CEP Past Awards

Germline mutations in the NF1 gene, encoding the p21Ras GTPase-activating protein (GAP) neurofibromin, cause NF1. Clinical manifestations are diverse for NF1 patients, but a predominant lesion is plexiform neurofibroma (PNF), which arises from the Schwann cell (SC) lineage. There are currently no approved targeted therapies for PNF or MPNST. Comprehensive understanding of the molecular and genetic underpinnings of the progression of PNF to atypical neurofibroma (ANF) to MPNST is essential for rationally designed therapeutic strategies. The goal of this proposal is to definitively characterize the transcriptome and kinome during PNF to MPNST progression in the DNSC models to identify potentially targetable vulnerabilities. Dr. Angus will analyze murine model systems with defined genetic lesions that faithfully recapitulate PNF to MPNST evolution. This study will provide molecular profiles for the genetic determinants of PNF progression to MPNST and directly test the efficacy of Aurora kinase inhibitors. Dr. Angus hypothesizes that models of PNF progression to MPNST harbor distinct, genotype-dependent, and therapeutically targetable kinome profiles. He will test this hypothesis as follows: 1) Define the functional kinome and gene expression profiles underlying progression from PNF to ANF to MPNST in genetically defined model systems; 2) Determine the relative sensitivity of distinct DNSC genotypes to targeted Aurora kinase inhibition; and 3) Identify kinome-specific signatures of ANF and MPNST progression via DNSC engraftment.

Clinical Impact. The leading cause of death for NF1 patients is MPNST, the majority of which arise from PNF precursor lesions. Even after surgical resection, the 5-year survival rates for MPNST are ~20-40% and there are no approved therapies. With nearly 50 kinase inhibitors approved for use in cancer and other diseases, the identification of targetable kinases involved in PNF to MPNST progression has the potential to lead to new therapies. Ongoing DHART SPORE studies have strengthened the rationale for use of the MEK inhibitor selumetinib and the multi-RTK inhibitor cabozantinib. These studies will directly contribute to the stated goals of Projects 1 and 2, providing a rich molecular characterization of PNF to MPNST development and test the potential utility of Aurora kinase inhibitors (currently in Phase I/II trials for numerous tumor types).

Currently, no viable therapies for NF1 associated HGG exist, underscoring the need for preclinical evaluation of different promising therapies. Dr. DeRaedt has developed a mouse model for NF1-associated HGG that involves injecting mouse high-grade glioma stem cells - derived from HGGs of the Nf1/p53 genetically engineered mouse model - into the brains of syngeneic mice. These HGG cells express luciferase to allow tracking of tumor growth using the IVIS imager. Preliminary data generated by Dr. DeRaedt show successful targeting using PD1 checkpoint blockade combination immunotherapies. Analysis of publicly available expression data of NF1-associated brain tumors shows that B7-H3 is highly expressed in these lesions. Moreover, clinical trials have shown that targeting B7-H3 is safe. Dr. DeRaedt hypothesizes that NF1-associated HGGs are sensitive to a combined inhibition of MEK, B7-H3 and PD1. He will test this hypothesis using a MEK inhibitor therapy combined with immune checkpoint blockade (B7-H3). They will also assess inhibitory signals of the immune system to evaluate, quantify, and plot CD8 infiltration.

Clinical Impact. Positive results in this preclinical trial could be quickly translated clinically, especially given that the CHOP neuro-oncology department has extensive experience and participates in multiple clinical trials for pediatric HGG. This project will greatly enhance our understanding of how NF1-associated gliomas can evade immune detection and if targeting B7-H3 is a viable therapy for these tumors. Moreover, the B7-H3 monoclonal antibodies are safe and currently being evaluated in clinical trials, and both MEK inhibitors and PD1 checkpoint blockade are approved.

The Nf1+/- tumor microenvironment (TME) impacts tumor growth and alters the immune landscape. Immunosuppressive cells, including Tregs and MDSCs, are abundant in the MPNST microenvironment and can contribute to their poor immunotherapy responses by blocking antitumor immunity. New therapeutic strategies that target immunosuppressive cells are critical to improving immunotherapy outcomes in MPNST. The cytokine IL-6 contributes to immunosuppression and accelerates tumor growth through both paracrine and autocrine mechanisms. While IL-6-directed therapy has not been explored in NF1-related tumors, there is strong rationale for targeting IL-6 in MPNSTs. Data from our group demonstrates IL-6 is upregulated in MPSNT cells and the Nf1+/- tumor microenvironment. Additionally, Dr. Dodd has generated preclinical data supporting a role for IL6-producing myeloid cells in MPNST progression and immunosuppression. These data inform the hypothesis that targeting the autocrine and paracrine sources of IL-6 may slow MPNST growth and attenuate the immunosuppressive TME, thus improving response to immunotherapy. The overall goal of this proposal is to determine the therapeutic benefit of blocking IL-6 signaling in MPNSTs using a combination of pharmacological, genetic, and mouse model approaches. This project will implement in vivo CRISPR/Cas9 tools to determine the impact of autocrine IL-6 blockade on MPNST growth (Aim 1) and the effects of paracrine IL-6 loss on MPNST growth and infiltration of immunosuppressive cells (Aim 2). This study will fill a critical gap in our understanding of IL-6 signaling in NF1-deficient tumors and potentially determine a new therapeutic target in the Nf1+/- TME.

Clinical Impact. The goal of this project is to generate preclinical rationale to support Phase I/II clinical trials of anti-IL-6 therapy in MPNSTs. These studies are highly translational and, if successful, can be rapidly transitioned into clinical trials. Importantly, multiple anti-IL6 agents are in clinical pipelines, and the anti-IL-6R agent tocilizumab is commonly used to treat rheumatoid arthritis (RA).

Researchers have started to identify the genetic determinants that drive progression of plexiform neurofibromas (PNF) towards malignant peripheral nerve sheath tumors (MPNST). These studies suggest that atypical neurofibromas (ANF), the intermediate step in this malignant transformation, arise as a clonal outgrowth of tumorigenic Schwann cells that have escaped senescence through inactivation of the INK4/ARF (CDKN2A/B) tumor suppressor axis. This project seeks to test a therapeutic approach in a preclinical ANF/MPNST mouse model that combines doxorubicin, one of the cytotoxic backbones of current MPNST treatment, with navitoclax, a newer senolytic agent that acts as an antagonist of the antiapoptotic Bcl-2 family of proteins, in an effort to drive tumor cells into senescence and then selectively eradicate the senescent cells. Additionally, the cyclin-dependent kinase (CDK) inhibitor dinaciclib induces cytotoxicity ín vitro and in vivo in CDKN2A-defective squamous cell lung cancer-derived cells and has been found to synergize with navitoclax. This project will assess the combination of navitoclax with doxorubicin and with dinaciclib to treat ANF and MPNST in a preclinical mouse model.

Aim 1. Evaluate the anti-tumor efficacy of navitoclax + doxorubicin in a preclinical ANF/MPNST mouse model with supportive in vitro testing of cell proliferation, survival, and senescence in DRG/nerve root neurosphere cells (DNSCs).

Aim 2. Evaluate the anti-tumor efficacy of navitoclax + dinaciclib in a preclinical ANF/MPNST mouse model with supportive in vitro testing of cell proliferation, survival, and senescence in DNSCs.

Clinical Impact. MPNST is leading cause of mortality for patients with NF1 and current treatment regimens are largely ineffective. Dr. Armstrong hypothesizes that navitoclax combined with doxorubicin or with dinaciclib will reduce tumor burden in a preclinical ANF/MPNST mouse model and inform a clinical trial for NF1 patients with ANF or MPNST. All 3 agents being tested are either commonly used in everyday practice or being employed in current clinical trials, which will facilitate clinical translation.

RAS proto-oncogenes and the NF1 tumor suppressor encode core components of the Ras/GTPase activating protein (Ras/GAP) molecular switch. No mechanism-based treatments exist for cancers with RAS and NF1 mutations. SHP2 is a ubiquitously expressed non-receptor protein tyrosine phosphatase and scaffolding protein that integrates upstream growth factor signals to promote activation of Ras proteins. Pharmacological inhibition of SHP2 with RMC-4550, a potent and selective SHP2 allosteric inhibitor, efficiently downregulate RAS-GTP levels and suppress the proliferation of various human cancer cell lines harboring KRASG12C, NF1LOF, or BRAFG466V/+ mutations. I have previously utilized mouse models of Nf-/- AML to perform controlled preclinical trials of novel targeted anti-cancer agents. The overall goal of this proposal is to deploy these reagents to establish the efficacy of RMC-4550 in accurate mouse models of AML.

Aim 1. To characterize the specificity of RMC-4550 in isogeneic cell lines driven by NF1 inactivation or oncogenic RAS mutations.

Aim 2. To establish the maximally tolerated dose (MTD), pharmacokinetics, bone marrow exposure, and efficacy of RMC-4550 in genetically accurate mouse models of AML.

Clinical Impact. Aberrant signaling through Ras proteins plays a central role in many cancers, including myeloproliferative neoplasms (MPNs) and acute myeloid leukemia (AML). Juvenile myelomonocytic leukemia (JMML) is an MPN that predominantly affects young children and is characterized by disease-initiating NRAS, KRAS, NF1, and PTPN11 mutations. Similarly, a comprehensive sequencing study recently uncovered somatic Ras pathway mutations in ~50% of pediatric AMLs. Germline PTPN11 mutations are the most frequent cause of Noonan syndrome, a common Rasopathy disorder. Together, these data infer that agents targeting hyperactive Ras signaling could provide a rational and effective therapeutic approach for children and adults with JMML and AML.

SHP2 is a protein tyrosine phosphatase that functions as a positive signal transducer linking multiple receptor tyrosine kinases (RTKs) to RAS. It thus serves as a physiological mediator for RAS activation. SHP2 inhibitors (SHP2i) are in clinical trials and have demonstrated efficacy in preclinical models of RAS and RAF mutant tumors. This approach has not been explored in NF1 mutant tumors. We hypothesize that SHP2i will reduce RAS-GTP loading mediated by SOS and inhibit RAS-mediated downstream MAPK signaling in NF1 mutant MPNST and abrogate tumor growth. Due to the inherent genomic complexity of MPNST tumors and multiple feedback loops regulating the activation of Raf/MEK/ERK signaling, combinatorial approaches targeting multiple disease-related survival pathways will likely be most effective. MEK inhibitors (MEKi) have produced exciting results in NF1 mutant plexiform neurofibroma. However, MEKi blocks MPNST progression to a limited extent, like RAS-mutant cancers where adaptive resistance to MEKi is mediated by induction of RTK signaling pathways. Thus, novel approaches and complementary treatment strategies that prevent the development of adaptive resistance and sensitize MPNST are urgently needed. We propose that SHP2i will efficiently block signals from multiple activated RTKs to overcome adaptive resistance to MEKi treatment and sensitize MPNST cells to MEKi.

Aim 1. Establish the functional role of SHP2 in tumor proliferation of NF1 mutant MPNST.

Aim 2. Validate the anti-tumor efficacy of combined SHP2i and MEKi in NF1 mutant MPNST and investigate the specific adaptive resistance mechanisms that arise upon MEKi treatment.

Clinical Impact. These results can be translated into a clinical trial to evaluate a combination of SHP2i and MEKi as a novel treatment paradigm in NF1 mutant MPNSTs. The goal of these studies is to glean critical insights that will help refine and develop second generation SHP2i to overcome resistance mechanisms. If SHP2i prove ineffective, these studies will nonetheless provide important insights into MEKi resistance mechanisms, including monitoring genomic and non-genomic adaptive bypass mechanisms such as transcriptional upregulation of RTKs in clinical settings of acquired MEKi resistance.

Presentations/Publications/New Support Facilitated by This CDP Award. No manuscripts to date. Dr. Sait will present an abstract for presentation at the NF conference in San Francisco from September 21-24, 2019.

Loss of function NF1 mutations cannot be directly “drugged” and there are no approved therapies that are specifically effective in NF1 mutant tumors. Dr. Gilbert performed a genome-scale CRISPR screen that identified a synthetic lethal relationship between NF1 inactivation and chemical inhibition of the ER- localized HSP70 chaperone, HSPA5 in the chronic myeloid leukemia cell line K562. His CDP project addresses the hypothesis that NF1 deficient tumor cells are uniquely sensitive to ER stress induced by inhibition of HSPA5 activity. If this chemical genetic interaction is a general phenomenon, it would define a conditional “non-oncogene” addiction in NF1 mutant tumors. The aims of this project are:

Aim 1. Are NF1 deficient myeloid cancers addicted to HSPA5 activity?

Aim 2. Determine the mechanism of synthetic lethality between NF1 loss and inhibition of HSPA5 in AML.

Clinical Impact. New therapeutic strategies that selectively target NF1 deficient tumors will have tremendous therapeutic application in myeloid malignancies and many other cancers. These experiments have the goal of selectively inhibiting the growth of NF1 deficient myeloid cancer cells. Furthermore, this general strategy may also be efficacious in other tissue contexts

Patients with NF1 are at an increased risk for developing therapy-induced second malignant neoplasms (SMN). By profiling genome-wide DNA methylation in a unique cohort of individuals with and without NF1 and with and without SMN, Dr. Singh seeks to uncover a methylation signature associated with an increased risk of SMN development among NF1 patients treated with chemotherapy and/or radiation for a primary cancer. She hopes to integrate the methylation profiles and genomic variants in key regulatory pathways, such as tumor suppressor genes and oncogenes, to understand the biologic late effects of radiation and chemotherapy in NF1 patients. The aims of this project are:

Aim 1. To determine if gene-specific DNA methylation status is associated with SNs in children with and without NF1 and primary neoplasia by conducting genome-wide DNA methylation profiling.

Aim 2.To identify SMN susceptibility SNPs through OncoArray genotyping. Due to limitations in resources – we decided to not undertake this aim at this time.

Clinical Impact. The identification of NF1 patients who are at the greatest risk of developing SMNs through the discovery of novel epigenetic biomarkers will aid in risk stratification in the development of potential new therapeutic strategies targeting epigenetic patterns. This work might also identify patients with deleterious predisposing mutations who will benefit from new therapeutic interventions.

Constitutional RAS gene mutations cause several RASopathy syndromes including Costello syndrome (HRAS G12A, G12S, G12V, G13D), Noonan syndrome (KRAS T58I, V14I, P34R and NRAS I24N, P34L, T50I, G60E) and cardiofaciocutaneous (CFC) syndrome (KRAS G60R, D153V). Understanding the structural details of how these mutations contribute to aberrant signaling may enable new strategies for treating or managing these syndromes, including cancers arising in affected individuals. The Westover lab is performing detailed structural characterization of a panel of germline KRAS mutations found in Costello, Noonan and CFC syndromes that cluster around the terminal phosphates of GTP and GDP. Previous studies showed that all of these mutations result in a high proportion of GTP-bound RAS and insensitivity to GAP-stimulated GTP hydrolysis. This CDP project addresses two distinct but highly related specific aims:

Aim 1. To determine the three-dimensional x-ray crystal structures of GDP-bound H-Ras G12S, K-Ras P34R, and K-Ras G60R.

Aim 2. To determine the three-dimensional x-ray crystal structures of GTP-bound H-Ras G12S, K-Ras P34R, and K-Ras G60R.

Clinical Impact. These new structural models will provide a reliable and valuable publicly available asset for understanding the molecular pathogenesis of RASopathy syndromes and may also suggest new therapeutic strategies.

Birth Defects Res. 2020 Jun;112(10):708-717. doi: 10.1002/bdr2.1647. Epub 2020 Mar 18.


J Biol Chem 2019 Sep 20;294(38):13964-13972. doi: 10.1074/jbc.RA119.009131. Epub 2019 Jul 24.


NF1 is a RASopathy that represents a major risk for the development of malignancies, particularly malignant peripheral nerve sheath tumor (MPNST). To date, surgery is the only treatment modality proven to have survival benefit for MPNST and even when maximal surgery is feasible, most of these tumors are incurable. Like other aggressive cancers, MPNSTs develop large hypoxic areas, which limits the efficacy of traditional antineoplastic agents. Although this has made MPNST a highly treatment-resistant tumor, it also offers the perfect niche for the growth of the oncolytic spore-forming bacterium Clostridium novyi-NT (C. novyi-NT). Dr. Staedtke recently created a next generation C. novyi-NT strain with significantly less toxicity, and she demonstrated robust anti-tumor responses when injected into MPNSTs of a transgenic Nf1 mouse model. C. novyi-NT-induced lysis of tumor cells transforms the non-immunogenic MPNST into an immunogenic phenotype, with local release of tumor antigens, recruitment of antigen-presenting cells and antigen-specific CD4+ and CD8+ T cells as well as suggestions of upregulations in the checkpoint machinery that could confer susceptibility to checkpoint blockade. Despite large success in other cancers, checkpoint blockade has not been effective in MPNST due to low expressions of checkpoint-related proteins and a “non-immunogenic” tumor microenvironment. As changes to the tumor immune microenvironment associated with bacterial anti-neoplastic therapy have not been investigated, Dr. Staedtke propose to immunologically phenotype MPNSTs during C. novyi-NT treatment to identify upregulated immune checkpoint targets and develop a rationale for the combinatorial use of checkpoint blockade with C. novyi-NT.

Aim 1. To immunologically phenotype MPNSTs during C. novyi-NT therapy via characterization of inhibitory immune checkpoint molecules, including PD-1, PD-L1/2, and other relevant immune checkpoint ligands and receptors (LAG-3, Tim-3, CTLA-4, TIGIT, BTLA) and distribution of leukocyte subpopulations using immunohistochemistry (IHC).

Aim 2. To evaluate the safety and efficacy of the combinatorial use of the most promising immune checkpoint inhibitor(s) with C. novyi-NT for the treatment of MPNSTs in NPcis mouse model.

Clinical Impact. Defining the immunophenotype of MPNSTs, tumor immune-environment, and endogenous factors that shape the anti-tumor response are prerequisites for successfully implementing rational new immune-based therapies. In addition, our study establishes a novel treatment strategy for these lethal tumors.

Nature. 2018 Dec;564(7735):273-277. doi: 10.1038/s41586-018-0774-y. Epub 2018 Dec 12.


British J Cancer 2021 Mar 3. doi: 10.1038/s41416-021-01270-8.


DRP Past Awards

The NF1 phenotype is clinically variable; however, a few variants have been found to be compatible with partial protein production and correlate with specific phenotypes. These distinct phenotypes involve either the nerve sheath (Schwann cells (SC)) with minimal melanocytic involvement or melanocytes with no nerve sheath involvement. A severe “spinal NF” phenotype is associated with G848R and R1276Q and is characterized by a massive internal tumor burden, with neurofibromas (originating from SC) at each spinal nerve root and extreme enlargement of most peripheral nerves. These individuals are at great risk of spinal cord compression, pain, and malignant change but, have a mild pigmentary phenotype. In contrast, relatively mild phenotypes are associated with delM992 and R1809C which are characterized by CALMs (originating from melanocytes) and no neurofibromas (no SC involvement) or risk for malignancy. Hence, distinct cell populations are affected by each mutation, resulting in different clinical phenotypes with different implications for later development of malignancy. Our overall goal is to determine how these genotypes affect the function of neurofibromin and its interactions with other proteins. We will test the hypothesis that neurofibromin differentially interacts with binding partners in a cell type-specific manner, and that mutations differentially disrupt those interactions. The proposed research will lead to identification of new neurofibromin functions and associated therapeutic targets.

Clinical Impact. This proposal directly addresses the role of NF1 mutations in cancer. We compare mutations that are and are not associated with malignancy in two different clinically relevant cell types. Identifying binding partners and determining those that are associated with malignancy may provide new therapeutic targets for cancers driven by NF1 mutations.

This study will build on our novel discovery of somatic aberrant splicing in MPNSTs. Dr. Rios has developed a novel computational algorithm to discover somatic aberrant splicing events (not caused by DNA sequence variation) from transcriptome sequencing (RNAseq). Preliminary results indicate that a subset of MPNSTs express an oncogenic splice-isoform of the MET receptor (METΔJMD). Somatic expression of the METΔJMD isoform is mechanistically different from other previously-identified somatic activating mutations in MET and may introduce a therapeutic vulnerability that can be exploited in this subset of MPNSTs. In Aim 1 of this study, Dr. Rios’ lab will apply a novel computational approach to comprehensively survey somatic aberrant splice-isoform expression in MPNSTs, a class of somatic variation that has yet to be investigated in these tumors. Furthermore, they will identify differentially-expressed genes associated with expression of these aberrant splice-isoforms. Second, the Rios lab has generated and conducted preliminary characterization of a novel MetΔJMD mouse model that allows in vivo investigation of the oncogenic potential of the MetΔJMD allele. In Aim 2 of this study, they will: (a) investigate the role of MetΔJMD splicing in the transformation of plexiform neurofibromas into MPNST using the Postn-cre;Nf1flox/flox mouse model; and, (b) investigate the oncogenic contribution of MetΔJMD to progression of MPNSTs by inter-crossing with cis-NP (cis-p53+/- Nf1+/-) mice. Together, this study will comprehensively survey a new class of oncogenic somatic variation and will characterize a novel aberrantly-spliced oncogenic MET ΔJMD allele in the pathogenesis of MPNSTs.

Clinical Impact. This study ushers in a new paradigm to study somatic aberrant splicing in MPNSTs. Preliminary results to date show that such oncogenic splicing events occur in MPNSTs, as have identified a significant proportion of NF1-associated MPNSTs harboring somatic METΔJMD splicing.

By forming a complex at the plasma membrane, NF1 and SPRED1 inactivate Ras after its activation by receptor tyrosine kinases. The NF1 and SPRED1 genes were recently identified as tumor suppressors in several distinct subtypes of melanoma that are characterized by their occurrence at different anatomic sites, the distinct mutational forces that act on their genomes, and their different spectra of driver mutations. Preliminary genetic and functional data from the Yeh lab suggest that NF1 and SPRED1 inactivation cooperates with other mutations to initiate melanocytic tumors. They will study the role of NF1 and SPRED1 inactivation in the context of wild-type or mutant KIT in immortalized mouse melanocytes, and hypothesize that that neither inactivation of the NF1/SPRED1 Ras inactivating complex or expression of mutant KIT will result in melanocyte transformation, but that the combination of the two will. Dr. Yeh will test this hypothesis using both in vitro and in vivo models. To translate this finding to the clinic, they have identified human melanoma cell lines with loss of NF1 or SPRED1 and expression of mutant KIT. Preliminary studies in vitro indicate that combination therapy with MEK and KIT inhibitors is synergistic in this genetic context. Dr. Yeh’s lab will extend these data by testing the synergy of MEK and KIT inhibitors in a xenograft model in order to bring this therapeutic strategy another step closer to clinical testing.

Clinical Impact. This proposal builds on a solid foundation of pre-existing genetic and functional data. NF1 is inactivated in up to a quarter of melanomas. This project will investigate the role of NF1 inactivation (both through direct mutation of NF1 and due to inactivation of SPRED1) in melanoma development and hypothesize that NF1 inactivation cooperates with other genetic alterations to transform melanocytes. This initial study is examining how the loss of NF1 or SPRED1 impacts KIT mutant melanocytes and seeking treatments that are effective in KIT mutant melanomas with NF1 inactivation. This work will have relevance to understanding potential mechanisms that lead to melanoma in the context of NF1 and Legius syndrome as well as developing treatments. The work here will establish a foundation for understanding how multiple weakly MAPK activating mutations cooperate in melanoma progression, applicable to patients with 'Rasopathies' and melanomas in which somatic “Rasopathy” associated mutations have occurred.

Individuals with NF1 develop neoplasms in multiple tissues types, including the central and peripheral nervous system. Many NF1 patients are treated with radiotherapy as children, and may receive multiple treatments over the course of their lives. The interpretation of their MRI imaging changes and the correlation to biologic and clinical processes is challenging. This proposal will apply state-of-the-art quantitative imaging and statistical analysis to compare irradiated Nf1 mutant mouse brains to those of controlwildtype mice to test the hypothesis that Nf1 mutant mice are susceptible to radiation-induced brain injury. This project seeks to determine whether early MRI-based alterations in the irradiated brain predicts and correlates to the development of cerebral radiation injury.

Aim 1. To quantify statistically significant differences in radiomic feature changes post-irradiation between irradiated and unirradiated Nf1 mutant and wildtype mice.

Aim 2.To correlate post-radiotherapy radiomic alterations to histologic alterations in irradiated murine brains.

Clinical Impact. Our studies represent the first application of state-of-the-art radiomic analysis to prospectively characterize the brain injury in the setting of radiation therapy. If successful, this novel image analysis approach may have utility in predicting and studying radiation injury in the brain and also other organs.The findings from this work may serve as pre-clinical data that can be further investigated and validated in NF1 patients with collaborators from the SPORE member institutions.

Neurofibromatosis type 1 (NF1) is associated with highly morbid skeletal conditions, including severe tibia dysplasia. Significant anterolateral bowing of the tibia often leads to fracture with subsequent pseudoarthrosis (PA - persistent lack of bone healing) requiring amputation. This study tests whether loss of NF1 in Leptin receptor (LepR)-expressing bone marrow skeletal stem cells (SSCs) is associated with persistent fractures in children with NF PA. To complement our genetic assessment of patient-derived LepR-expressing SSCs, this study will develop and evaluate fracture healing in vivo using a conditional LepR-cre;Nf1F/F mouse model. This study will, for the first time, provide critical progress toward identifying the key cell population responsible for PA in NF1 patients while also providing a novel Nf1-deficient mouse model for studies of osteoblastogenesis defects following fracture.

Aim 1. To determine whether LEPR-expressing MSCs harbor second-hit NF1 mutations

Aim 2. To characterize fracture healing in a novel LepR-cre;Nf1F/F osteoprogenitor mouse model

Clinical Impact. In many ways, tibia PA may be considered a benign bone tumor caused by somatic NF1 deficiency. Since tibia PA is localized, treatment requires aggressive resection; however, resection is often insufficient and requires amputation. This study represents the initial steps toward understanding disease mechanisms at a cellular level, which Dr. Rios expects will provide evidence for targeted pharmacologic therapies that will achieve life-changing outcomes and improve limb salvage for children with NF1.

Children with NF1 are strongly predisposed to juvenile myelomonocytic leukemia (JMML) and leukemia cells from these patients frequently inactivate the normal NF1 allele. Similarly, somatic CBL, KRAS, NRAS, and PTPN11 mutations are found in most patients with JMML, providing strong genetic evidence that hyperactive Ras signaling plays a central role in this aggressive myeloproliferative neoplasm (MPN). Using a model of JMML driven by Shp2D61Y expression, the Chan lab found that myeloid cells from these mice are hyper-inflammatory and produce exaggerated levels of reactive oxygen species (ROS). They also found that inhibition of PI3K p110δ normalizes GM-CSF hypersensitivity of Shp2D61Y-expressing myeloid cells. The overall goal of this DRP project is to ask if inhibition of the proinflammatory protein, PI3K p110δ, improves homing, engraftment, expansion, and myeloid differentiation of wild-type donor cells transplanted into diseased, Shp2D61Y-expressing recipient mice through the following specific aims:

Aim 1. To compare bone marrow niche composition and the homing, engraftment, differentiation, and tissue distribution of transplanted wild-type (WT) hematopoietic cells in WT, Shp2D61Y, and Shp2D61Y;p110δD910A/D910A compound mutant recipient mice.

Aim 2. To investigate the effects of treating Shp2D61Y recipients with a PI3K p110δ inhibitor (GS-9820) on bone marrow niche composition.

Clinical Impact. This DRP project addresses how hyperactive innate immunity may contribute to JMML pathogenesis and relapse after HSCT by damaging the bone marrow microenvironment, thus limiting normal donor cell engraftment after transplantation and promoting leukemia relapse.

FDA-approved drugs targeting the underlying molecular lesions in lung adenocarcinoma only apply to about 15% of patients. Put another way, 85% of lung ADC patients currently lack targeted options. This proposal grows from three observations: (1) A comprehensive genomic analysis of over 700 lung ADC cases performed by Dr. Collisson and his colleagues in the TCGA nominated NF1 loss/mutation as a novel candidate driver of lung ADC cases that otherwise lack activated oncoproteins in the RAS-RAF-MAPK pathway; (2) experiments performed by the Collisson lab in Nf1 mutant mice mouse showed Nf1 loss alone is insufficient to causes lung ADC; and, (3) additional studies in cultured ADC cell lines indicated that some, but not all, additional oncogenic events cooperate to induce transformation in the setting of NF1 loss. Together, these observations support the hypothesis that NF1 loss cooperates with other genomic events in the development of lung ADC. In this DRP project, the Collisson lab is modeling genomic aberrations found in human lung ADC in a customized, patient-centric manner using preclinical systems (mouse and cell line models) to pursue two aims:

Aim 1. To define the role that NF1 loss plays alongside other, co-incident genomic events in both tumorigenesis and response to targeted therapy in preclinical cellular systems.

Aim 2. To define the role that NF1 loss plays alongside other, co-incident genomic events in response to targeted therapy in a human/mouse-lung cancer co-clinical trial.

Clinical Impact. This work addresses the hypothesis that the ~15,000 annual cases of lung ADC might benefit from treatment with a MEK inhibitor, with or without concurrent therapies.

Over 140 non-synonymous recurrent somatic mutations in NF1 have now been identified in ~10% of non-small cell lung cancer (NSCLC) patients, but their functional impact and therapeutic implications in NSCLC are largely unknown. This is a key knowledge gap to fill because the effects of genetic alteration of NF1 are mutation and cell context-specific. The aims of this DRP project are:

Aim 1. To define the functions of patient-derived NF1 mutant variants present in human NSCLC.

Aim 2. To identify a rational therapeutic strategy for NF1-mutant NSCLC using preclinical models.

Clinical Impact. Defining the biochemical and functional consequences of novel NF1 mutations in NSCLC is a prerequisite for implementing rational new therapies. Ongoing studies in engineered isogenic cell lines with and without NF1 mutations are also investigating growth dependencies and candidate vulnerabilities.

As loss of Ras GTPase activating protein (Ras-GAP) function results in constitutive Ras activity, a logical therapeutic approach for tumors with loss of NF1 is pharmacologic inhibition of Ras-GTP. Direct inhibition of hyperactive Ras however, has not proven feasible. As Raf/MEK/ERK signaling is a critical downstream effector of activated Ras, MEK inhibitors have been investigated NF1 mutant cancer models, including models of MPNST. While these data support a rationale for testing MEK inhibitors in patients with NF1-associated cancers, the preclinical responses to these agents were partial at best and suggest a need for a better understanding of the role of ERK signaling in MPNST. Crosstalk from upstream RTKs and multiple Ras effector pathways, as well as release-of-negative-feedback adaptive changes in response to MEK/ERK inhibition, may limit the success of MEK inhibition in treating these tumors. The goals of this DRP project, therefore, are to demonstrate the concept of “adaptive resistance” and describe the adaptation of the signaling network to inhibition of RAS effector pathways in NF1-associated cancer.

Aim 1. Elucidate patterns of tumor evolution to MPNST and identify additional genomic alterations that may contribute to Ras signaling dependency.

Aim 2. Determine the dependency of NF1-associated malignancies on ERK signaling and the adaptive signaling response to pharmacologic MEK or ERK inhibition.

Aim 3. Test rational combination strategies to target tumors with loss of NF1.

Clinical Impact. NF1 inactivation in MPNST and other tumors deregulates Ras signaling. Complex feedback circuits are activated in response to mutational activation of Ras signaling networks. A novel element to the proposed research, therefore, includes a primary focus on signaling adaptation and release-of-negative-feedback changes in ERK signaling that condition a tumor’s response to targeted MEK inhibition.

Cancer Res. 2021 Feb 1;81(3):747-762. doi: 10.1158/0008-5472.CAN-20-1992. Epub 2020 Nov 17.


Optic pathway glioma (OPG) is the second most common tumor in patients with NF1. Although ~30% of all OPG cases are attributable to germline NF1 mutation, the genetic etiology of the remaining 70% of cases remains unexplained. This proposal is formally testing the hypothesis that both rare and common germline variants in Ras pathway genes may contribute to OPG risk. To achieve this, a population-based case-control study, nested within the California Birth Cohort, has been developed. By linking the California Cancer Registry to the California Department of Public Health’s newborn bloodspot archive, Dr. Walshhave obtained pre-diagnostic blood specimens from 160 patients that developed OPG. By leveraging these unique and mature resources, this registry-based approach can study genetic predisposition to OPG in a large and ethnically-diverse sample of Californian children. The aims of this DRP project are:

Aim 1. Identify deleterious germline gene mutations in a multi-ethnic California cohort of OPG patients using a custom Ras-pathway gene sequencing platform to: (A) Determine the proportion of patients in a large population-based sample whose OPG is attributable to germline mutations of NF1; and, (B) identify high-penetrance OPG predisposition genes other than NF1.

Aim 2. Identify genetic modifiers of OPG risk in NF1 patients by performing a genome-wide association study (GWAS) of NF1 patients with OPG compared with 2,920 ethnicity-matched controls.

Clinical Impact. The identification of both rare and common variants underlying OPG risk can reveal information leading to improved care and risk-stratification for children and adolescents diagnosed with OPG. Additionally, our results can inform reproductive genetic counseling for adult OPG survivors.

Despite the constitutive activation or amplification of many receptor tyrosine kinases in malignant peripheral nerve sheath tumors (MPNSTs), pharmacologic inhibition of these RTKs has not resulted in clinical benefit. This DRP project is using potent and selective small molecule inhibitors to investigate STAT3 as a candidate therapeutic target in MPNST. The underlying scientific rationale is that position downstream of several of these signaling molecules and in light of recent reports demonstrating STAT3 as a driver of MPNST. Our long-term goal is to improve patient outcome by therapeutically modulating STAT3 and its downstream pathways. The central hypothesis is that STAT3 exerts transcriptional control on critical pathways for MPNST cell survival.

Aim 1. To validate STAT3 as a target in MPNST using in vitro systems and determine which STAT3 targets are critical in the blockade of cellular proliferation and invasion.

Aim 2. To determine the efficacy of STAT3 targeted disruption in xenograft models of human MPNST.

Clinical Impact. This project aligns with the overall goal of the DHART SPORE to develop of better treatments for tumors characterized by NF1 inactivation or driven by hyperactive Ras signaling by interrogating STAT3 as a therapeutic target in MPNST.