Recommendations for the collection and annotation of biosamples for analysis of biomarkers in neurofibromatosis and schwannomatosis clinical trials.

INTRODUCTION: Neurofibromatosis 1 and schwannomatosis are characterized by potential lifelong morbidity and life-threatening complications. To date, however, diagnostic and predictive biomarkers are an unmet need in this patient population. The inclusion of biomarker discovery correlatives in neurofibromatosis 1/schwannomatosis clinical trials enables study of low-incidence disease. The implementation of a common data model would further enhance biomarker discovery by enabling effective concatenation of data from multiple studies. METHODS: The Response Evaluation in Neurofibromatosis and Schwannomatosis biomarker working group reviewed published data on emerging trends in neurofibromatosis 1 and schwannomatosis biomarker research and developed recommendations in a series of consensus meetings. RESULTS: Liquid biopsy has emerged as a promising assay for neurofibromatosis 1/schwannomatosis biomarker discovery and validation. In addition, we review recommendations for a range of biomarkers in clinical trials, neurofibromatosis 1/schwannomatosis-specific data annotations, and common data models for data integration. CONCLUSION: These Response Evaluation in Neurofibromatosis and Schwannomatosis consensus guidelines are intended to provide best practices for the inclusion of biomarker studies in neurofibromatosis 1/schwannomatosis clinical trials, data, and sample annotation and to lay a framework for data harmonization and concatenation between trials.

Advances in Targeted Therapy for Neurofibromatosis Type 2 (NF2)-Associated Vestibular Schwannomas.

PURPOSE OF REVIEW: Neurofibromatosis 2 (NF2) is an autosomal-dominant genetic disorder characterized by bilateral vestibular schwannomas (VS), meningiomas, ependymomas, spinal and peripheral schwannomas, optic gliomas, and juvenile cataracts. Ongoing studies provide new insight into the role of the NF2 gene and merlin in VS tumorigenesis. RECENT FINDINGS: As NF2 tumor biology becomes increasingly understood, therapeutics targeting specific molecular pathways have been developed and evaluated in preclinical and clinical studies. NF2-associated VS are a source of significant morbidity with current treatments including surgery, radiation, and observation. Currently, there are no FDA-approved medical therapies for VS, and the development of selective therapeutics is a high priority. This manuscript reviews NF2 tumor biology and current therapeutics undergoing investigation for treatment of patients with VS.

Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin.

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

Genetic disruption of the small GTPase RAC1 prevents plexiform neurofibroma formation in mice with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1-/- Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) in NF1-/- Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre- littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox ;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.

Cdkn2a (Arf) loss drives NF1-associated atypical neurofibroma and malignant transformation.

Plexiform neurofibroma (PN) tumors are a hallmark manifestation of neurofibromatosis type 1 (NF1) that arise in the Schwann cell (SC) lineage. NF1 is a common heritable cancer predisposition syndrome caused by germline mutations in the NF1 tumor suppressor, which encodes a GTPase-activating protein called neurofibromin that negatively regulates Ras proteins. Whereas most PN are clinically indolent, a subset progress to atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBP) and/or to malignant peripheral nerve sheath tumors (MPNSTs). In small clinical series, loss of 9p21.3, which includes the CDKN2A locus, has been associated with the genesis of ANNUBP. Here we show that the Cdkn2a alternate reading frame (Arf) serves as a gatekeeper tumor suppressor in mice that prevents PN progression by inducing senescence-mediated growth arrest in aberrantly proliferating Nf1-/- SC. Conditional ablation of Nf1 and Arf in the neural crest-derived SC lineage allows escape from senescence, resulting in tumors that accurately phenocopy human ANNUBP and progress to MPNST with high penetrance. This animal model will serve as a platform to study the clonal development of ANNUBP and MPNST and to identify new therapies to treat existing tumors and to prevent disease progression.

A proteasome-resistant fragment of NIK mediates oncogenic NF-κB signaling in schwannomas.

Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.

Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours.

BACKGROUND: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. METHODS: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. RESULTS: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. CONCLUSIONS: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.

A molecular basis for neurofibroma-associated skeletal manifestations in NF1.

