Xianlin Yang

Nf1-Dependent Tumors Require a Microenvironment Containing Nf1+/−- and c-kit-Dependent Bone Marrow

Interactions between tumorigenic cells and their surrounding microenvironment are critical for tumor progression yet remain incompletely understood. Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a common genetic disorder characterized by complex tumors called neurofibromas. Genetic studies indicate that biallelic loss of Nf1 is required in the tumorigenic cell of origin in the embryonic Schwann cell lineage. However, in the physiologic state, Schwann cell loss of heterozygosity is not sufficient for neurofibroma formation and Nf1 haploinsufficiency in at least one additional nonneoplastic lineage is required for tumor progression. Here, we establish that Nf1 heterozygosity of bone marrow-derived cells in the tumor microenvironment is sufficient to allow neurofibroma progression in the context of Schwann cell Nf1 deficiency. Further, genetic or pharmacologic attenuation of c-kit signaling in Nf1+/- hematopoietic cells diminishes neurofibroma initiation and progression. Finally, these studies implicate mast cells as critical mediators of tumor initiation.

In vivo demonstration that human parathyroid hormone 1-38 inhibits the expression of osteoprotegerin in bone with the kinetics of an immediate early gene.

Osteoprotegerin (OPG) is a potent inhibitor of osteoclast formation and function. To elucidate how OPG is regulated in bone, we examined (1) the expression and localization of OPG protein in bone tissue, (2) the effect of human parathyroid hormone 1–38 (hPTH 1–38) on OPG messenger RNA (mRNA) levels in rat femur metaphyseal and diaphyseal bone, and (3) the effect of hPTH(1–38) on expression of OPG mRNA in cultured osteoblast‐like cells derived from the metaphysis and diaphysis, and in ROS 17/2.8 osteosarcoma cells. Because PTH has been shown to stimulate osteoblast activity via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal transduction pathway we also investigated whether PTH action on OPG in vivo is dependent on activation of cAMP/PKA pathway. Immunohistochemistry was used to evaluate OPG protein expression and Northern blot hybridization was used to analyze OPG mRNA expression both in vivo and in vitro. Immunohistochemistry of OPG protein expression in the rat distal femur metaphysis revealed that it was localized predominantly in preosteoblasts, osteoblasts, lining cells, and the osteoid layer, with occasional immunoreactivity in osteocytes and cells of the bone marrow. Subcutaneous (sc) administration of a single injection of hPTH(1–38) at 80 μg/kg induced a rapid and transient decrease in OPG mRNA expression in both metaphyseal and diaphyseal bone. The decrease in OPG message was evident by 1 h and mRNA levels returned to baseline after 3 h. PTH analog PTH(1–31), which stimulates intracellular cAMP accumulation, inhibited OPG expression, whereas PTH analogs (3–34 and 7–34) that do not stimulate cAMP production had no effect on expression. In contrast to PTH, prostaglandin E2 (PGE2) had no effect on OPG mRNA expression in vivo in the metaphyseal bone cells, under conditions in which PGE2 does promote expression of the c‐fos gene. The in vivo effects of hPTH(1–38) on OPG mRNA were confirmed in isolated primary osteoblast cultures derived from either metaphyseal or diaphyseal bone as well as in ROS 17/2.8 osteosarcoma cells. We propose that the rapid and transient decrease in OPG expression may initiate a cascade of events resulting in the differentiation of osteoclast progenitor. Such a spatially and temporally programmed effect of PTH might contribute to bone turnover. (J Bone Miner Res 2000;15:863–871)

Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice.

ASXL1 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. De novo ASXL1 mutations cause Bohring-Opitz syndrome characterized by multiple congenital malformations. We show that Asxl1 deletion in mice led to developmental abnormalities including dwarfism, anophthalmia, and 80% embryonic lethality. Surviving Asxl1(-/-) mice lived for up to 42 days and developed features of myelodysplastic syndrome (MDS), including dysplastic neutrophils and multiple lineage cytopenia. Asxl1(-/-) mice had a reduced hematopoietic stem cell (HSC) pool, and Asxl1(-/-) HSCs exhibited decreased hematopoietic repopulating capacity, with skewed cell differentiation favoring granulocytic lineage. Asxl1(+/-) mice also developed mild MDS-like disease, which could progress to MDS/myeloproliferative neoplasm, demonstrating a haploinsufficient effect of Asxl1 in the pathogenesis of myeloid malignancies. Asxl1 loss led to an increased apoptosis and mitosis in Lineage(-)c-Kit(+) (Lin(-)c-Kit(+)) cells, consistent with human MDS. Furthermore, Asxl1(-/-) Lin(-)c-Kit(+) cells exhibited decreased global levels of H3K27me3 and H3K4me3 and altered expression of genes regulating apoptosis (Bcl2, Bcl2l12, Bcl2l13). Collectively, we report a novel ASXL1 murine model that recapitulates human myeloid malignancies, implying that Asxl1 functions as a tumor suppressor to maintain hematopoietic cell homeostasis. Future work is necessary to clarify the contribution of microenvironment to the hematopoietic phenotypes observed in the constitutional Asxl1(-/-) mice.

