Karl Staser

Imatinib mesylate for plexiform neurofibromas in patients with neurofibromatosis type 1: a phase 2 trial

BACKGROUND Plexiform neurofibromas are slow-growing chemoradiotherapy-resistant tumours arising in patients with neurofibromatosis type 1 (NF1). Currently, there are no viable therapeutic options for patients with plexiform neurofibromas that cannot be surgically removed because of their proximity to vital body structures. We undertook an open-label phase 2 trial to test whether treatment with imatinib mesylate can decrease the volume burden of clinically significant plexiform neurofibromas in patients with NF1. METHODS Eligible patients had to be aged 3-65 years, and to have NF1 and a clinically significant plexiform neurofibroma. Patients were treated with daily oral imatinib mesylate at 220 mg/m(2) twice a day for children and 400 mg twice a day for adults for 6 months. The primary endpoint was a 20% or more reduction in plexiform size by sequential volumetric MRI imaging. Clinical data were analysed on an intention-to-treat basis; a secondary analysis was also done for those patients able to take imatinib mesylate for 6 months. This trial is registered with ClinicalTrials.gov, number NCT01673009. FINDINGS Six of 36 patients (17%, 95% CI 6-33), enrolled on an intention-to-treat basis, had an objective response to imatinib mesylate, with a 20% or more decrease in tumour volume. Of the 23 patients who received imatinib mesylate for at least 6 months, six (26%, 95% CI 10-48) had a 20% or more decrease in volume of one or more plexiform tumours. The most common adverse events were skin rash (five patients) and oedema with weight gain (six). More serious adverse events included reversible grade 3 neutropenia (two), grade 4 hyperglycaemia (one), and grade 4 increases in aminotransferase concentrations (one). INTERPRETATION Imatinib mesylate could be used to treat plexiform neurofibromas in patients with NF1. A multi-institutional clinical trial is warranted to confirm these results. FUNDING Novartis Pharmaceuticals, the Indiana University Simon Cancer Centre, and the Indiana University Herman B Wells Center for Pediatric Research.

Mast cells and the neurofibroma microenvironment.

Neurofibromatosis type 1 (NF1) is the most common genetic disorder with a predisposition to malignancy and affects 1 in 3500 persons worldwide. NF1 is caused by a mutation in the NF1 tumor suppressor gene that encodes the protein neurofibromin. Patients with NF1 have cutaneous, diffuse, and plexiform neurofibromas, tumors comprised primarily of Schwann cells, blood vessels, fibroblasts, and mast cells. Studies from human and murine models that closely recapitulate human plexiform neurofibroma formation indicate that tumorigenesis necessitates NF1 loss of heterozygosity in the Schwann cell. In addition, our most recent studies with bone marrow transplantation and pharmacologic experiments implicate haploinsufficiency of Nf1 (Nf1(+/-)) and c-kit signaling in the hematopoietic system as required and sufficient for tumor progression. Here, we review recent studies implicating the hematopoietic system in plexiform neurofibroma genesis, delineate the physiology of stem cell factor-dependent hematopoietic cells and their contribution to the neurofibroma microenvironment, and highlight the application of this research toward the first successful, targeted medical treatment of a patient with a nonresectable and debilitating neurofibroma. Finally, we emphasize the importance of the tumor microenvironment hypothesis, asserting that tumorigenic cells in the neurofibroma do not arise and grow in isolation.

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.

Pathogenesis of Plexiform Neurofibroma: Tumor-Stromal/Hematopoietic Interactions in Tumor Progression

Neurofibromatosis type 1 (NF1) is a genetic disease that results from either heritable or spontaneous autosomal dominant mutations in the NF1 gene. A second-hit mutation precedes the predominant NF1 neoplasms, which include myeloid leukemia, optic glioma, and plexiform neurofibroma. Despite this requisite NF1 loss of heterozygosity in the tumor cell of origin, nontumorigenic cells contribute to both generalized and specific disease manifestations. In mouse models of plexiform neurofibroma formation, Nf1 haploinsufficient mast cells promote inflammation, accelerating tumor formation and growth. These recruited mast cells, hematopoietic effector cells long known to permeate neurofibroma tissue, mediate key mitogenic signals that contribute to vascular ingrowth, collagen deposition, and tumor growth. Thus, the plexiform neurofibroma microenvironment involves a tumor/stromal interaction with the hematopoietic system that depends, at the molecular level, on a stem cell factor/c-kit-mediated signaling axis. These observations parallel findings in other NF1 disease manifestations and are clearly relevant to medical management of these neurofibromas.

Normal hematopoiesis and neurofibromin-deficient myeloproliferative disease require Erk

Neurofibromatosis type 1 (NF1) predisposes individuals to the development of juvenile myelomonocytic leukemia (JMML), a fatal myeloproliferative disease (MPD). In genetically engineered murine models, nullizygosity of Nf1, a tumor suppressor gene that encodes a Ras-GTPase-activating protein, results in hyperactivity of Raf/Mek/Erk in hematopoietic stem and progenitor cells (HSPCs). Activated Erk1/2 phosphorylate kinases and transcription factors with myriad mitogenic roles in diverse cell types. However, genetic studies examining Erk1/2s differential and/or combined control of normal and Nf1-deficient myelopoiesis are lacking. Moreover, prior studies relying on chemical Mek/Erk inhibitors have reached conflicting conclusions in normal and Nf1-deficient mice. Here, we show that while single Erk1 or Erk2 disruption did not grossly compromise myelopoiesis, dual Erk1/2 disruption rapidly ablated granulocyte and monocyte production in vivo, diminished progenitor cell number, and prevented HSPC proliferation in vitro. Genetic disruption of Erk1/2 in the context of Nf1 nullizygosity (Mx1Cre(+)Nf1(flox/flox)Erk1(-/-)Erk2(flox/flox)) fully protects against the development of MPD. Collectively, we identified a fundamental requirement for Erk1/2 signaling in normal and Nf1-deficient hematopoiesis, elucidating a critical hematopoietic function for Erk1/2 while genetically validating highly selective Mek/Erk inhibitors in a leukemia that is otherwise resistant to traditional therapy.