PURPOSE: Plexiform neurofibromas (pNF) develop in children with neurofibromatosis type 1 (NF1) and can be associated with several skeletal comorbidities. Preclinical mouse studies revealed Nf1 deficiency in osteoprogenitor cells disrupts, in a MEK-dependent manner, pyrophosphate (PPi) homeostasis and skeletal mineralization. The etiology of NF-associated skeletal manifestations remains unknown. METHODS: We used mouse models of NF1 neurofibromas to assess bone mineralization of skeletal structures adjacent to tumors. Expression of genes involved in pyrophosphate homeostasis was assessed in mouse and human NF tumors and Schwann cell cultures. We used dual-energy X-ray absorptiometry (DXA) to assess tumor-associated changes in bone mineral density (BMD) in an individual with NF1 following treatment with the MEK inhibitor selumetinib. RESULTS: We detected increased nonmineralized bone surfaces adjacent to tumors in mouse models of NF1 neurofibromas. Expression of Enpp1, a PPi-generating ectophosphatase, and ANKH, a PPi transporter, was increased in mouse and human neurofibroma-derived tissues and Schwann cells, respectively. In one patient, tumor-associated reductions in BMD were partially rescued following therapy with selumetinib. CONCLUSION: Results indicate that NF-associated skeletal pathologies in NF1 are associated with dysregulated pyrophosphate homeostasis in adjacent NF tumors and suggest that treatment of NFs with MEK inhibitors may improve skeletal manifestations of the disease.

Early administration of imatinib mesylate reduces plexiform neurofibroma tumor burden with durable results after drug discontinuation in a mouse model of neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by plexiform neurofibromas (pNF), which are thought to be congenital tumors that arise in utero and enlarge throughout life. Genetic studies in murine models delineated an indispensable role for the stem cell factor (SCF)/c-kit pathway in pNF initiation and progression. A subsequent phase 2 clinical trial using imatinib mesylate to inhibit SCF/c-kit demonstrated tumor shrinkage in a subset of preexisting pNF; however, imatinib's role on preventing pNF development has yet to be explored. PROCEDURE: We evaluated the effect of imatinib dosed at 10-100 mg/kg/day for 12 weeks to one-month-old Nf1flox/flox ;PostnCre(+) mice, prior to onset of pNF formation. To determine durability of response, we then monitored for pNF growth at later time points, comparing imatinib- with vehicle-treated mice. We assessed gross and histopathological analysis of tumor burden. RESULTS: Imatinib administered preventatively led to a significant decrease in pNF number, even at doses as low as 10 mg/kg/day. Tumor development continued to be significantly inhibited after cessation of imatinib dosed at 50 and 100 mg/kg/day. In the cohort of treated mice that underwent prolonged follow-up, the size of residual tumors was significantly reduced as compared with age-matched littermates that received vehicle control. CONCLUSIONS: Early administration of imatinib inhibits pNF genesis in vivo, and effects are sustained after discontinuation of therapy. These findings may guide clinical use of imatinib in young NF1 patients prior to the substantial development of pNF.

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.

An abnormal bone marrow microenvironment contributes to hematopoietic dysfunction in Fanconi anemia.

Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation.

Preclinical Evidence for the Use of Sunitinib Malate in the Treatment of Plexiform Neurofibromas.

PURPOSE: Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c-Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c-Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1(flox/-) pNF murine model. EXPERIMENTAL DESIGN: Proliferation, ß-hexosaminidase release (degranulation), and Erk1/2 phosphorylation were assessed in sunitinib treated Nf1(+/-) mast cells and fibroblasts, respectively. Krox20;Nf1(flox/-) mice with established pNF were treated sunitinib or PBS-vehicle control for a duration of 12 weeks. pNF metabolic activity was monitored by serial [(18)F]DG-PET/CT imaging. RESULTS: Sunitinib suppressed multiple in vitro gain-in-functions of Nf1(+/-) mast cells and fibroblasts and attenuated Erk1/2 phosphorylation. Sunitinib treated Krox20;Nf1(flox/-) mice exhibited significant reductions in pNF size, tumor number, and FDG uptake compared to control mice. Histopathology revealed reduced tumor cellularity and infiltrating mast cells, markedly diminished collagen deposition, and increased cellular apoptosis in sunitinib treated pNF. CONCLUSIONS: Collectively, these results demonstrate the efficacy of sunitinib in reducing tumor burden in Krox20;Nf1(flox/-) mice. These preclinical findings demonstrate the utility of inhibiting multiple RTKs in pNF and provide insights into the design of future clinical trials.

