1beta and that, on an architectural level, HP1alpha is the most polymorphic variant of the HP1 family. These data provide evidence for HP1 plasticity under shifting microenvironmental conditions and offer a new conceptual framework for understanding chromatin dynamics at the molecular level. Show less

We have examined HP1beta-chromatin interactions in different molecular contexts in vitro and in vivo. Employing purified components we show that HP1beta exhibits selective, stoichiometric, and salt-resistant binding to recombinant histone H3, associating primarily with the helical "histone fold" domain. Furthermore, using "bulk" nucleosomes released by MNase digestion, S-phase extracts, and fragments of peripheral heterochromatin, we demonstrate that HP1beta associates more tightly with destabilized or disrupted nucleosomes (H3/H4 subcomplexes) than with intact particles. Western blotting and mass spectrometry data indicate that HP1beta-selected H3/H4 particles and subparticles possess a complex pattern of posttranslational modifications but are not particularly enriched in me3K9-H3. Consistent with these results, mapping of HP1beta and me3K9-H3 sites in vivo reveals overlapping, yet spatially distinct patterns, while transient transfection assays with synchronized cells show that stable incorporation of HP1beta-gfp into heterochromatin requires passage through the S-phase. The data amassed challenge the dogma that me3K9H3 is necessary and sufficient for HP1 binding and unveil a new mode of HP1-chromatin interactions. Show less