Nestin expression in osteocytes following myeloablation and during bone marrow metastasis.

Nestin is an intermediate filament protein, which was originally detected in neuroepithelial stem cells. Besides its use as a phenotypic marker of mesenchymal stem cells in the hematopoeitic stem cell niche, the functional interpretation of nestin+ cells remains elusive. We investigated the cellular expression of nestin in bone marrow trephine biopsies of MPN patients, following myeloablation at a stage of hypocellularity during early regeneration. Here, nestin is highly expressed in mature osteocytes, arteriolar endothelial and perivascular cells and small capillaries within the bone marrow space, but not in sinusoid lining cells. This is in stark contrast to nestin expression pattern in myeloproliferative neoplasms that show hypercellularity due to oncogenic driver mutations. Here, nestin is expressed exclusively in endothelial cells of arterioles, but not in osteocytes or small capillaries. Thus, the pattern of nestin expression following myeloablation inversely correlates with cellularity in the bone marrow. This nestin expression pattern is mimicking early postnatal transcriptional programming during bone marrow development. We show that nestin expression in osteocytes occurs across different species following transplant and also in bone marrow metastasis.

Blastic plasmacytoid dendritic-cell neoplasia: a challenging case report.

Blastic plasmacytoid dendritic-cell neoplasm (BPDCN) is an extremely rare disease that originates from dendritic cells and is associated with a poor overall survival (OS). Diagnostic and therapeutic standards are less well-established in comparison to other leukemic conditions and standards of care are lacking. Morphologic and molecular similarities to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) are hard to distinguish. We here report a BPDCN patient with a long, challenging diagnostic period. While bone marrow biopsies initially failed to prove the correct diagnosis, a cutaneous biopsy finally identified a CD45+/CD56+/CD4+/CD123+/CD33+/MPO- population suggestive of BPDCN which was confirmed by flow cytometry. Molecular analysis revealed an ASXL-1, TET2 and SRSF2-mutation, cytogenetic analysis showed a normal karyotype. Treatment with the recently approved CD123-cytotoxin Tagraxofusp showed initially a very good response. This case reflects diagnostic and therapeutic difficulties in BPDCN as very rare, easily misdiagnosed neoplasia and the need for precise diagnostic care.

Quantification of unperturbed phosphoprotein levels in immune cell subsets with phosphoflow to assess immune signaling in autoimmune disease.

Activation of innate immune sensors by endogenous DNA and RNA can lead to autoimmune and autoinflammatory diseases. Quantification of the unperturbed phosphoprotein content in immune cells provides insight into the spontaneous activity of immune signaling pathways triggered by nucleic acid recognition. Here, we present a phosphoflow protocol for measuring phosphoproteins in mouse models of autoimmunity that incorporates strategies to preserve native phosphoprotein levels during sample collection and to reliably detect low signaling activity common in chronic disease states. For complete details on the use and execution of this protocol, please refer to Jütte et al. (2021).

Intercellular cGAMP transmission induces innate immune activation and tissue inflammation in Trex1 deficiency.

Intercellular transmission of the second messenger 2',3'-cGAMP, synthesized by the viral DNA sensor cGAMP synthase (cGAS), is a potent mode of bystander activation during host defense. However, whether this mechanism also contributes to cGAS-dependent autoimmunity remains unknown. Here, using a murine bone marrow transplantation strategy, we demonstrate that, in Trex1 -/- -associated autoimmunity, cGAMP shuttling from radioresistant to immune cells induces NF-κB activation, interferon regulatory factor 3 (IRF3) phosphorylation, and subsequent interferon signaling. cGAMP travel prevented myeloid cell and lymphocyte death, promoting their accumulation in secondary lymphoid tissue. Nonetheless, it did not stimulate B cell differentiation into autoantibody-producing plasmablasts or aberrant T cell priming. Although cGAMP-mediated bystander activation did not induce spontaneous organ disease, it did trigger interface dermatitis after UV light exposure, similar to cutaneous lupus erythematosus. These findings reveal that, in Trex1-deficiency, intercellular cGAMP transfer propagates cGAS signaling and, under conducive conditions, causes tissue inflammation.

Treatment Response Monitoring in Patients with Advanced Malignancies Using Cell-Free SHOX2 and SEPT9 DNA Methylation in Blood: An Observational Prospective Study.

Patients with incurable cancer usually receive palliative treatment with significant toxicity and limited efficacy. Methylation analysis of circulating cell-free DNA (ccfDNA) in blood from cancer patients represents a promising approach for minimally invasive, real-time monitoring of treatment response. Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) methylation was analyzed in N = 8865 malignant and N = 746 normal adjacent tissues across 33 different malignancies from The Cancer Genome Atlas. Furthermore, we performed quantitative SHOX2 and SEPT9 ccfDNA methylation analysis in plasma obtained before and consecutively during treatment from prospectively enrolled N = 115 patients with various advanced cancers. SHOX2 and/or SEPT9 hypermethylation in malignant tissues is present in various carcinomas, sarcoma, melanoma, brain tumors, mesothelioma, and hematopoietic malignancies. Among the prospectively enrolled cancer patients, 61% (70/115) of patients had a baseline-positive blood cumulative ccfDNA methylation score (CMS) and were eligible for response monitoring. Dynamic changes of CMS during treatment were strongly associated with treatment response. A CMS increase indicated response up to 80 days before conventional monitoring. SHOX2 and SEPT9 ccfDNA methylation represents a pan-cancer biomarker and has the potential to be a powerful tool for monitoring treatment response in patients with solid tumors and lymphomas. The early identification of nonresponders might allow for a timely change of treatment regimen.

Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM. METHODS: The MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235). RESULTS: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs. CONCLUSIONS: Our results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.