Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes

HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy

Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor protein MYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential of MYD88(L265P)-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based on in silico predictions, we identified potential MYD88(L265P)-containing HLA ligands for several HLA class I restrictions. A set of HLA class I MYD88(L265P)-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential of MYD88(L265P) mutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients with MYD88(L265P+) NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

MyD88 mutations in B cell tumors: constitutive signaling by TIR domain oligomerization

Myeloid differentiation primary response 88 (MyD88) is the key adaptor protein that mediates signals upon activation of Toll-like receptors (TLR) and the IL-1 receptor (IL-1R). MyD88 consists of a Toll/IL-1R (TIR) domain that interacts with TIR domains of receptors or other adaptor proteins, whereas the death domain (DD) recruits downstream IL-1R-associated kinases (IRAKs) to form the so-called Myddosome signaling complex. A leucine to proline substitution of the amino acid residue 265 (L265P) within the TIR domain of MyD88 has recently been described as a very frequent somatic mutation in several types of B cell non-Hodgkin lymphomas, all of which are associated with considerable mortality; i.e. diffuse large B cell lymphoma (DLBCL), Waldenström’s macroglobulinemia (WM), mucosa-associated lymphoid tissue lymphoma (MALT) and chronic lymphocytic leukemia (CLL). Recent reports revealed MyD88 L265P strongly increases binding and activation of IRAK1 as well as Bruton’s tyrosine kinase (BTK), thus these interactions are suggested to sustain cancer cell survival by constitutive activation of the transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells). However, the precise molecular events leading to NF-κB hyperactivation and whether other B cell-intrinsic pathways are affected still remains unknown. In an effort to address these questions we found that the hyperactive phenotype of MyD88 L265P is caused by increased oligomerization propensity of the mutated TIR domain. L265P TIR domains in contrast to WT TIRs were alone able to trigger cell activation by strongly interacting with and therefore utilizing WT MyD88, possibly explaining why heterozygous mutation could be sufficient to trigger tumorigenesis. Confocal microscopy revealed cytosolic aggregation of TIR-mutants. MyD88 L265P colocalized with IRAKs indicating that L265P mutant assembly of oligomeric Myddosome post-receptor complexes leads to cell activation. Furthermore, cell lysates of L265P-mutated lymphoma cell lines contained complexes of high molecular weight including MyD88 and IRAK1, indicative for Myddosomes and confirming the aggregation phenotype of the mutant. Consequently blocking MyD88 oligomerization induced death of L265P-mutated but not WT DLBCL cells. Preliminary data indicate that L265P alters binding of MyD88 to several proteins which are important for regulatory circuits in B cell carcinogenesis. We plan to corroborate these findings in primary tumor cells and in vitro murine models, and propose to evaluate and utilize novel insights of how L265P may modulate tumor cell persistence by using small molecule inhibitors targeting the newly discovered interaction partners.MyD88 (myeloid differentiation primary response 88) ist ein wichtiges Adapter-Protein das Signale von aktivierten Toll-like Rezeptoren (TLRs) und dem Interleukin-1-Rezeptor (IL-1R) weiterleitet. MyD88 besteht aus einer Toll/IL-1R-Proteindomäne (TIR) welche mit TIR-Domänen von Rezeptoren oder weiteren Adapter-Proteinen interagiert. Über die sog. DD-Proteindomäne (death domain) von MyD88 werden IL-1R-assoziierte Kinasen (IRAKs) rekrutiert wobei alle Proteine zusammen sog. Myddosomen bilden, aktivierte Signalkomplexe von hohem Molekulargewicht. In Studien wurde gezeigt, dass die MyD88 TIR-Domäne häufig in diversen B-Zell Non-Hodgkin-Lymphomen mutiert ist. Dazu gehören Neoplasien mit hoher Todesfolge wie z.B. das diffuse großzellige B-Zell-Lymphom (DLBCL), Waldenströms Makroglobulinämie (WM) oder die Chronische lymphatische Leukämie (CLL). Meistens entsteht durch Punktmutation im MYD88-Gen ein Austausch der 265. Aminosäure von Leucin zu Prolin (L265P). Studien konnten belegen, dass die L265P-Mutation eine starke Bindung von MyD88 mit IRAK- und BTK (Bruton-Tyrosinkinase) verursacht. Beide Kinasen waren dabei phosphoryliert und somit aktiv, was eine konstitutive Aktivierung des Transkriptionsfaktors NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) auslöste. Es ist davon auszugehen, dass letztendlich die erhöhte Expression anti-apoptotischer Gene durch hyperaktives NF-κB zur Persistenz von malignen Tumorzellen führt. Jedoch sind die genauen Vorgänge die zu dieser Hyperaktivierung führen nicht im Detail verstanden. Die vorliegende Arbeit beschreibt einen bisher unbekannten Mechanismus der Krebsentstehung: Expression von L265P-mutierten TIR-Domänen waren im Vergleich zu WT TIRs alleine ausreichend um NF-κB zu aktivieren. Die Mutante offenbarte dabei eine starke Bindungsaffinität zu sowohl mutierten, als auch WT TIR-Domänen, was eine wirksame Oligomerisierung von MyD88 Molekülen zur Folge hatte. Dieser Umstand wäre auch eine Erklärung dafür, dass eine heterozygot vorliegende MyD88-Mutation, wie es in den allermeisten Fällen von Lymphomen der Fall ist, ausreichend für die Krebsentstehung wäre. Fluoreszenzmikroskopische Untersuchungen zeigten Protein-Aggregation von L265P-mutierten TIR-Domänen im Zytoplasma. Weiterhin konnten wir Co-Lokalisation von MyD88 L265P mit verschiedenen IRAK Kinasen nachweisen, was vermuten lässt dass es sich bei beschriebenen Aggregaten um Myddosomen handelt, welche letztendlich zur Aktivierung von Signalwegen führen. Nach Zentrifugation von DLBCL Zelllysaten konnten wir mutiertes MyD88 in Komplexen mit hohem Molekulargewicht nachweisen. Diese Komplexe in L265P-mutierten Zellen enthielten auch mehr IRAK1 im Vergleich zu den MyD88 WT Zellen. Das weist darauf hin, dass eine L265P-bedingte Bildung von aktiven Myddosom-Aggregaten auch in DLBCL stattfindet. Folgerichtig konnten wir durch Blockieren der TIR-Oligomerisierung ein gezieltes Abtöten von MyD88 L265P-mutierten DLBCL Zelllinien erreichen. Weiterhin deuten vorläufige Daten an, dass die MyD88 L265P-Mutation auch die Affinität zu weiteren Proteinen verändert, welche eine wichtige Funktion in der B-Zell Onkogenese einnehmen. Diese Erkenntnisse sollen sowohl in primären Tumorzellen als auch in einem in vitro Mausmodell bestätigt werden. Wir hoffen, dass sich unsere Neuentdeckungen als nützlich erweisen und planen deshalb zur Evaluierung, kon-stitutiv-aktivierte Signalwege in MyD88-mutierten Lymphomen pharmakologisch zu inhibieren