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.

Plexiform neurofibroma genesis: questions of Nf1 gene dose and hyperactive mast cells

Purpose of reviewTumorigenic cells can co-opt normal functions of nonmalignant hematopoietic cells, promoting tumor progression. Recent mouse and human studies indicate that mast cells underpin inflammation in the plexiform neurofibroma microenvironment of neurofibromatosis type 1. In this model, Nf1 homozygous-deficient Schwann cells recruit hyperactive mast cells, promoting tumorigenesis. Here, we discuss the importance of Nf1 gene dosage, delineate hematopoietic contributions to the plexiform neurofibroma microenvironment, and highlight applications to human treatment. Recent findingsPrevious studies found that plexiform neurofibroma formation in a mouse model requires biallelic loss of Nf1 in Schwann cells and an Nf1 heterozygous cellular background. Now, transplantation and pharmacological experiments have indicated that tumor formation specifically requires Nf1 heterozygosity of c-kit-dependent bone marrow. SummaryNeurofibromatosis type 1 results from autosomal dominant mutations of the NF1 tumor suppressor gene. Although unpredictable second-hit mutations in the remaining NF1 allele precede local manifestations such as tumor formation, human and mouse data indicate that NF1/Nf1 gene haploinsufficiency modulates cellular physiology and disease pathogeneses. In particular, Nf1 haploinsufficient mast cells demonstrate multiple gain-in-functions, and mast cells permeate neurofibroma tissue. Transplantation experiments have shown that these aberrant mast cells critically underpin the tumor microenvironment. Using these findings, clinicians have medically treated a patient with a debilitating plexiform neurofibroma.

Multiple increased osteoclast functions in individuals with neurofibromatosis type 1

Skeletal abnormalities including scoliosis, tibial dysplasia, sphenoid wing dysplasia, and decreased bone mineral density (BMD) are associated with neurofibromatosis type 1 (NF1). We report the cellular phenotype of NF1 human‐derived osteoclasts and compare the in vitro findings with the clinical phenotype. Functional characteristics (e.g., osteoclast formation, migration, adhesion, resorptive capacity) and cellular mechanistic alterations (e.g., F‐actin polymerization, MAPK phosphorylation, RhoGTPase activity) from osteoclasts cultured from peripheral blood of individuals with NF1 (N = 75) were assessed. Osteoclast formation was compared to phenotypic, radiologic, and biochemical data. NF1 osteoprogenitor cells demonstrated increased osteoclast forming capacity. Human NF1‐derived osteoclasts demonstrated increased migration, adhesion, and in vitro bone resorption. These activities coincided with increased actin belt formation and hyperactivity in MAPK and RhoGTPase pathways. Although osteoclast formation was increased, no direct correlation of osteoclast formation with BMD, markers of bone resorption, or the clinical skeletal phenotype was observed suggesting that osteoclast formation in vitro cannot directly predict NF1 skeletal phenotypes. While NF1 haploinsufficiency produces a generalized osteoclast gain‐in‐function and may contribute to increased bone resorption, reduced BMD, and focal skeletal defects associated with NF1, additional and perhaps local modifiers are likely required for the development of skeletal abnormalities in NF1.

The Plexiform Neurofibroma Microenvironment

Dynamic interactions between tumorigenic cells and surrounding cells, including immunomodulatory hematopoietic cells, can dictate tumor initiation, progression, and transformation. Hematopoietic-stromal interactions underpin the plexiform neurofibroma, a debilitating tumor arising in individuals afflicted with Neurofibromatosis type 1 (NF1), a common genetic disorder resulting from mutations in the NF1 tumor suppressor gene. At the tissue level, plexiform neurofibromas demonstrate a complex microenvironment composed of Schwann cells, fibroblasts, perineural cells, mast cells, secreted collagen, and blood vessels. At the cellular level, specific interactions between these cells engender tumor initiation and progression. In this microenvironment hypothesis, tumorigenic Schwann cells secrete pathological concentrations of stem cell factor, which recruit c-kit expressing mast cells. In turn, activated mast cells release inflammatory effectors stimulating the tumorigenic Schwann cells and their supporting fibroblasts and blood vessels, thus promoting tumor expansion in a feed-forward loop. Bone marrow transplantation experiments in plexiform neurofibroma mouse models have shown that tumorigenesis requires Nf1 haploinsufficiency in the hematopoietic compartment, suggesting that tumor microenvironments can depend on intricate interactions at both cellular and genetic levels. Overall, our continued understanding of critical tumor-stromal interactions will illuminate novel therapeutic targets, as shown by the first-ever successful medical treatment of a plexiform neurofibroma by targeted inhibition of the stem cell factor/c-kit axis.