First-hit SETBP1 mutations cause a myeloproliferative disorder with bone marrow fibrosis.

ABSTRACT: SETBP1 mutations are found in various clonal myeloid disorders. However, it is unclear whether they can initiate leukemia, because SETBP1 mutations typically appear as later events during oncogenesis. To answer this question, we generated a mouse model expressing mutated SETBP1 in hematopoietic tissue: this model showed profound alterations in the differentiation program of hematopoietic progenitors and developed a myeloid neoplasm with megakaryocytic dysplasia, splenomegaly, and bone marrow fibrosis, prompting us to investigate SETBP1 mutations in a cohort of 36 triple-negative primary myelofibrosis (TN-PMF) cases. We identified 2 distinct subgroups, one carrying SETBP1 mutations and the other completely devoid of somatic variants. Clinically, a striking difference in disease aggressiveness was noted, with patients with SETBP1 mutation showing a much worse clinical course. In contrast to myelodysplastic/myeloproliferative neoplasms, in which SETBP1 mutations are mostly found as a late clonal event, single-cell clonal hierarchy reconstruction in 3 patients with TN-PMF from our cohort revealed SETBP1 to be a very early event, suggesting that the phenotype of the different SETBP1+ disorders may be shaped by the opposite hierarchy of the same clonal SETBP1 variants.

THBS1 and THBS2 Enhance the In Vitro Proliferation, Adhesion, Migration and Invasion of Intrahepatic Cholangiocarcinoma Cells.

In intrahepatic cholangiocarcinoma (iCCA), thrombospondin 1 (THBS1) and 2 (THBS2) are soluble mediators released in the tumor microenvironment (TME) that contribute to the metastatic spreading of iCCA cells via a lymphatic network by the trans-differentiation of vascular endothelial cells to a lymphatic-like phenotype. To study the direct role of THBS1 and THBS2 on the iCCA cells, well-established epithelial (HuCCT-1) and mesenchymal (CCLP1) iCCA cell lines were subjected to recombinant human THBS1 and THBS2 (rhTHBS1, rhTHBS2) for cellular function assays. Cell growth, cell adhesion, migration, and invasion were all enhanced in both CCLP1 and HuCCT-1 cells by the treatment with either rhTHBS1 or rhTHBS2, although they showed some variability in their intensity of speeding up cellular processes. rhTHBS2 was more intense in inducing invasiveness and in committing the HuCCT-1 cells to a mesenchymal-like phenotype and was therefore a stronger enhancer of the malignant behavior of iCCA cells compared to rhTHBS1. Our data extend the role of THBS1 and THBS2, which are not only able to hinder the vascular network and promote tumor-associated lymphangiogenesis but also exacerbate the malignant behavior of the iCCA cells.

Modeling skeletal dysplasia in Hurler syndrome using patient-derived bone marrow osteoprogenitor cells.

Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet-based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.

A pathogenic role for brain-derived neurotrophic factor (BDNF) in fibrous dysplasia of bone.

Brain derived neurotrophic factor (BDNF) is a neurotrophin, expressed in the central nervous system and in peripheral tissues, that is regulated by the Gsα/cAMP pathway. In bone, it regulates osteogenesis and stimulates RANKL secretion and osteoclast formation in osteolytic tumors such as Multiple Myeloma. Fibrous dysplasia (FD) of bone is a rare genetic disease of the skeleton caused by gain-of-function mutations of the Gsα gene in which RANKL-dependent enhanced bone resorption is a major cause of bone fragility and clinical morbidity. We observed that BDNF transcripts are expressed in human FD lesions. Specifically, immunolocalization studies performed on biopsies obtained from FD patients revealed the expression of BDNF in osteoblasts and, to a lower extent, in the spindle-shaped cells within the fibrous tissue. Therefore, we hypothesized that BDNF can play a role in the pathogenesis of FD by stimulating RANKL secretion and bone resorption. To test this hypothesis, we used the EF1α-GsαR201C mouse model of the human disease (FD mice). Western blot analysis revealed a higher expression of BDNF in bone segments of FD mice compared to WT mice and the immunolabeling pattern within mouse FD lesions was similar to that observed in human FD. Treatment of FD mice with a monoclonal antibody against BDNF reduced the fibrous tissue along with the number of osteoclasts and osteoblasts within femoral lesions. These results reveal BDNF as a new player in the pathogenesis of FD and a potential molecular mechanism by which osteoclastogenesis may be nourished within FD bone lesions. They also suggest that BDNF inhibition may be a new approach to reduce abnormal bone remodeling in FD.

Reduced expression of secretogranin VGF in laryngeal squamous cell carcinoma.

Laryngeal cancer accounts for one-third of all head and neck tumors, with squamous cell carcinoma (SCC) being the most predominant type, followed by neuroendocrine tumors. Chromogranins, are commonly used as biomarkers for neuroendocrine tumors, including laryngeal cancer. It has been reported that secretogranin VGF, a member of the chromogranin family, can be also used as a significant biomarker for neuroendocrine tumors. However, the expression and role of VGF in laryngeal carcinomas have not been previously investigated. Therefore, the present study aimed to determine the expression levels of VGF in laryngeal SCC (LSCC). The present study collected tumor tissues, as well as serum samples, from a cohort of 15 patients with LSCC. The results of reverse transcription-quantitative PCR, western blot analysis and immunofluorescence assays showed that the selective VGF precursor was downregulated in patients with LSCC. Notably, in tumor tissue, the immunoreactivity for VGF was found in vimentin-positive cells, probably corresponding to T lymphocytes. The current preliminary study suggested that the reduced expression levels of VGF observed in tumor tissue and at the systemic level could sustain LSCC phenotype. Overall, VGF could be a potential biomarker for detecting neoplastic lesions with a higher risk of tumor invasiveness, even in non-neuroendocrine tumors.

AML alters bone marrow stromal cell osteogenic commitment via Notch signaling.

Introduction: Acute myeloid leukemia (AML) is a highly heterogeneous malignancy caused by various genetic alterations and characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). This abnormal growth of AML cells disrupts normal hematopoiesis and alters the BM microenvironment components, establishing a niche supportive of leukemogenesis. Bone marrow stromal cells (BMSCs) play a pivotal role in giving rise to essential elements of the BM niche, including adipocytes and osteogenic cells. Animal models have shown that the BM microenvironment is significantly remodeled by AML cells, which skew BMSCs toward an ineffective osteogenic differentiation with an accumulation of osteoprogenitors. However, little is known about the mechanisms by which AML cells affect osteogenesis. Methods: We studied the effect of AML cells on the osteogenic commitment of normal BMSCs, using a 2D co-culture system. Results: We found that AML cell lines and primary blasts, but not normal hematopoietic CD34+ cells, induced in BMSCs an ineffective osteogenic commitment, with an increase of the early-osteogenic marker tissue non-specific alkaline phosphatase (TNAP) in the absence of the late-osteogenic gene up-regulation. Moreover, the direct interaction of AML cells and BMSCs was indispensable in influencing osteogenic differentiation. Mechanistic studies identified a role for AML-mediated Notch activation in BMSCs contributing to their ineffective osteogenic commitment. Inhibition of Notch using a γ-secretase inhibitor strongly influenced Notch signaling in BMSCs and abrogated the AML-induced TNAP up-regulation. Discussion: Together, our data support the hypothesis that AML infiltration produces a leukemia-supportive pre-osteoblast-rich niche in the BM, which can be partially ascribed to AML-induced activation of Notch signaling in BMSCs.