Aberrant Myeloid Differentiation Contributes to the Development of Osteoporosis in Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1), also known as von Recklinghausen disease, is a common autosomal dominant genetic disorder affecting approximately 1 in 3000 individuals worldwide. NF1 results from heritable or spontaneous mutations of the NF1 tumor suppressor gene. NF1 encodes the protein neurofibromin, which functions to negatively regulate Ras-activity. Approximately 50 % of NF1 patients develop osteopenia or osteoporosis, resulting in significantly increased rates of long-bone fracture and morbidity. While defective osteoblast bone anabolism has been implicated as a central factor in the pathogenesis of NF1 associated skeletal deficits, recent data suggest that NF1 (Nf1) haploinsufficiency within the hematopoietic compartment, particularly in osteoclasts and myeloid progenitors, plays a pivotal role in engendering NF1 osseous manifestations. In this chapter, we review the latest data from clinical studies and murine models delineating a critical role for hematopoietic compartment, myeloid progenitors of NF1 (Nf1) haploinsufficient and their progeny-osteoclasts, in the pathogenesis of NF1 associated osteopenia/osteoporosis and discuss putative targets for future therapeutics.

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.

Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1(+/-)) mice. Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1(+/-) MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.

The NF1+/- Immune Microenvironment: Dueling Roles in Neurofibroma Development and Malignant Transformation.

Neurofibromatosis type 1 (NF1) is a common genetic disorder resulting in the development of both benign and malignant tumors of the peripheral nervous system. NF1 is caused by germline pathogenic variants or deletions of the NF1 tumor suppressor gene, which encodes the protein neurofibromin that functions as negative regulator of p21 RAS. Loss of NF1 heterozygosity in Schwann cells (SCs), the cells of origin for these nerve sheath-derived tumors, leads to the formation of plexiform neurofibromas (PNF)-benign yet complex neoplasms involving multiple nerve fascicles and comprised of a myriad of infiltrating stromal and immune cells. PNF development and progression are shaped by dynamic interactions between SCs and immune cells, including mast cells, macrophages, and T cells. In this review, we explore the current state of the field and critical knowledge gaps regarding the role of NF1(Nf1) haploinsufficiency on immune cell function, as well as the putative impact of Schwann cell lineage states on immune cell recruitment and function within the tumor field. Furthermore, we review emerging evidence suggesting a dueling role of Nf1+/- immune cells along the neurofibroma to MPNST continuum, on one hand propitiating PNF initiation, while on the other, potentially impeding the malignant transformation of plexiform and atypical neurofibroma precursor lesions. Finally, we underscore the potential implications of these discoveries and advocate for further research directed at illuminating the contributions of various immune cells subsets in discrete stages of tumor initiation, progression, and malignant transformation to facilitate the discovery and translation of innovative diagnostic and therapeutic approaches to transform risk-adapted care.

Efficacy of the Allosteric MEK Inhibitor Trametinib in Relapsed and Refractory Juvenile Myelomonocytic Leukemia: a Report from the Children's Oncology Group.

Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of young children caused by mutations that increase Ras signaling output. Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment, but patients with relapsed or refractory (advanced) disease have dismal outcomes. This phase II trial evaluated the safety and efficacy of trametinib, an oral MEK1/2 inhibitor, in patients with advanced JMML. Ten infants and children were enrolled, and the objective response rate was 50%. Four patients with refractory disease proceeded to HSCT after receiving trametinib. Three additional patients completed all 12 cycles permitted on study and continue to receive off-protocol trametinib without HSCT. The remaining three patients had progressive disease with two demonstrating molecular evolution by the end of cycle 2. Transcriptomic and proteomic analyses provided novel insights into the mechanisms of response and resistance to trametinib in JMML. ClinicalTrials.gov Identifier: NCT03190915. Significance: Trametinib was safe and effective in young children with relapsed or refractory JMML, a lethal disease with poor survival rates. Seven of 10 patients completed the maximum 12 cycles of therapy or used trametinib as a bridge to HSCT and are alive with a median follow-up of 24 months.