Michael Gerg

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Significance Diphthamide is a conserved modification on eukaryotic translation elongation factor 2 (eEF2). Analyses of genetically defined diphthamide-deficient cell lines indicate that this modification determines not only sensitivity of cells to the ADP-ribosylating toxins Pseudomonas exotoxin A and diphtheria toxin, but it also modulates nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and TNF receptor signaling pathways. The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1–DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

A Role for IGF-1R-Targeted Therapies in Small-Cell Lung Cancer?

BACKGROUND Small-cell lung cancer (SCLC) is an aggressive disease with a poor prognosis. The insulin-like growth factor-1 receptor (IGF-1R) is an autocrine growth factor and an attractive therapeutic target in many solid tumors, but particularly in lung cancer. PATIENTS AND METHODS This study examined tumor samples from 23 patients diagnosed with SCLC, 11 resected specimens and 12 nodal biopsies obtained by mediastinoscopy, for expression of IGF-1R using the monoclonal rabbit anti-IGF-1R (clone G11, Ventana Medical Systems, Tucson, AZ) and standard immunohistochemistry (IHC). RESULTS All 23 tumor samples expressed IGF-1R with a range of stain intensity from weak (1+) to strong (3+). Ten tumors had a score of 3+, 7 tumors 2+, and 6 tumors 1+. Patient survival data were available for all 23 patients. Two patients died < 30 days post biopsy, therefore, the intensity of anti-IGF-1R immunostaining for 21 patients was correlated to survival. Patients with 3+ immunostaining had a poorer prognosis (P = .003). The overall survival of patients who underwent surgical resection was significantly better (median survival not reached) than patients who were not resected (median survival, 7.4 months) (P = .006). CONCLUSION IGF-1R targeted therapies may have a role in the treatment of SCLC in combination with chemotherapy or as maintenance therapy. Further studies on the clinical benefit of targeting IGF-1R in SCLC are needed.

Pharmacodynamic Monitoring of RO5459072, a Small Molecule Inhibitor of Cathepsin S

Major histocompatibility complex class II (MHCII)-restricted antigen priming of CD4+ T cells is both involved in adaptive immune responses and the pathogenesis of autoimmune diseases. Degradation of invariant chain Ii, a protein that prevents premature peptide loading, is a prerequisite for nascent MHCII–peptide complex formation. A key proteolytic step in this process is mediated by cathepsin S. Inhibition of this cysteine protease is known to result in the intracellular accumulation of Lip10 in B cells. Here, we describe the development and application of a neoepitope-based flow cytometry assay measuring accumulation of Lip10. This novel method enabled the investigation of cathepsin S-dependent MHCII maturation in professional antigen-presenting cell (APC) subsets. Inhibition of cathepsin S by a specific inhibitor, RO5459072, in human PBMC ex vivo resulted in accumulation of Lip10 in B cells and myeloid dendritic cells, but not in plasmacytoid dendritic cells and only to a minor degree in monocytes. We qualified Lip10 as a pharmacodynamic biomarker by showing the cathepsin S inhibitor-dependent accumulation of Lip10 in vivo in cynomolgus monkeys treated with RO5459072. Finally, dosing of RO5459072 in a first-in-human clinical study (www.ClinicalTrials.gov, identifier NCT02295332) exhibited a dose-dependent increase in Lip10, confirming target engagement and demonstrating desired pharmacologic inhibition in vivo. The degree of cathepsin S antagonist-induced maximum Lip10 accumulation in APCs varied significantly between individuals both in vitro and in vivo. This finding has not been reported previously using alternative, less sensitive methods and demands further investigation as to the potential of this biomarker to predict response to treatment. These results will help guide subsequent clinical studies investigating the pharmacokinetic and pharmacodynamic relationship of cathepsin S inhibitor RO5459072 after multiple dosing.

Deciphering the Stepwise Binding Mode of HRG1β to HER3 by Surface Plasmon Resonance and Interaction Map

For the development of efficient anti-cancer therapeutics against the HER receptor family it is indispensable to understand the mechanistic model of the HER receptor activation upon ligand binding. Due to its high complexity the binding mode of Heregulin 1 beta (HRG1β) with its receptor HER3 is so far not understood. Analysis of the interaction of HRG1β with surface immobilized HER3 extracellular domain by time-resolved Surface Plasmon Resonance (SPR) was so far not interpretable using any regular analysis method as the interaction was highly complex. Here, we show that Interaction Map (IM) made it possible to shed light on this interaction. IM allowed deciphering the rate limiting kinetic contributions from complex SPR sensorgrams and thereby enabling the extraction of discrete kinetic rate components from the apparently heterogeneous interactions. We could resolve details from the complex avidity-driven binding mode of HRG1β with HER3 by using a combination of SPR and IM data. Our findings contribute to the general understanding that a major conformational change of HER3 during its activation is induced by a complex sequential HRG1β docking mode.