Signaling response to transient redox stress in human isolated T cells: Molecular sensor role of syk kinase and functional involvement of IL2 receptor and L-selectine

Reactive oxygen species (ROS) are central effectors of inflammation and play a key role in cell signaling. Previous reports have described an association between oxidative events and the modulation of innate immunity. However, the role of redox signaling in adaptive immunity is still not well understood. This work is based on a novel investigation of diamide, a specific oxidant of sulfhydryl groups, and it is the first performed in purified T cell tyrosine phosphorylation signaling. Our data show that ex vivo T cells respond to –SH group oxidation with a distinctive tyrosine phosphorylation response and that these events elicit specific cellular responses. The expression of two essential T-cell receptors, CD25 and CD62L, and T-cell cytokine release is also affected in a specific way. Experiments with Syk inhibitors indicate a major contribution of this kinase in these phenomena. This pilot work confirms the presence of crosstalk between oxidation of cysteine residues and tyrosine phosphorylation changes, resulting in a series of functional events in freshly isolated T cells. Our experiments show a novel role of Syk inhibitors in applying their anti-inflammatory action through the inhibition of a ROS-generated reaction

Telomere length shortening is associated with treatment-free remission in chronic myeloid leukemia patients

We studied telomere length in 32 CML patients who discontinued imatinib after achieving complete molecular remission and 32 age-sex-matched controls. The relative telomere length (RTL) was determined by q-PCR as the telomere to single copy gene (36B4) ratio normalized to a reference sample (K-562 DNA). Age-corrected RTL (acRTL) was also obtained. The 36-month probability of treatment-free remission (TFR) was 59.4 %. TFR patients showed shorter acRTL compared to relapsed (mean ± SD = 0.01 ± 0.14 vs 0.20 ± 0.21; p = 0.01). TFR was significantly higher in CML patients with acRTL ≤0.09 (78.9 vs 30.8 %, p = 0.002). CML stem cells harboring longer telomeres possibly maintain a proliferative potential after treatment discontinuation

po 049 egfr blockade induces a paneth cell like phenotype with rewired signalling dependencies in crc tumoursat maximal response

Introduction Anti-EGFR therapies with the monoclonal antibodies cetuximab and panitumumab have improved survival in colorectal cancer (CRC) patients; nevertheless, incomplete mass obliteration and eventual relapse are a common setback, even after a plateau of maximal response. Preclinical data suggest that tumour recurrence may be fueled by a reservoir of so-called 'drug-tolerant persisters' that engage non-mutational routes of adaptation to therapy. Yet, the molecular underpinnings that sustain residual disease, as well as the strategies to oppose it, are largely unexplored. Material and methods The effects of targeted therapies were evaluated in patient-derived xenografts. The biochemical and biological consequences of drug exposure were gauged by immunohistochemistry and morphometric analyses (in vivo), and by time-lapse imaging, Western Blot, Cell Titer-Glo and Caspase-Glo assays (in vitro). Transcriptional perturbations were assessed by microarray analysis and/or RT-qPCR. The activity of transcriptional modulators was measured by reporter assays in vitro. Results and discussions Residual tumours surviving cetuximab treatment exhibited a quiescent, Wnt-high, and secretory/Paneth cell-like state as a distinctive trait. This pattern outlines that of EGFR-inhibited quiescent stem cells of the normal intestine, suggesting that developmental trajectories are somehow coopted by cancer cells to face external insults. Such phenotype was reversible with drug suspension, pointing to non-genetic plasticity as a determinant of cancer cell reprogramming. Residual tumours also displayed lower expression of EGFR-activating ligands, congruent with reduced EGFR dependency, and showed rewired reliance on compensatory HER2/HER3 activity, as well as persistent PI3K signalling. Mechanistically, the acquisition of Paneth cell-like features was mediated, at least partly, by inactivation of YAP – a key driver of intestinal cell regeneration. Therapeutically, combined blockade of EGFR and PI3K/AKT lessened residual disease burden, but did not lead to long-term disease control. However, treatment with panHER, a mixture of antibodies concurrently targeting EGFR, HER2, and HER3, reduced tumour volumes and delayed tumour relapse after therapy cessation. Conclusion Drug tolerance in cetuximab-sensitive CRC models involves a switch towards a Paneth-cell like state typified by sustained HER2/HER3 and PI3K signalling. Treatment with panHER effectively exhausted residual tumour burden and impeded/delayed late relapse

Intrinsic Resistance to MEK Inhibition in KRAS Mutant Lung and Colon Cancer through Transcriptional Induction of ERBB3

Summary There are no effective therapies for the ∼30% of human malignancies with mutant RAS oncogenes. Using a kinome-centered synthetic lethality screen, we find that suppression of the ERBB3 receptor tyrosine kinase sensitizes KRAS mutant lung and colon cancer cells to MEK inhibitors. We show that MEK inhibition results in MYC-dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. Drugs targeting both EGFR and ERBB2, each capable of forming heterodimers with ERBB3, can reverse unresponsiveness to MEK inhibition by decreasing inhibitory phosphorylation of the proapoptotic proteins BAD and BIM. Moreover, ERBB3 protein level is a biomarker of response to combinatorial treatment. These data suggest a combination strategy for treating KRAS mutant colon and lung cancers and a way to identify the tumors that are most likely to benefit from such combinatorial treatment

Colorectal cancer residual disease at maximal response to EGFR blockade displays a druggable Paneth cell–like phenotype

Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease

Separating Lentiviral Vector Injection and Induction of Gene Expression in Time, Does Not Prevent an Immune Response to rtTA in Rats

BACKGROUND: Lentiviral gene transfer can provide long-term expression of therapeutic genes such as erythropoietin. Because overexpression of erythropoietin can be toxic, regulated expression is needed. Doxycycline inducible vectors can regulate expression of therapeutic transgenes efficiently. However, because they express an immunogenic transactivator (rtTA), their utility for gene therapy is limited. In addition to immunogenic proteins that are expressed from inducible vectors, injection of the vector itself is likely to elicit an immune response because viral capsid proteins will induce "danger signals" that trigger an innate response and recruit inflammatory cells. METHODOLOGY AND PRINCIPAL FINDINGS: We have developed an autoregulatory lentiviral vector in which basal expression of rtTA is very low. This enabled us to temporally separate the injection of virus and the expression of the therapeutic gene and rtTA. Wistar rats were injected with an autoregulatory rat erythropoietin expression vector. Two or six weeks after injection, erythropoietin expression was induced by doxycycline. This resulted in an increase of the hematocrit, irrespective of the timing of the induction. However, most rats only responded once to doxycycline administration. Antibodies against rtTA were detected in the early and late induction groups. CONCLUSIONS: Our results suggest that, even when viral vector capsid proteins have disappeared, expression of foreign proteins in muscle will lead to an immune respons

A common variant of the MACC1 gene is significantly associated with overall survival in colorectal cancer patients

<p>Abstract</p> <p>Background</p> <p>The newly discovered metastasis-associated in colon cancer-1 (MACC1) gene is a key regulator of the HGF/MET pathway. Deregulation of HGF/MET signaling is reported as a prognostic marker for tumorigenesis, early stage invasion, and metastasis. High expression levels of MACC1 have been associated with colon cancer metastasis and reduced survival. Potential links between the genetic diversity of the MACC1 locus and overall survival are unknown. We therefore investigated the association between MACC1 tagging single nucleotide polymorphisms (SNPs) and overall survival in a large cohort of colorectal cancer patients.</p> <p>Methods</p> <p>The study included 318 subjects with histopathologically proven colorectal cancer at the Academic Teaching Hospital Feldkirch, Austria. Survival data were provided by the federal agency for statistics in Austria. Genomic DNA was isolated from formalin-fixed paraffin-embedded specimens; six tagging SNPs (rs1990172, rs3114446, rs10275612, rs3095007, rs3095009, and rs7780032), capturing most of the common variants of the MACC1 locus, were genotyped by SNaPshot assays.</p> <p>Results</p> <p>Over a mean follow up period of 5.3 (± 1.0) years, 94 deaths were recorded. Carriers of the G-allele of SNP rs1990172 showed a significantly decreased overall survival (additive HR = 1.38 [1.05-1.82]; <it>p </it>= 0.023). Multivariate analysis adjusted for age and UICC tumor stage confirmed this result (HR = 1.49 [1.12-1.98]; <it>p </it>= 0.007). Other investigated genetic variants of the MACC1 gene were not significantly associated with overall survival (<it>p</it>-values > 0.05).</p> <p>Conclusions</p> <p>For the first time, our study investigated the influence of MACC1 tagging polymorphisms on overall survival suggesting SNP rs1990172 as a predictor for reduced overall survival in colorectal cancer patients. Further studies will be required to validate our findings.</p