Peter H. Schafer

Bio: Peter H. Schafer is an academic researcher from Celgene. The author has contributed to research in topics: Lenalidomide & Apremilast. The author has an hindex of 52, co-authored 204 publications receiving 9683 citations.

Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide.

Abstract: Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN–DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBNYW/AA. Overexpression of CRBN wild-type protein, but not CRBNYW/AA mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21WAF-1 expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Prolonged Bacterial Culture to Identify Late Periprosthetic Joint Infection: A Promising Strategy

Abstract: BACKGROUND: The value of microbiological culture to diagnose late periprosthetic infection is limited, especially because standard methods may fail to detect biofilm-forming sessile or other fastidious bacteria. There is no agreement on the appropriate cultivation period, although this period is a crucial factor. This study was designed to assess the duration of culture that is necessary for reliable detection. PATIENTS AND METHODS: Ten periprosthetic tissue specimens each were obtained during revision from 284 patients with suspected late hip or knee arthroplasty infection. Five samples were examined by microbiological culture over a 14-day period, and 5 were subjected to histologic analysis. To define infection, a pre-established algorithm was used; this included detection of indistinguishable organisms in >/=2 tissue samples or growth in 1 tissue sample and a positive result of histologic analysis (>5 neutrophils in at least 10 high-power fields). The time to detection of organisms was monitored. RESULTS: Infection was diagnosed in 110 patients. After 7 days (the longest incubation period most frequently reported), the detection rate via culture was merely 73.6%. Organisms indicating infection were found for up to 13 days. "Early"-detected species (mostly staphylococci) emerged predominantly during the first week, whereas "late"-detected agents (mostly Propionibacterium species) were detected mainly during the second week. In both populations, an unequivocal correlation between the number of culture-positive tissue samples and positive results of histologic analysis was noted, which corroborated the evidence that true infections were detected over the entire cultivation period. CONCLUSIONS: Prolonged microbiological culture for 2 weeks is promising because it yields signs of periprosthetic infection in a significant proportion of patients that would otherwise remain unidentified.

Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

Abstract: Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

Lenalidomide Enhances Natural Killer Cell and Monocyte-Mediated Antibody-Dependent Cellular Cytotoxicity of Rituximab-Treated CD20+ Tumor Cells

Abstract: Purpose: Lenalidomide has significant activity in myelodysplastic syndromes, multiple myeloma, and non-Hodgkin9s lymphoma (NHL). In previous studies, natural killer (NK) cell expansion by lenalidomide was shown to enhance the cytotoxic effect of rituximab. This study assessed the ability of lenalidomide to enhance antibody-dependent cellular cytotoxicity (ADCC) in rituximab-treated NHL cell lines and primary tumor cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) in vitro . Experimental Design: An in vitro ADCC system was used to assess the ability of lenalidomide to enhance human NK cell and monocyte function in response to rituximab. Results: Lenalidomide directly enhanced IFN-γ production via Fc-γ receptor-mediated signaling in response to IgG. It was also a potent enhancer of NK cell-mediated and monocyte-mediated tumor cell ADCC for a variety of rituximab-treated NHL cell lines in vitro , an effect that was dependent on the presence of antibody and either interleukin-2 or interleukin-12. Lenalidomide also enhanced the ability of NK cells to kill primary tumor cells derived from three patients with B-CLL who have been treated previously with fludarabine plus cyclophosphamide. Enhanced NK cell ADCC was associated with enhanced granzyme B and Fas ligand expression and could be inhibited by a granzyme B inhibitor and partially inhibited by antibody to FasL. Enhanced NK cell Fc-γ receptor signaling is associated with enhanced phosphorylated extracellular signal-related kinase levels leading to enhanced effector function. Conclusions: These findings suggest that lenalidomide has the potential to enhance the rituximab-induced killing of NHL cell lines and primary B-cell chronic lymphocytic leukemia cells via a NK cell-mediated and monocyte-mediated ADCC mechanism in vitro , providing a strong rationale for the combination of lenalidomide with IgG1 antibodies to target tumor-specific antigens in patients with cancer.

Toll-like receptor signalling

Abstract: One of the mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms is through the Toll-like receptors (TLRs), which recognize specific molecular patterns that are present in microbial components. Stimulation of different TLRs induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also instructs the development of antigen-specific acquired immunity. Here, we review the rapid progress that has recently improved our understanding of the molecular mechanisms that mediate TLR signalling.

Toll-like receptors.

Abstract: The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.

Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

Abstract: We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.

Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes

Abstract: A novel scoring function to estimate protein-ligand binding affinities has been developed and implemented as the Glide 4.0 XP scoring function and docking protocol. In addition to unique water desolvation energy terms, protein-ligand structural motifs leading to enhanced binding affinity are included: (1) hydrophobic enclosure where groups of lipophilic ligand atoms are enclosed on opposite faces by lipophilic protein atoms, (2) neutral-neutral single or correlated hydrogen bonds in a hydrophobically enclosed environment, and (3) five categories of charged-charged hydrogen bonds. The XP scoring function and docking protocol have been developed to reproduce experimental binding affinities for a set of 198 complexes (RMSDs of 2.26 and 1.73 kcal/mol over all and well-docked ligands, respectively) and to yield quality enrichments for a set of fifteen screens of pharmaceutical importance. Enrichment results demonstrate the importance of the novel XP molecular recognition and water scoring in separating active and inactive ligands and avoiding false positives.