Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors.

Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.

Identification of Neural Mechanisms in First Single-Sweep Analysis in oVEMPs and Novel Normative Data.

BACKGROUND: Bone-conducted (BC) VEMPs provide important tools for measuring otolith function. However, two major drawbacks of this method are encountered in clinical practice-small n10 amplitude and averaging technique. In this study, we present the results of a new VEMP setup measuring technique combined with a novel single-sweep analysis. METHODS: The study included BC oVEMP data from 92 participants for the evaluation of normative data using a novel analysis technique. For evaluating test-retest reliability, the intraclass correlation coefficient (ICC) was used. RESULTS: We found significant n10 amplitude differences in single-sweep analyses after the first and second measurements. Thereby, mathematical analyses of the head movement did not show any differences in the first or second measurements. The normative n10 amplitude was 20.66 µV with an asymmetric ratio (AR) of 7%. The new value of late shift difference (LSD) was 0.01 ms. The test retest-reliability showed good to excellent ICC results in 9 out of 10 measurements. CONCLUSIONS: Our results support a phenomenon in single-sweep analysis of the first stimuli independent of head movement and signal morphology. Furthermore, the values obtained with the new measurement method appear to be more sensitive and may allow an extended diagnostic range due to the new parameter LSD.

KRasG12C inhibitors in clinical trials: a short historical perspective.

KRas is the most frequently mutated oncogene in human cancer, and even 40 years after the initial discovery of Ras oncogenes in 1982, no approved drug directly targets Ras in Ras-driven cancer. New information and approaches for direct targeting of mutant Ras have fueled hope for the development of direct KRas inhibitors. In this review, we provide a comprehensive historical perspective of the development of promising KRasG12C inhibitors that covalently bind to the mutated cysteine residue in the switch-II pocket and trap the protein in the inactive GDP bound state. After decades of failure, three covalent G12C-specific inhibitors from three independent companies have recently entered clinical trials and therefore represent new hope for patients suffering from KRasG12C driven cancer.

Characterization of Covalent Pyrazolopyrimidine-MKK7 Complexes and a Report on a Unique DFG-in/Leu-in Conformation of Mitogen-Activated Protein Kinase Kinase 7 (MKK7).

Pyrazolopyrimidines are well-established as covalent inhibitors of protein kinases such as the epidermal growth factor receptor or Bruton's tyrosine kinase, and we recently described their potential in targeting mitogen-activated protein kinase kinase 7 (MKK7). Herein, we report the structure-activity relationship of pyrazolopyrimidine-based MKK7 inhibitors and solved several complex crystal structures to gain insights into their binding mode. In addition, we present two structures of apo-MKK7, exhibiting a DFG-out and an unprecedented DFG-in/Leu-in conformation.

POLE Score: a comprehensive profiling of programmed death 1 ligand 1 expression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) being characterized by a pronounced stromal compartment is commonly diagnosed at an advanced stage limiting curative treatment options. Although therapeutical targeting of immune checkpoint regulators like programmed death 1 ligand 1 (PD-L1) represent a promising approach that substantially improved survival of several highly aggressive malignancies, convincing indicators for response prediction are still lacking for PDAC which might be attributed to the insufficient characterization of PD-L1 status. Therefore, we investigated PD-L1 expression by immunohistochemistry in a well characterized cohort of 59 PDAC and 18 peritumoral tissues. Despite the histopathological homogeneity within our cohort, tumor tissues exhibited a great heterogeneity regarding PD-L1 expression. Considering distinct PD-L1 expression patterns, we established the novel POLE Score that incorporates overall PD-L1 expression (P), cellular Origin of PD-L1 (O), PD-L1 level in tumor-associated Lymph follicles (L) and Enumerated local PD-L1 distribution (E). We show that tumoral PD-L1 expression is higher compared to peritumoral areas. Furthermore, POLE Score parameters correlated with overall survival, tumor grade, Ki67 status, local proximity of tumor cells and particular stroma composition. For the first time, we demonstrate that PD-L1 is mostly expressed by stroma and rarely by tumor cells in PDAC. Moreover, our in situ analyses on serial tissue sections and in vitro data suggest that PD-L1 is prominently expressed by tumor-associated macrophages. In conclusion, POLE Score represents a comprehensive characterization of PD-L1 expression in tumor and stroma compartment and might provide the basis for improved patient stratification in future clinical trials on PD-1/PD-L1 targeting therapies in PDAC.

Structure-Guided Development of Covalent and Mutant-Selective Pyrazolopyrimidines to Target T790M Drug Resistance in Epidermal Growth Factor Receptor.

Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile.

CD4+ T cells potently induce epithelial-mesenchymal-transition in premalignant and malignant pancreatic ductal epithelial cells-novel implications of CD4+ T cells in pancreatic cancer development.