Oncogenic MYD88 mutations in lymphoma: novel insights and therapeutic possibilities

Oncogenic MYD88 mutations, most notably the Leu 265 Pro (L265P) mutation, were recently identified as potential driver mutations in various B-cell non-Hodgkin Lymphomas (NHLs). The L265P mutation is now thought to be common to virtually all NHLs and occurs in between 4 and 90% of cases, depending on the entity. Since it is tumor-specific, the mutation, and the pathways it regulates, might serve as advantageous therapeutic targets for both conventional chemotherapeutic intervention, as well as immunotherapeutic strategies. Here, we review recent progress on elucidating the molecular and cellular processes affected by theL265P mutation of MYD88, describe a new in vivo model for MyD88 L265P-mediated oncogenesis, and summarize how these findings could be exploited therapeutically by specific targeting of signaling pathways. In addition, we summarize current and explore future possibilities for conceivable immunotherapeutic approaches, such as L265P-derived peptide vaccination, adoptive transfer of L265P-restricted T cells, and use of T-cell receptor-engineered T cells. With clinical trials regarding their efficacy rapidly expanding to NHLs, we also discuss potential combinations of immune checkpoint inhibitors with the described targeted chemotherapies of L265P signaling networks, and/or with the above immunological approaches as potential ways of targeting MYD88-mutated lymphomas in the future

A Third Dose of the COVID-19 Vaccine, CVnCoV, Increased the Neutralizing Activity against the SARS-CoV-2 Wild-Type and Delta Variant

A third dose of CVnCoV, a former candidate mRNA vaccine against SARS-CoV-2, was previously shown to boost neutralizing antibody responses against SARS-CoV-2 wild-type in adults aged 18–60 and >60 years in a phase 2a clinical study. In the present study, we report the neutralizing antibody responses to a wild-type and a variant of concern, Delta, after a third dose of the vaccine on day (D)57 and D180. Neutralization activity was assessed using a microneutralization assay. Comparable levels of neutralizing antibodies against the wild-type and Delta were induced. These were higher than those observed after the first two doses, irrespective of age or pre-SARS-CoV-2-exposure status, indicating that the first two doses induced immune memory. Four weeks after the third dose on D180, the neutralizing titers for wild-type and Delta were two-fold higher in younger participants than in older participants; seroconversion rates were 100% for wild-type and Delta in the younger group and for Delta in the older group. A third CVnCoV dose induced similar levels of neutralizing responses against wild-type virus and the Delta variant in both naïve and pre-exposed participants, aligning with current knowledge from licensed COVID-19 vaccines that a third dose is beneficial against SARS-CoV-2 variants

Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes