Nf1 Haploinsufficiency Research Articles

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.

Splicing annotation of endometrial cancer GWAS risk loci reveals potentially causal variants and supports a role for NF1 and SKAP1 as susceptibility genes.

Alternative splicing contributes to cancer development. Indeed, splicing analysis of cancer genome-wide association study (GWAS) risk variants has revealed likely causal variants. To systematically assess GWAS variants for splicing effects, we developed a prioritization workflow using a combination of splicing prediction tools, alternative transcript isoforms, and splicing quantitative trait locus (sQTL) annotations. Application of this workflow to candidate causal variants from 16 endometrial cancer GWAS risk loci highlighted single-nucleotide polymorphisms (SNPs) that were predicted to upregulate alternative transcripts. For two variants, sQTL data supported the predicted impact on splicing. At the 17q11.2 locus, the protective allele for rs7502834 was associated with increased splicing of an exon in a NF1 alternative transcript encoding a truncated protein in adipose tissue and is consistent with an endometrial cancer transcriptome-wide association study (TWAS) finding in adipose tissue. Notably, NF1 haploinsufficiency is protective for obesity, a well-established risk factor for endometrial cancer. At the 17q21.32 locus, the rs2278868 risk allele was predicted to upregulate a SKAP1 transcript that is subject to nonsense-mediated decay, concordant with a corresponding sQTL in lymphocytes. This is consistent with a TWAS finding that indicates decreased SKAP1 expression in blood increases endometrial cancer risk. As SKAP1 is involved in Tcell immune responses, decreased SKAP1 expression may impact endometrial tumor immunosurveillance. In summary, our analysis has identified potentially causal endometrial cancer GWAS risk variants with plausible biological mechanisms and provides a splicing annotation workflow to aid interpretation of other GWAS datasets.

Modeling Relapsed, Refractory Acute Lymphoblastic Leukemia from a Child with Neurofibromatosis

Neurofibromatosis type 1 (NF-1) is an autosomal dominant disorder affecting approximately 1:3000 individuals globally. While approximately 50% are familial, with over 3000 causative germline variants in the neurofibromatosis (NF1) gene identified, the remainder occur sporadically. These mutations lead to haploinsufficiency of NF1 and neurofibromin, a tumor suppressor and important negative regulator of RAS signaling. Children with NF-1 have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop acute lymphoblastic leukemia (ALL).A 9-year-old male presented in 2015 with persistent migratory subcutaneous swellings and multiple bony aches with lytic lesions on bone imaging. He had a high white cell count with eosinophilia (WCC 43.4 x 10 9/L, eosinophils 23.87 x 10 9/L) with no circulating blasts, 10% marrow blasts (CD10+/CD19+/CD34+) and was CNS negative. Although previously undiagnosed, NF-1 was clinically suspected due to typical skin changes.He was diagnosed with iAMP21 ALL and NF-1 was confirmed with the identification of a germline NF1 donor splice site mutation (c.1845G>A:p.L615=). Bone marrow cells were sorted by flow cytometry on CD19 positivity and underwent transcriptomic sequencing. This revealed a P2RY8-CRLF2 gene fusion, with no other clinically relevant variants, while a custom Taqman low density array indicated high-risk B-ALL subtype Ph-like ALL. Multiplex ligation-dependent probe amplification (MLPA) confirmed iAMP21 and also identified IKZF1 exon 2-3 and BTG1 deletions.Treatment followed the high-risk B-ALL arm of the AEIOP-BFM ALL2009 protocol due to persistent end-consolidation MRD in addition to iAMP21 and the Ph-like phenotype. He relapsed three years later off treatment and was refractory to both salvage chemotherapy and blinatumomab. The iAMP21, P2RY8-CRLF2 gene fusion, IKZF1 exon 2-3 and BTG1 deletions remained detectable. Whole exome sequencing of CD19 positive samples from diagnosis, relapse and mesenchymal stem cells (germline control) was performed, identifying a NF1 c.7400dupT:p.L2467 frameshift (fs) mutation only at relapse.To understand the implications of NF1 p.L2467fs, the P2RY8-CRLF2 gene fusion was first transduced into the interleukin 3 (IL3) dependent murine pro-B cell line Ba/F3. P2RY8-CRLF2 alone is not transforming and is thought to be a secondary event in iAMP21 ALL, providing an ideal model to study the cumulative effect of the NF1fs. The NF1fs was then introduced to the P2RY8-CRLF2 cells by CRISPR/Cas9. A proliferation assay was performed without IL3 and demonstrated the P2RY8-CRLF2+NF1fs cell line was IL3 independent, indicative of leukemic transformation, whereas all other lines were not (vs Ba/F3, p = 0.001).Neurofibromin can be constitutively phosphorylated at the c-terminus, negatively regulating NF1-GAP activity, suppressing RAS signaling and inducing cell cycle arrest. Therefore, to demonstrate loss of function due to the c-terminus NF1 p.L2467fs and increased RAS signaling, western blotting for pERK was performed. Significant upregulation of pERK was confirmed in P2RY8-CRLF2+NF1fs in comparison to Ba/F3 control cells (p=0.007) (Figure 1).The MEK inhibitors trametinib and mirdametinib are in clinical trials for NF-1 patients and have shown efficacy in ALL models with RAS mutations. In a 3-day cell death assay, only P2RY8-CRLF2+NF1fs demonstrated sensitivity to trametinib (LD 50 P2RY8-CRLF2 = >6.4 µM, NF1fs = >6.4 µM, P2RY8-CRLF2+NF1fs =1.7µM; p < 0.001) and mirdametinib (LD 50 P2RY8-CRLF2 = >16 µM, NF1fs = >16 µM, P2RY8-CRLF2+NF1fs = 8.3 µM; p < 0.0001) (Figure 2).Here, we have demonstrated a LOF NF1fs mutation using an in-vitro model of ALL. Germline NF1 haploinsufficiency and a second hit NF1 mutation in ALL is limited to one report of monozygotic twins with neurofibromatosis. We propose that NF1 p.L2467fs caused bi-allelic LOF and therefore contributed to relapse in this patient. An understanding of the genomic complexities that lead to relapse may also inform personalized treatment strategies. While this patient subsequently achieved remission with inotuzomab and underwent successful stem cell transplantation, the sensitivity to MEK inhibitors is an exciting development for neurofibromatosis patients with ALL. [Display omitted] DisclosuresYeung: Amgen: Honoraria; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. White: BMS: Honoraria, Research Funding; Novartis: Research Funding.

Sustained fetal hematopoiesis causes juvenile death from leukemia: evidence from a dual-age–specific mouse model

AbstractIt is not clear whether disrupted age-specific hematopoiesis contributes to the complex manifestations in leukemia patients who carry identical mutations, particularly in pediatric and adult patients with similar clinical characteristics. By studying a dual-age–specific mouse model, we demonstrate that (1) loss of Pten during the fetal-to-adult hematopoiesis switch (hematopoiesis switch) causes sustained fetal hematopoiesis, resulting in death in juvenile leukemia; (2) myeloid-biased hematopoiesis in juvenile mice is associated with the sustained fetal properties of hematopoietic stem cells (HSCs); (3) the age specificity of juvenile myelomonocytic leukemia depends on the copy number of Pten and Nf1; (4) single-allelic Pten deletion during the hematopoiesis switch causes constitutive activation of MAPK in juvenile mice with Nf1 loss of heterozygosity (LOH); and (5) Nf1 LOH causes monocytosis in juvenile mice with Pten haploinsufficiency but does not cause lethality until adulthood. Our data suggest that 1 copy of Pten is sufficient to maintain an intact negative-feedback loop of the Akt pathway and HSC function in reconstitution, despite MAPK being constitutively activated in juvenile Pten+/ΔNf1LOH mice. However, 2 copies of Pten are required to maintain the integrity of the MAPK pathway in juvenile mice with Nf1 haploinsufficiency. Our data indicate that previous investigations of Pten function in wild-type mice may not reflect the impact of Pten loss in mice with Nf1 mutations or other genetic defects. We provide a proof of concept that disassociated age-specific hematopoiesis contributes to leukemogenesis and pediatric demise.

Cooperative Epigenetic Regulation By ASXL1 and NF1 Loss on Leukemogenesis

Introduction:Additional sex combs-like 1 (ASXL1) is frequently mutated in a wide range of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasm, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). Notably, ASXL1 mutations are generally associated with the poor clinical outcomes. We and others have established Asxl1 mouse models and demonstrated that loss of Asxl1 leads to MDS-like disease, which can transform to myeloid leukemia in aged mice. These studies suggest that additional mutations may cooperate with Asxl1 loss to induce the leukemia transformation. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated.Methods: To identify cooperating events with ASXL1 mutations in myeloid malignancies, we recruited a cohort of 138 ASXL1 mutated patients and performed targeted exome sequencing. We then characterized the hematopoietic features using mouse models. A serial hematopoietic phenotypic analyses were used, including peripheral blood counts, flow cytometry, colony assay, morphology and transplantation assays. To decipher the molecular mechanisms by which loss of Asxl1 and Nf1 cooperate in promoting myeloid leukemia transformation, we performed RNA-seq and ChIP-seq to identify the differentially expressed genes and their associated histone modifications in four genotypes of mice, WT, Asxl1+/-, Nf1+/-, and Asxl1+/-;Nf1+/- mice. Finally, the leukemic mice were treated with pharmacologic inhibitors targeting both MAPK pathway and BET bromodomain in vivo as a proof-of-concept approach.Results: We analyzed the gene mutation profiles of 138 ASXL1 mutated patients based on targeted sequencing and found that 35 of these patients have gene mutations involving in RAS/MAPK signaling pathway, including NF1, NRAS, KRAS, PTPN11 or CBL. The incidence of AML was significantly higher in patients with RAS pathway mutations (48.6%) than in the cases without RAS pathway mutations (29.1%, p = 0.036, chi-square test). These data suggest that concomitant mutations of ASXL1 and RAS pathway genes associate with poor prognosis in myeloid malignancies. To validate the functional significance of cooperative mutations of ASXL1 and NF1, a negative regulator of the RAS signaling pathway, in the disease progression of myeloid leukemia, we generated Asxl1+/-;Nf1+/- and Mx1Cre;Asxl1fl/fl;Nf1fl/fl (Asxl1Δ/Δ;Nf1Δ/Δ) mice and performed hematopoietic phenotypic analyses. Asxl1 loss cooperates with haploinsufficiency of Nf1 to accelerate the development of myeloid leukemia in mice. Asxl1Δ/Δ;Nf1Δ/Δ mice displayed a rapid, progressive leukocytosis with severe anemia and thrombocytopenia 3-6 months after pIpC injection, indicative of aggressive myeloid leukemia with a 100% penetrance. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells lead to transcriptional activation of multiple pathways critical for leukemogenesis, such as MYC, NRAS, and BRD4. Convergent analysis of RNA-seq and ChIP-seq data reveal that the hyperactive MYC and BRD4 transcription program is correlated with elevated H3K4 tri-methylation at the promoter regions of genes involving these pathways. Importantly, pharmacological inhibition of both MAPK pathway and BET bromodomain prevents leukemia initiation and inhibits disease progression in Asxl1Δ/Δ;Nf1Δ/Δ mice, and significantly prolonged the survival of leukemic Asxl1+/-;Nf1+/- mice.Conclusion: This study sheds lights on the understanding of the cooperative effect between epigenetic alterations and signaling pathways in accelerating the progression of myeloid malignancies and provides a rationale therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations. DisclosuresNo relevant conflicts of interest to declare.

Chromatin regulator Asxl1 loss and Nf1 haploinsufficiency cooperate to accelerate myeloid malignancy.

ASXL1 is frequently mutated in myeloid malignancies and is known to co-occur with other gene mutations. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Here, we report that Asxl1 loss cooperated with haploinsufficiency of Nf1, a negative regulator of the RAS signaling pathway, to accelerate the development of myeloid leukemia in mice. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells resulted in a gain-of-function transcriptional activation of multiple pathways such as MYC, NRAS, and BRD4 that are critical for leukemogenesis. The hyperactive MYC and BRD9 transcription programs were correlated with elevated H3K4 trimethylation at the promoter regions of genes involving these pathways. Furthermore, pharmacological inhibition of both the MAPK pathway and BET bromodomain prevented leukemia initiation and inhibited disease progression in Asxl1Δ/Δ Nf1Δ/Δ mice. Concomitant mutations of ASXL1 and RAS pathway genes were associated with aggressive progression of myeloid malignancies in patients. This study sheds light on the effect of cooperation between epigenetic alterations and signaling pathways on accelerating the progression of myeloid malignancies and provides a rational therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations.

Abstract PR09: Modeling the tumor microenvironment in sarcoma: The impact on MPNST biology and chemotherapeutic response

Abstract NF1 haploinsufficiency is a hallmark of neurofibromatosis Type 1 and is critical for neurofibroma development. The role of the supporting stroma (endothelial cells, immune cells, fibroblasts, etc.) in the development of patient neurofibromas is well established. However, the impact of NF1 haploinsufficiency on MPNST biology is less clear. Sporadic MPNSTs arise in patients with NF1 wild-type stroma (NF1 +/+), while NF1-associated MPNSTs arise in neurofibromatosis patients with NF1 haploinsufficient stroma (NF1 -/+). Patients with NF1-associated MPNSTs have worse outcome than patients with sporadic MPNSTs, but the cause of this difference is under debate. To define the impact of stromal genetics on tumor biology, we have developed unique mouse models that reflect the genetics of patient-associated MPNSTs. Using paired littermate mice, we model the genetic status of NF1 in the tumor microenvironment of different MPNST patient populations. We use Adenovirus-Cre injections to generate MPNSTs in NF1 Flox/Flox; CDKN2A Flox/Flox and NF1 Null/Flox; CDKN2A Flox/Flox paired littermate mice to model tumors from NF1 wild-type and NF1-assoicated patients, respectively. We find that NF1 haploinsufficiency (NF1 +/- stroma) accelerates tumor onset, which is accompanied by an increase in immune cells comprising CD11B+ cells, monocytes, and mast cells. Furthermore, mast cells are also enriched in human NF1-associated MPNSTs. We used these mouse models in a preclinical trial to examine how the tumor microenvironment influences response to multiagent chemotherapy and found that the stromal NF1 status does not impact the response of MPNSTs to chemotherapy. Taken together, these studies help clarify the role of the NF1 haploinsufficient tumor microenvironment in MPNSTs. This abstract is also being presented as Poster B15. Citation Format: Rebecca Dodd, Chang-Lung Lee, Mary Overton, Wesley Huang, William Eward, Davis Ingram, Diana Cordona, Alexander Lazar, David Kirsch. Modeling the tumor microenvironment in sarcoma: The impact on MPNST biology and chemotherapeutic response [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR09.

Characterization of early communicative behavior in mouse models of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder caused by mutation of the NF1 gene, in which 80% of affected children exhibit cognitive and behavioral issues. Based on emerging evidence that NF1 may be an autism predisposition gene, we examined autism spectrum disorder (ASD)-relevant early communicative behavior in Nf1 mouse models and observed alterations in both models. The changes in early communicative behavior in Nf1 mutant mice should motivate further studies into the causative factors and potential treatments for ASD arising in the context of NF1.

NF1+/- Hematopoietic Cells Accelerate Malignant Peripheral Nerve Sheath Tumor Development without Altering Chemotherapy Response.

Haploinsufficiency in the tumor suppressor NF1 contributes to the pathobiology of neurofibromatosis type 1, but a related role has not been established in malignant peripheral nerve sheath tumors (MPNST) where NF1 mutations also occur. Patients with NF1-associated MPNST appear to have worse outcomes than patients with sporadic MPNST, but the mechanism underlying this correlation is not understood. To define the impact of stromal genetics on the biology of this malignancy, we developed unique mouse models that reflect the genetics of patient-associated MPNST. Specifically, we used adenovirus-Cre injections to generate MPNST in Nf1Flox/Flox; Ink4a/ArfFlox/Flox and Nf1Flox/-; Ink4a/ArfFlox/Flox paired littermate mice to model tumors from NF1-wild-type and NF1-associated patients, respectively. In these models, Nf1 haploinsufficiency in hematopoietic cells accelerated tumor onset and increased levels of tumor-infiltrating immune cells comprised of CD11b+ cells, monocytes, and mast cells. We observed that mast cells were also enriched in human NF1-associated MPNST. In a coclinical trial to examine how the tumor microenvironment influences the response to multiagent chemotherapy, we found that stromal Nf1 status had no effect. Taken together, our results clarify the role of the NF1-haploinsufficient tumor microenvironment in MPNST. Cancer Res; 77(16); 4486-97. ©2017 AACR.

CRISPR/Cas9 editing of Nf1 gene identifies CRMP2 as a therapeutic target in neurofibromatosis type 1-related pain that is reversed by (S)-Lacosamide.

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the Nf1 gene. Patients with NF1 commonly experience severe pain. Studies on mice with Nf1 haploinsufficiency have been instructive in identifying sensitization of ion channels as a possible cause underlying the heightened pain suffered by patients with NF1. However, behavioral assessments of Nf1 mice have led to uncertain conclusions about the potential causal role of Nf1 in pain. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) genome editing system to create and mechanistically characterize a novel rat model of NF1-related pain. Targeted intrathecal delivery of guide RNA/Cas9 nuclease plasmid in combination with a cationic polymer was used to generate allele-specific C-terminal truncation of neurofibromin, the protein encoded by the Nf1 gene. Rats with truncation of neurofibromin, showed increases in voltage-gated calcium (specifically N-type or CaV2.2) and voltage-gated sodium (particularly tetrodotoxin-sensitive) currents in dorsal root ganglion neurons. These gains-of-function resulted in increased nociceptor excitability and behavioral hyperalgesia. The cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) regulates activity of these channels, and also binds to the targeted C-terminus of neurofibromin in a tripartite complex, suggesting a possible mechanism underlying NF1 pain. Prevention of CRMP2 phosphorylation with (S)-lacosamide resulted in normalization of channel current densities, excitability, as well as of hyperalgesia following CRISPR/Cas9 truncation of neurofibromin. These studies reveal the protein partners that drive NF1 pain and suggest that CRMP2 is a key target for therapeutic intervention.

Timing of the loss of Pten protein determines disease severity in a mouse model of myeloid malignancy

AbstractJuvenile myelomonocytic leukemia (JMML) is an aggressive pediatric mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN). JMML leukemogenesis is linked to a hyperactivated RAS pathway, with driver mutations in the KRAS, NRAS, NF1, PTPN11, or CBL genes. Previous murine models demonstrated how those genes contributed to the selective hypersensitivity of JMML cells to granulocyte macrophage–colony-stimulating factor (GM-CSF), a unifying characteristic in the disease. However, it is unclear what causes the early death in children with JMML, because transformation to acute leukemia is rare. Here, we demonstrate that loss of Pten (phosphatase and tensin homolog) protein at postnatal day 8 in mice harboring Nf1 haploinsufficiency results in an aggressive MPN with death at a murine prepubertal age of 20 to 35 days (equivalent to an early juvenile age in JMML patients). The death in the mice was due to organ infiltration with monocytes/macrophages. There were elevated activities of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) in cells at physiological concentrations of GM-CSF. These were more pronounced in mice with Nf1 haploinsufficiency than in littermates with wild-type Nf1, but this model is insufficient to cause cells to be GM-CSF hypersensitive. This new model represents a murine MPN model with features of a pediatric unclassifiable mixed MDS/MPN and mimics many clinical manifestations of JMML in terms of age of onset, aggressiveness, and organ infiltration with monocytes/macrophages. Our data suggest that the timing of the loss of PTEN protein plays a critical role in determining the disease severity in myeloid malignancies. This model may be useful for studying the pathogenesis of pediatric diseases with alterations in the Ras pathway.

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.

Capturing the wide variety of impaired fracture healing phenotypes in Neurofibromatosis Type 1 with eight key factors: a computational study.

Congenital pseudarthrosis of the tibia (CPT) is a rare disease which normally presents itself during early childhood by anterolateral bowing of the tibia and spontaneous tibial fractures. Although the exact etiology of CPT is highly debated, 40–80% of CPT patients are carriers of a mutation in the Neurofibromatosis Type 1 (NF1) gene, which can potentially result in an altered phenotype of the skeletal cells and impaired bone healing. In this study we use a computational model of bone regeneration to examine the effect of the Nf1 mutation on bone fracture healing by altering the parameter values of eight key factors which describe the aberrant cellular behaviour of Nf1 haploinsufficient and Nf1 bi-allelically inactivated cells. We show that the computational model is able to predict the formation of a hamartoma as well as a wide variety of CPT phenotypes through different combinations of altered parameter values. A sensitivity analysis by “Design of Experiments” identified the impaired endochondral ossification process and increased infiltration of fibroblastic cells as key contributors to the degree of severity of CPT. Hence, the computational model results have added credibility to the experimental hypothesis of a genetic cause (i.e. Nf1 mutation) for CPT.

Cooperative Effect of Haploinsufficient ASXL1 and NF1 in Acute Myeloid Leukemia Transformation

Objective: The advent of high-throughput whole genome or exome sequencing has led to the identification of almost all genetic aberrations in acute myeloid leukemia (AML). These genetic aberrations have been classified into at least three classes (class I: activate signal transduction pathways and confer a proliferation advantage; class II: affect transcription factors and impair hematopoietic differentiation; class III: concern with epigenetic regulation. AML development is considered to be a multistep process that requires the collaboration of at least two classes of mutations, the so-called "two-hit model". Additional Sex Combs Like 1 (ASXL1) gene, encoding polycomb protein ASXL1, is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. Hyperactive RAS signaling is also common in myeloid malignancies, which is caused by mutations in the RAS genes or an inactivation of the neurofibromatosis (NF1) gene. The objective of this project is to determine the cooperative effects of NF1 loss (class I) and ASXL1 loss (class III) on the AML development using a novel mouse model with combined haploinsufficiency of Asxl1 and Nf1.Methods: In the current study, we intercrossed Asxl1+/- mice with Nf1+/- mice and characterized the hematopoietic features of Asxl1+/-;Nf1+/- mice in vivo. A serial analyses were used for hematopoietic analysis, including peripheral blood counts, blood smear, morphology, flow cytometry and histology. In vivo tumor transfer was performed to evaluate the malignant nature of the infiltrated cells in moribund/diseased compound Asxl1+/-;Nf1+/- mice. To define the molecular mechanisms by which haploinsufficient Asxl1 and Nf1 cooperate in promoting AML transformation,western blotting was performed to examine the dysregulated histone modifications and RNA-seq was performed to survey the differentially expressed genes in each genotype of mice.Results: A worse survival rate was seen in Asxl1+/-;Nf1+/- mice as compared to all other three groups of control mice, including WT, Nf1+/-, and Asxl1+/- mice. WT and Nf1+/- mice remain disease free till the end of the observed period of time (17 months), while a proportion of aged Asxl1+/- mice developed MDS-like diseases as characterized by multilineage cytopenias and dysplasia, which is consistent with our previous report (Wang et al. Blood 2014). Interestingly, haploinsufficiency of both Asxl1 and Nf1 results in progressive and lethal AML, as reflected by >20% of blasts in the bone marrow. Asxl1+/-;Nf1+/- mice also exhibited an elevated white blood cell counts with anemia and thrombocytopenia, as well as spleno-hepatomegaly. Infiltration of immatured myeloid cells was promident in the spleens and livers of Asxl1+/-;Nf1+/- mice. Tumor transfer assay revealed that the leukemia was transferable to the sub-lethal irradiated recipients, and the secondary transplanted mice displaying a similar phenotype to the primary AML mouse as early as 7 weeks after the tumor transfer. Furthermore, biochemistry study revealed a reduction of the global levels of H3K27me3 in Asxl1+/- c-Kit+ cells. Heterozygous of Nf1 in Asxl1+/- c-Kit+ cellsfurther diminished globle level of H3K27me3. Interestingly, the mRNA expression of Jarid2, an accessory component of polycomb repressive complex-2 (PRC2), was significantly lower in c-Kit+ cells of Asxl1+/-;Nf1+/- micethan that in c-Kit+ cells of other genotypes of mice. The reduced level of Jarid2 may result in further reduction in recruitment of the PRC2 component to the chromatin, dminishing H3K27me3 level.Conclusion: Mutations of NF1, encoding neurofibromin that functions as a negative regulator of RasGTP, are found co-exist with ASXL1 mutations in patients with myeloid malignancies. Our data indicate that combined haploinsufficiency of ASXL1 and NF1 trigger AML transformation through an additive effects to reduce H3K27me3 levels. Together, our data reinforce the impact of combined genetic abnormalities in class I and class III genes in the progression of myeloid malignancies into AML. DisclosuresNo relevant conflicts of interest to declare.

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.

Nf1 Haploinsufficiency Alters Myeloid Lineage Commitment and Function, Leading to Deranged Skeletal Homeostasis.

Although nullizygous loss of NF1 leads to myeloid malignancies, haploinsufficient loss of NF1 (Nf1) has been shown to contribute to osteopenia and osteoporosis which occurs in approximately 50% of neurofibromatosis type 1 (NF1) patients. Bone marrow mononuclear cells of haploinsufficient NF1 patients and Nf1(+/-) mice exhibit increased osteoclastogenesis and accelerated bone turnover; however, the culprit hematopoietic lineages responsible for perpetuating these osteolytic manifestations have yet to be elucidated. Here we demonstrate that conditional inactivation of a single Nf1 allele within the myeloid progenitor cell population (Nf1-LysM) is necessary and sufficient to promote multiple osteoclast gains-in-function, resulting in enhanced osteoclastogenesis and accelerated osteoclast bone lytic activity in response to proresorptive challenge in vivo. Surprisingly, mice conditionally Nf1 heterozygous in mature, terminally differentiated osteoclasts (Nf1-Ctsk) do not exhibit any of these skeletal phenotypes, indicating a critical requirement for Nf1 haploinsufficiency at a more primitive/progenitor stage of myeloid development in perpetuating osteolytic activity. We further identified p21Ras-dependent hyperphosphorylation of Pu.1 within the nucleus of Nf1 haploinsufficient myelomonocytic osteoclast precursors, providing a novel therapeutic target for the potential treatment of NF1 associated osteolytic manifestations.

Differential Effects of Neurofibromin Gene Dosage on Melanocyte Development

Mutations in neurofibromin (NF1) cause the dominant genetic disorder neurofibromatosis type 1. Neurofibromatosis is characterized by Schwann cell-based tumors and skin hyperpigmentation, resulting from both haploinsufficiency and loss of heterozygosity. The fact that some pigment cells (melanocytes) arise from Schwann cell precursors suggests that neurofibromin could be required during the common precursor stage. In this study, we found a missense mutation in neurofibromin in Dark skin 9 (Dsk9) mutant mice, revealing that Nf1 mutations cause skin hyperpigmentation in mice, as they do in humans. Using tissue-specific knockouts, we found that haploinsufficiency of neurofibromin in melanocytes via Mitf-cre is insufficient to cause darker skin, whereas haploinsufficiency in bipotential Schwann cell-melanoblast precursors via Plp1-creER is sufficient. These findings suggest that there is a narrow developmental window during which Nf1 haploinsufficiency acts on pigment cells. Using fate mapping, we discovered differences in the colonization of the dermis and epidermis by melanocytes that arise from Schwann cell precursors, an unexpected complexity of melanocyte development. As homozygous knockout of Nf1 via Mitf-cre is sufficient to cause darker skin, we conclude that reduced gene dosage can act by a mechanism different from complete gene loss, even when the end result of both is very similar.

Haploinsufficiency of Nf1 in Myeloid Lineages Contributes to Neurofibromatosis Type 1 Associated Skeletal Deficits

Abstract 830Myelopoiesis influences multiple physiological processes including skeletal remodeling. Osteoclasts (OCs) are specialized myeloid cells responsible for bone resorption. Dysregulated myeloid differentiation and OC bone lytic activity can lead not only to hematopoietic deficits, but also to skeletal diseases such as osteoporosis and osteopetrosis. Haploinsufficient loss of the Nf1 tumor suppressor gene leads to Ras mediated cytokine hypersensitivity in multiple myeloid lineages, including marrow-derived myeloid progenitor cells, mast cells, and OCs. Aside from neoplasia, patients with the genetic disorder neurofibromatosis type 1 (NF1) exhibit a spectrum of osseous defects including osteoporosis and pseudarthrosis – severe non-union fractures that respond poorly to treatment and often require amputation. Detailed investigation of the cellular and molecular mechanisms underlying NF1 pathological bone repair is needed to identify targeted therapies.In a series of adoptive bone marrow transfer experiments, we recently established that Nf1 haploinsufficiency in the hematopoietic system plays a critical role in the pathogenesis of tibial fracture non-union in the NF1 murine model. Since Nf1+/− OCs exhibit multiple gain-in-fuctions, we reasoned that haploinsufficient loss of Nf1 in myeloid OC progenitors is required for the genesis of osseous phenotypes. To test this hypothesis, we generated Nf1f/+;LysMCre+ mice harboring conditional inactivation of a single Nf1 allele in myeloid lineages. Here, we report that Nf1f/+;LysMCre+ mice exhibit increased frequency of osteoclast progenitors (CFU-M), enhanced osteoclastogenesis, and accelerated bone resorption following ovariectomy induced resorptive stress in vivo. Interestingly, recombination of the floxed Nf1 allele under CtskCre, restricted to mature osteoclasts, did not recapitulate these phenotypes in analogous experiments. These data provide direct genetic evidence that haploinsufficiency of Nf1 in myeloid progenitors, but not terminally differentiated OCs alone, is required to expand the pool of OC progenitors, thereby inducing increased bone resorption in vivo.To further investigate the contribution of Nf1 hapolinsufficient myeloid cells in a NF1 psuedarthrosis mouse model, we transplanted bone marrow (BM) cells from WT and Nf1f/+;LysMCre+ mice and monitored tibial fracture repair after stable hemapoietic reconstitution. Compared to WT BM cells, mice reconstituted with Nf1f/+;LysMCre BM cells exhibited deficient cortical bridging and significantly reduced callus bone volume fraction (BV/TV) as determined by micro-computed tomography (μCT), indicating substantial deficits in fracture repair. Histological analysis revealed fibrous infiltrates and excessive osteoclast numbers within the fracture callus. To determine the molecular mechanisms by which Nf1 status affects the proliferation and commitment of OC progenitors, we considered transcription factors that are important for early myeloid differentiation. Here, we show that Nf1+/− OC gain-in functions are associated with increased nuclear levels of phosphorylated Pu.1. These data implicate Ras as a molecular switch regulating macrophage and OC development by controlling both the expression and post-translational modification of the critical myeloid transcription factor Pu.1. In sum, our genetic and bone marrow transplantation studies further demonstrate that Nf1+/− OCs and their progenitors are the culprit hematopoietic cell lineage responsible for the pathogenesis of NF1 non-union fracture. Thus, therapies targeting the myeloid lineage, especially OCs and their progenitors, may be required for the treatment of NF1 skeletal deficits. Disclosures:No relevant conflicts of interest to declare.

Partial Blindness to Submicron Topography in NF1 Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric

Cells sense physical properties of their extracellular environment and translate them into biochemical signals. In this study, cell responses to surfaces with submicron topographies were investigated in cultured human NF1 haploinsufficient fibroblasts. Age-matched fibroblasts from 8 patients with neurofibromatosis type 1 (NF1^+/–) and 9 controls (NF1^+/+) were cultured on surfaces with grooves of 200 nm height and lateral distance of 2 µm. As cellular response indicator, the mean cell orientation along microstructured grooves was systematically examined. The tested NF1 haploinsufficient fibroblasts were significantly less affected by the topography than those from healthy donors. Incubation of the NF1^+/– fibroblasts with the farnesyltransferase inhibitor FTI-277 and other inhibitors of the neurofibromin pathway ameliorates significantly the cell orientation. These data indicate that NF1 haploinsufficiency results in an altered response to specific surface topography in fibroblasts. We suggest a new function of neurofibromin in the sensoric mechanism to topographies and a partial mechanosensoric blindness by NF1 haploinsufficiency.

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.