Erk1 positively regulates osteoclast differentiation and bone resorptive activity.

The extracellular signal-regulated kinases (ERK1 and 2) are widely-expressed and they modulate proliferation, survival, differentiation, and protein synthesis in multiple cell lineages. Altered ERK1/2 signaling is found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, Neurofibromatosis type 1, and Cardio-facio-cutaneous syndrome, suggesting that MEK-ERK signals regulate human skeletal development. Here, we examine the consequence of Erk1 and Erk2 disruption in multiple functions of osteoclasts, specialized macrophage/monocyte lineage-derived cells that resorb bone. We demonstrate that Erk1 positively regulates osteoclast development and bone resorptive activity, as genetic disruption of Erk1 reduced osteoclast progenitor cell numbers, compromised pit formation, and diminished M-CSF-mediated adhesion and migration. Moreover, WT mice reconstituted long-term with Erk1−/− bone marrow mononuclear cells (BMMNCs) demonstrated increased bone mineral density as compared to recipients transplanted with WT and Erk2−/− BMMNCs, implicating marrow autonomous, Erk1-dependent osteoclast function. These data demonstrate Erk1 plays an important role in osteoclast functions while providing rationale for the development of Erk1-specific inhibitors for experimental investigation and/or therapeutic modulation of aberrant osteoclast function.

The tumor suppressor CDKN3 controls mitosis

A genome-wide screen of phosphatases that control mitosis identified CDKN3, which acts through the CDC2 signaling axis.

Hyperactive transforming growth factor-β1 signaling potentiates skeletal defects in a neurofibromatosis type 1 mouse model.

Dysregulated transforming growth factor beta (TGF‐β) signaling is associated with a spectrum of osseous defects as seen in Loeys‐Dietz syndrome, Marfan syndrome, and Camurati‐Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF‐β1 signaling pivotally underpins osseous defects in Nf1flox/−;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF‐β1 levels are fivefold to sixfold increased both in Nf1flox/−;Col2.3Cre mice and in a cohort of NF1 patients. Nf1‐deficient osteoblasts, the principal source of TGF‐β1 in bone, overexpress TGF‐β1 in a gene dosage–dependent fashion. Moreover, Nf1‐deficient osteoblasts and osteoclasts are hyperresponsive to TGF‐β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21‐Ras–dependent hyperactivation of the canonical TGF‐β1–Smad pathway. Reexpression of the human, full‐length neurofibromin guanosine triphosphatase (GTPase)‐activating protein (GAP)‐related domain (NF1 GRD) in primary Nf1‐deficient osteoblast progenitors, attenuated TGF‐β1 expression levels and reduced Smad phosphorylation in response to TGF‐β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF‐β receptor 1 (TβRI) kinase inhibitor, SD‐208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1flox/−;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF‐β1 signaling in the pathogenesis of NF1‐associated osteoporosis and pseudarthrosis, thus implicating the TGF‐β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies.

A murine model of neurofibromatosis type 2 that accurately phenocopies human schwannoma formation

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder resulting from germline mutations in the NF2 gene. Bilateral vestibular schwannomas, tumors on cranial nerve VIII, are pathognomonic for NF2 disease. Furthermore, schwannomas also commonly develop in other cranial nerves, dorsal root ganglia and peripheral nerves. These tumors are a major cause of morbidity and mortality, and medical therapies to treat them are limited. Animal models that accurately recapitulate the full anatomical spectrum of human NF2-related schwannomas, including the characteristic functional deficits in hearing and balance associated with cranial nerve VIII tumors, would allow systematic evaluation of experimental therapeutics prior to clinical use. Here, we present a genetically engineered NF2 mouse model generated through excision of the Nf2 gene driven by Cre expression under control of a tissue-restricted 3.9kbPeriostin promoter element. By 10 months of age, 100% of Postn-Cre; Nf2(flox/flox) mice develop spinal, peripheral and cranial nerve tumors histologically identical to human schwannomas. In addition, the development of cranial nerve VIII tumors correlates with functional impairments in hearing and balance, as measured by auditory brainstem response and vestibular testing. Overall, the Postn-Cre; Nf2(flox/flox) tumor model provides a novel tool for future mechanistic and therapeutic studies of NF2-associated schwannomas.