Chronic pancreatitis (CP) is a risk factor of pancreatic ductal adenocarcinoma (PDAC) and characterized by a pronounced desmoplastic reaction with CD4+ T cells accounting for the majority of the stromal T cell infiltrate. Epithelial-mesenchymal-transition (EMT) is a critical process for metastasis by which epithelial/carcinoma cells become enabled to disseminate probably prior to tumor formation. To investigate whether CD4+ T cells induce EMT in human pancreatic ductal epithelial cells, premalignant H6c7 cells were mono- or co-cultured with human CD4+CD25+CD127-CD49d- regulatory T cells (T-regs) or CD4+CD25- T-effector cells (T-effs) being isolated by negative magnetic bead separation from blood of healthy donors. Particularly in the presence of activated T-effs, H6c7 cells acquired a spindle-shaped morphology, reduced E-cadherin expression, and elevated expression of the mesenchymal proteins vimentin, L1CAM, and ZEB-1. This was accompanied by an increased invasive behavior. Moreover, activated T-effs exerted similar effects in the PDAC cell line T3M4. Blocking of TNF-α and IL-6 being released at greater amounts into supernatants during co-cultures with activated T-effs attenuated the EMT-associated alterations in H6c7 cells. Supporting these findings, EMT-associated alterations (exemplified by reduced E-cadherin expression and enhanced expression of vimentin and L1CAM) were predominantly detected in ductal epithelium of CP tissues surrounded by a dense stroma enriched with CD4+ T cells. Overall this study points to a novel role of CD4+ T cells beyond their immune function in pancreatic tumorigenesis and underscores the view that EMT induction in pancreatic ductal epithelial cells represents an early event in PDAC development being essentially promoted by inflammatory processes.

Comparative characterization of stroma cells and ductal epithelium in chronic pancreatitis and pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive stroma being also present in chronic pancreatitis (CP). Using immunohistochemistry, the stroma of CP and PDAC was comprehensively analyzed and correlated with epithelial/carcinoma-related alterations and clinicopathological patient characteristics. While there were no significant differences between CP and PDAC regarding the distribution of CD3+ T cells and α-SMA+ fibroblasts, proportions of CD4+ and CD8+ T cells were significantly lower and numbers of CD25+(CD4+) and FoxP3+(CD4+) regulatory T cells were greater in PDAC compared with CP. Macrophages were more prevalent in CP, but localized more closely to carcinoma cells in PDAC, as were γδ-T cells. Duct-related FoxP3 and L1CAM expression increased from CP to PDAC, while vimentin expression was similarly abundant in both diseases. Moreover, stromal and epithelial compartments of well-differentiated tumors and CPs shared considerable similarities, while moderately and poorly differentiated tumors significantly differed from CP tissues. Analysis of 27 parameters within each pancreatic disease revealed a significant correlation of i) CD4+ and FoxP3+CD4+ T cells with FoxP3 expression in PDAC cells, ii) α-SMA+ fibroblasts with L1CAM expression and proliferation in PDAC cells, iii) CD3 and CD8 expression with γδ-TCR expression in both pancreatic diseases and iv) CD68+ and CD163+ macrophages with vimentin expression in PDAC cells. High expression of FoxP3, vimentin and L1CAM in PDAC cells as well as a tumor-related localization of macrophages each tended to correlate with higher tumor grade. Multivariate survival analysis revealed a younger age at time of surgery as a positive prognostic marker for PDAC patients with the most frequently operated disease stage T3N1M0. Overall this study identified several interrelationships between stroma and epithelial/carcinoma cells in PDACs but also in CP, which in light of previous experimental data strongly support the view that the inflammatory stroma contributes to malignancy-associated alterations already in precursor cells during CP.

L1CAM promotes enrichment of immunosuppressive T cells in human pancreatic cancer correlating with malignant progression.

Regulatory T cell (T-reg) enrichment in the tumor microenvironment is regarded as an important mechanism of tumor immune escape. Hence, the presence of T-regs in highly malignant pancreatic ductal adenocarcinoma (PDAC) is correlated with short survival. Likewise, the adhesion molecule L1CAM is upregulated during PDAC progression in the pancreatic ductal epithelium also being associated with poor prognosis. To investigate whether L1CAM contributes to enrichment of T-regs in PDAC, human CD4(+)CD25(+)CD127(-)CD49d(-) T-regs and CD4(+)CD25(-) T-effector cells (T-effs) were isolated by magnetic bead separation from blood of healthy donors. Their phenotype and functional behavior were analyzed in dependence on human premalignant (H6c7) or malignant (Panc1) pancreatic ductal epithelial cells, either exhibiting or lacking L1CAM expression. T cells derived from blood and tumors of PDAC patients were analyzed by flow cytometry and findings were correlated with clinical parameters. Predominantly T-regs but not T-effs showed an increased migration on L1CAM expressing H6c7 and Panc1 cells. Whereas proliferation of T-regs did not change in the presence of L1CAM, T-effs proliferated less, exhibited a decreased CD25 expression and an increased expression of CD69. Moreover, these T-effs exhibited a regulatory phenotype as they inhibited proliferation of autologous T cells. Accordingly, CD4(+)CD25(-)CD69(+) T cells were highly abundant in PDAC tissues compared to blood being associated with nodal invasion and higher grading in PDAC patients. Overall, these data point to an important role of L1CAM in the enrichment of immunosuppressive T cells in particular of a CD4(+)CD25(-)CD69(+)-phenotype in PDAC providing a novel mechanism of tumor immune escape which contributes to tumor progression.