Hyperactive Ras/MAPK signaling is critical for tibial nonunion fracture in neurofibromin-deficient mice

Neurofibromatosis type 1 (NF1) is a common genetic disorder affecting 1 in 3500 individuals. Patients with NF1 are predisposed to debilitating skeletal manifestations, including osteopenia/osteoporosis and long bone pseudarthrosis (nonunion fracture). Hyperactivation of the Ras/mitogen-activated protein kinase (MAPK) pathway in NF1 is known to underlie aberrant proliferation and differentiation in cell lineages, including osteoclast progenitors and mesenchymal stem cells (MSCs) also known as osteoblast progenitors (pro-OBLs). Our current study demonstrates the hyper Ras/MAPK as a critical pathway underlying the pathogenesis of NF1-associated fracture repair deficits. Nf1-deficient pro-OBLs exhibit Ras/MAPK hyperactivation. Introduction of the NF1 GTPase activating-related domain (NF1 GAP-related domain) in vitro is sufficient to rescue hyper Ras activity and enhance osteoblast (OBL) differentiation in Nf1(-/-) pro-OBLs and NF1 human (h) MSCs cultured from NF1 patients with skeletal abnormalities, including pseudarthrosis or scoliosis. Pharmacologic inhibition of mitogen-activated protein kinase kinase (MEK) signaling with PD98059 partially rescues aberrant Erk activation while enhancing OBL differentiation and expression of OBL markers, osterix and osteocalcin, in Nf1-deficient murine pro-OBLs. Similarly, MEK inhibition enhances OBL differentiation of hMSCs. In addition, PD98059 rescues aberrant osteoclast maturation in Nf1 haploinsufficient bone marrow mononuclear cells (BMMNCs). Importantly, MEK inhibitor significantly improves fracture healing in an NF1 murine model, Col2.3Cre;Nf1(flox/-). Collectively, these data indicate the Ras/MAPK cascade as a critical pathway in the pathogenesis of bone loss and pseudarthrosis related to NF1 mutations. These studies provide evidence for targeting the MAPK pathway to improve bone mass and treat pseudarthrosis in NF1.

Primary osteopathy of vertebrae in a neurofibromatosis type 1 murine model.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutation of the NF1 tumor suppressor gene. Spinal deformities are common skeletal manifestations in patients with NF1. To date, the mechanism of vertebral abnormalities remains unclear because of the lack of appropriate animal models for the skeletal manifestations of NF1. In the present study, we report a novel murine NF1 model, Nf1(flox/-);Col2.3Cre(+) mice. These mice display short vertebral segments. In addition, a significant reduction in cortical and trabecular bone mass of the vertebrae was observed in Nf1(flox/-);Col2.3Cre(+) mice as measured by dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). Peak stress and peak load were also significantly reduced in Nf1(flox/-);Col2.3Cre(+) mice as compared to controls. Furthermore, the lumbar vertebrae showed enlargement of the inter-vertebral canal, a characteristic feature of lumbar vertebrae in NF1 patients. Finally, histologic analysis demonstrated increased numbers of osteoclasts and decreased numbers of osteoblasts in the vertebrae of Nf1(flox/-);Col2.3Cre(+) mice in comparison to controls. In summary, Nf1(flox/-);Col2.3Cre(+) mice demonstrate multiple structural and functional abnormalities in the lumbar vertebrae which recapitulate the dystrophic vertebral changes in NF1 patients. This novel murine model provides a platform to understand the cellular and molecular mechanisms underlying the pathogenesis of spinal deficits in NF1 patients.

The haploinsufficient hematopoietic microenvironment is critical to the pathological fracture repair in murine models of neurofibromatosis type 1

Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a complex genetic disorder with a high predisposition of numerous skeletal dysplasias including short stature, osteoporosis, kyphoscoliosis, and fracture non-union (pseudoarthrosis). We have developed murine models that phenocopy many of the skeletal dysplasias observed in NF1 patients, including reduced bone mass and fracture non-union. We also show that the development of these skeletal manifestations requires an Nf1 haploinsufficient background in addition to nullizygous loss of Nf1 in mesenchymal stem/progenitor cells (MSCs) and/or their progenies. This is replicated in two animal models of NF1, PeriCre+;Nf1flox/− and Col2.3Cre+;Nf1flox/−mice. Adoptive transfer experiments demonstrate a critical role of the Nf1+/− marrow microenvironment in the impaired fracture healing in both models and adoptive transfer of WT bone marrow cells improves fracture healing in these mice. To our knowledge, this is the first demonstration of a non-cell autonomous mechanism in non-malignant NF1 manifestations. Collectively, these data provide evidence of a combinatory effect between nullizygous loss of Nf1 in osteoblast progenitors and haploinsufficiency in hematopoietic cells in the development of non-malignant NF1 manifestations.