Past Awards

Project Abstract. Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive form of soft tissue sarcoma that represents the leading cause of premature death in individuals with neurofibromatosis type 1 (NF1). The malignant transformation of benign plexiform neurofibromas (PNF) often occurs through the development of atypical neurofibroma/atypical neurofibromatosis neoplasms of uncertain biological potential (ANF/ANNUBP), which are characterized by distinct histopathological features and proclivity for CDKN2A copy number loss. Informed by these clinical observations, our laboratory has developed novel genetically engineered mouse models (GEMMs) harboring conditional inactivation of Nf1 and Cdkn2a (Ink4a/Arf) in Schwann cell precursors, which spontaneously develop ANF/ANNUBP reminiscent of human patients and transform to MPNST with high penetrance. Emerging data suggests that the Nf1+/- microenvironment may act as a double-edged sword, promoting outgrowth of benign NF1 associated tumors such as PNF, while on the other hand acting as a safeguard to forestall malignant transformation. Spatial profiling of a series of human NF1-associated peripheral nerve sheath tumors conducted collaboratively through the DHART SPORE suggests that ANNUBP are heavily infiltrated by T cells and exhibit enhanced signatures of immune surveillance. However as malignant transformation ensues, T cells become excluded from the microenvironment and dysfunctional. We hypothesize that the Nf1+/- microenvironment, and specifically Nf1+/- T cells, are critical to constraining the outgrowth of malignant clones of disease within PNF and ANF/ANNUBP precursor lesions harboring Nf1 and Cdkn2a (Ink4a/Arf) loss. To test this hypothesis, in Aim 1, we will establish the impact of a WT vs Nf1+/- microenvironment in modulating the rate of MPNST progression in our fully immunocompetent Nf1-Ink4a/Arf mutant GEMMs. In Aim 2, we will define the functional consequences of depletion of specific T cell subsets on progression of tumors along the neurofibroma to MPNST continuum in Nf1-Ink4a/Arf mutant mice using neutralizing antibodies to CD4, CD8 or the combination. Collectively, these data will provide a novel window of insight into the role of the Nf1+/- microenvironment and T cell subsets in governing the natural history of neurofibroma progression and malignant transformation in NF1.

Clinical Impact. MPNST is a rapidly fatal form of sarcoma that evolves from benign PNF and ANF/ANNUBP precursors in persons with NF1. Aside from wide marginal excision, there are no curative therapies. A primary objective of DHART SPORE Project 1 is to gain insight into how Nf1 and Cdkn2a inactivation in Schwann cell precursors prompts the progression of tumors along the neurofibroma to MPNST continuum to mechanistically inform the development of new and effective targeted therapies. The studies proposed here leverage GEMMs harboring these key genetic driver events developed by our laboratory, and further build upon recent findings by Dr. Lu Le and colleagues in Project 1 suggesting that the Nf1+/- microenvironment promotes PNF growth, yet paradoxically protects against malignant transformation. Collectively, these studies will directly contribute to the goals of Project 1 by characterizing at single cell resolution how Nf1 heterozygosity shapes the immune landscape across PNF, ANF/ANNUBP and MPNST. By establishing the functional role of Nf1+/- T cells in forestalling malignant transformation of PNF and ANF/ANNUBP precursors, the proposed translationally based studies have the potential to directly inform future investigation of immunotherapy-based approaches for MPNST chemoprevention in ANF/ANNUBP lesions that are not amenable to surgical resection.

Project Abstract. NF1 is a modulator of ERK signaling in NF1-associated and sporadic glioma. While targeted therapies have successfully impacted some ERK-dependent gliomas (those with BRAF mutations), current targeted therapies are inadequate for other ERK-dependent glioma. We and others have previously demonstrated somatic NF1 loss in glioma neurosphere models confers an ERK-dependent phenotype that is sensitive to MEK inhibition (MEKi), though the effects are not durable. Interestingly however, inhibition of ERK signaling may unmask other potentially-targetable vulnerabilities in NF1-deficient cancers. Specifically, ERK signaling appears to modulate DNA damage repair (DDR), particularly homologous recombination (HR). We have generated preliminary data suggesting HR is dependent on ERK signaling and downregulated in the presence of MEKi. Here, we propose to study the effect of MEKi on DDR pathways, with a focus on HR expression and the potential for exploitation of decreased HR expression in NF1-/- glioma cell lines using a combination of targeted therapies. We will identify the effect of MEKi on DDR pathway activity in NF1-mutant and wild-type glioma neurosphere lines. We will determine whether the addition of a PARP inhibitor to a MEK inhibitor effectively inhibits DNA repair and increases cell death in those same cell lines. Lastly, using in vivo models of NF1-/- glioma we will confirm the combination of a PARP and MEK inhibitor decreases tumor growth. Through a well-characterized panel of glioma neurospheres and adherent lines with varied expression of NF1 we will use molecular biology techniques to define the role of ERK dependence in maintaining DNA repair genes in glioma. The outcomes of these studies have direct implications for patients with NF1 mutant low and high grade glioma that is sporadic or NF1-associated.

Clinical Impact. The proposal “Exploiting ERK-dependence with combination targeted therapy in NF1-/- glioma” directly supports the goals of the DHART SPORE by interrogating mechanisms that may be specific to NF1 loss and associated cellular dysregulation that may enable novel therapies for tumors driven by NF1-loss leading to ERK-dependence. The proposal is specifically focused on NF1-associated gliomas, a rare and often fatal diagnosis in desperate need of new, rational therapies. The results from this work have the potential to be broadly applicable to gliomas with NF1 mutations (somatic or germline) as well as other cancers with NF1-loss that leads to ERK-dependence. The proposed experiments are complementary, but not overlapping, with the work ongoing in project 2, for which the project mentor, Dr. Jaishri Blakely, is a co-investigator. Finally, the proposal seeks to maximize the use of DHART SPORE cores via exploring the response to targeted therapy in glioma with NF1-loss utilizing the Omics Core in collaboration with Drs. Yiu and Angus, and commitment to share in vivo samples with the Biospecimen/Pathology Core.

Project Abstract. Neurofibromatosis Type 1 (NF1), which arises from mutation of the NF1 tumor suppressor, is one of the most common genetic cancer predisposition syndromes. Approximately half of individuals with NF1 develop benign peripheral nerve sheath tumors called plexiform neurofibromas (PNF), a subset of which undergo transformation into malignant peripheral nerve sheath tumors (MPNSTs). MPNST is a highly aggressive, treatment refractory form of sarcoma and is the leading cause of death in NF1 patients. There are currently no targeted therapies for MPNST, and complete surgical resection remains the sole curative option. Predictive biomarkers to identify PNF with malignant potential are urgently needed, as are molecular targets for the development of effective therapies to treat MPNSTs. Our recent work defining the spatial transcriptomic profile of the PNF-to-MPNST continuum revealed that malignant transformation of PNF coincides with a striking increase in the expression of the mitotic protein CENPF. Importantly, upregulation of CENPF has been found by others to promote tumor progression of various cancer types and portends poor prognosis. Further, recent studies have shown that tumor-derived CENPF can trigger production of CENPF-specific autoantibodies detectable in the sera of a subset of cancer patients. Together, these findings suggest that CENPF is a potential predictive biomarker to implement in the screening of NF1 patients for early detection of MPNST, either through immunohistochemical evaluation of biopsy samples or through minimally invasive serological detection of CENPF autoantibodies. In addition, we found that siRNA-mediated silencing of CENPF significantly impaired the growth of human MPNST cell lines, indicating that CENPF may represent a therapeutic target for the treatment of surgically unresectable or metastatic MPNST. The work outlined in this proposal will evaluate the utility of CENPF as a biomarker for the prediction of malignant potential of PNF and directly assess the efficacy of CENPF as a therapeutic target for the treatment of MPNST.

Clinical Impact. The dismal 5-year survival rates of 20-40% for MPNST highlight an urgent need for biomarkers to enable screening of NF1 patients for early detection of MPNST and for the development of targeted therapies. The goal of this proposal is to evaluate the potential of the CENPF protein to serve both these functions. We will (1) validate our transcriptomic data that implicate overexpression of CENPF in the malignant transformation of PNF and (2) evaluate the in vitro and in vivo therapeutic efficacy of targeting CENPF for the treatment of MPNST. Further, this work will evaluate CENPF autoantibodies as potential serological biomarkers for noninvasive detection of MPNST. At the mechanistic level, these studies will employ functional kinome profiling to identify therapeutically targetable kinases that mediate the antitumor effect of CENPF silencing that we have observed in MPNST cell lines. Together, these studies will directly address the goals of the DHART SPORE Projects 1 and 2, defining a genetic determinant of the PNF-to-MPNST progression and testing the utility of CENPF as a therapeutic target.

Project Abstract. Neurofibromatosis type 1 (NF1) is a cancer predisposing syndrome affecting 1 in 3000 people, which has diverse clinical manifestations including benign plexiform neurofibromas (PNs) and malignant peripheral nerve sheath tumors (MPNSTs). In 10% of cases, plexiform neurofibromas show nuclear and cellular abnormalities that accompany progression towards malignancy. Once they transform into MPNSTs, therapeutic options become severely limited. While mutations in various genes like CDKN2A, TP53, SUZ12, and EED may indicate completed transformation to MPNST, there are few early genetic indicators that characterize transition from PN to MPNST. Additionally, cis-regulation of expression in genes of interest have been studied in PN and MPNSTs extensively, but little is known about the trans-regulation of gene expression via microRNAs in these tumors. Preliminary gene expression analysis of publicly available PNs and MPNST data and previous research has identified genes that have greater expression in MPNSTs than PNs. Many of these genes are also subject to trans-regulation of expression by one or more microRNAs. One possibility is that the reduced repression of these target genes by mutated miRNAs lead to the observed expression changes. Moreover, since miRNAs have strict sequence prerequisites for viability, they may accumulate and manifest early mutations from evolving genomic instability in abnormally growing cells of PNs. We hypothesize that miRNA genes mutated due to early genomic instability accompanying malignant transformation of PNs to MPNSTs act as early indicators of this transformation. The goal of this proposal is to identify genomic variants in miRNA genes and their critical domains that are key differentiators between MPNSTs and PNs. We propose to use recently published miRNA-specific variant calling methods and supervised machine learning approaches to identify these key candidate miRNAs and their critical domains that are important for PN to MPNST transition. These candidates will provide important insights into the events underlying tumor transition and also serve as foundation for future experimental studies.

Clinical Impact. Malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of death in people with neurofibromatosis type 1, and currently have no approved targeted pharmacological therapies. Surgical interventions can reduce the tumor burden, but still lead to poor prognosis in patients. In many cases, the MPNSTs originate from benign plexiform neurofibromas (PNs). Identifying early indicators of malignant transformation as proposed in this study will be helpful in understanding the biology of this transition as well as identifying new therapeutic avenues for MPNSTs.

Project Abstract. Plexiform neurofibromas (PN) develop in up to half of patients with neurofibromatosis type 1 (NF1), and often cause significant complications including pain, disfigurement, and functional deficits. Treatment is indicated with the onset of functional deficits; however once developed, deficits such as weakness are often irreversible. The optimal time to initiate therapy prior to the onset of functional deficits is unclear, as there are no validated diagnostic or predictive biomarkers for nerve damage resulting in functional impairment with PN.

Tumor volume and conventional magnetic resonance imaging (MRI) characteristics do not correlate with functional deficits or predict future functional decline. There is a pressing need to identify non-invasive imaging techniques which quantify ongoing damage to the affected nerves in PN to guide optimal timing for therapeutic intervention. The proposed research uses diffusion tensor imaging (DTI) to characterize peripheral nerve axon microstructure and magnetoencephalography (MEG) to measure nerve axon conduction velocity in subjects with PN. This innovative combination of DTI and MEG provides non-invasive and sensitive measures of extremity nerve structure and function allowing diagnostic quantification of injury to the nerve. All aims will be accomplished with a prospective cross-sectional cohort study. Aim 1 will assess the correlation between DTI measures of extremity PN in subjects with and without PN-associated weakness. We hypothesize that DTI measures such as fractional anisotropy are significantly lower in extremities with weakness compared with intra-subject contralateral extremities and inter-subject extremities without weakness. Aim 2 will assess the correlation between DTI measures of nerve structure and MEG measures of conduction velocity to provide a functional and mechanistic correlate between DTI and PN with and without associated weakness. The quantification of changes in DTI and MEG in the PN-affected nerve will provide the preliminary data necessary for a larger prospective study evaluating the temporal relationship between DTI and MEG changes and the onset of weakness due to PN. This knowledge will ultimately provide an imaging tool to identify the ideal timing for initiation of therapy for patients with PN prior to the onset of irreversible functional deficits, thereby allowing earlier intervention and improved outcomes for patients with PN.

Clinical Impact. Plexiform neurofibroma are a major cause of morbidity for patients with NF1. A lack of diagnostic and predictive imaging biomarkers has prevented identification of patients at risk for the development of functional deficits and determination of the appropriate time for therapeutic intervention prior to the onset of irreversible morbidity. This study uses a novel and innovative combination of non-invasive imaging tools to address a critical gap in the diagnostic approach to NF1-PN. Further, this study promotes collaboration between researchers in the field of NF and radiology to correlate functional and imaging measures in patients affected by PN. The overall goal of this project is to provide tools to personalize treatment decisions based upon earlier identification of nerve damage with prompt initiation of therapy to lessen the negative impact of PN on quality of life in patients with NF1.

Project Abstract. RAS proto-oncogenes and the NF1 tumor suppressor encode core components of the Ras/GTPase activating protein (Ras/GAP) molecular switch. They are among a small group of genes mutated at high frequency in many human malignancies. Thus, developing effective therapeutic strategies for reversing the biochemical consequences of hyperactive Ras signaling is a fundamental obstacle to reducing the worldwide burden of NF1 and cancer. However, no effective mechanism-based treatments exist for cancers with RAS or NF1 mutations. Belvarafenib is a novel pan-Raf inhibitor that has shown promising safety and efficacy in Phase I studies of adults with solid NRAS-mutant tumors. However, it has not been studied in hematologic cancers, nor in NF1-mutant tumors. My preliminary data show that belvarafenib has activity in a panel of 6 NRAS and KRAS-mutant human acute myeloid leukemia (AML) cell lines at nanomolar potency. Furthermore, this novel Raf dimer inhibitor synergizes powerfully with the allosteric MEK inhibitor cobimetinib ex vivo. Given that NF1 is a direct negative regulator of Ras output, I hypothesize that this combination will also be effective in AML and other aggressive NF1-mutant cancers. Here, I propose a controlled murine preclinical trial of belvarafenib and cobimetinib using AMLs initiated by retroviral insertional mutagenesis, a technique with which our laboratory has extensive experience. The overall goal of the proposal is to investigate belvarafenib and cobimetinib in a biologically accurate and relevant mouse model to establish the efficacy of these agents, alone or in combination, in Nf1 and Nras-mutant AML. Based on this preclinical trial, we will be able to assess efficacy and mechanisms of response and resistance to belvarafenib and cobimetinib in AML.

Clinical Impact. Children with NF1 are at greatly increased risk of developing juvenile myelomonocytic leukemia (JMML) and other myeloid malignancies. JMML progresses to acute myeloid leukemia (AML) in ~25% of JMML patients and recurring somatic mutations in NF1, NRAS, KRAS and other Ras pathway genes are common in pediatric AML. Outcomes for these children are poor and studies of targeted agents have been disappointing to date. This is due, in part, to re-activation of downstream pathways that restore abnormal signaling. Co-targeting multiple distinct Ras effector proteins is a promising strategy for circumventing feedback reactivation. Here, I present promising efficacy and synergy data in RAS-mutant AML cell lines using belvarafenib, a novel pan-Raf inhibitor, and cobimetinib, a MEK inhibitor. In this project, I will extend these data to genetically accurate in vivo models. My goal is to identify effective new agents and drug combinations for cancers driven by NF1 inactivation and other mutations that result in hyperactive Ras signaling. Here I propose murine preclinical trials and studies to characterize resistance in an AML model that accurately recapitulates human biology. I expect that data from this tractable system will also inform future studies of Belvarafenib in other NF1-associated neoplasms and cancer.

Project Abstract. Neurofibromatosis type I (NF1) is a congenital disorder resulting from loss-of-function mutations in the NF1 gene that leads to the hyperactivation of RAS and the downstream RAF/MEK/ERK (MAPK) pathway. NF1 is characterized by dermatological, skeletal, and neurological manifestations in addition to predisposition to different types of tumors. Among these, plexiform neurofibromas are frequently developed in NF1 patients and grow along nerve structures; despite being considered benign tumors, plexiform neurofibromas cause severe morbidity, by disfiguring the patient or reducing the quality of life (motor disfunction and pain). These tumors can also become deadly by compressing vital structures or progressing to malignant invasive cancer. Recently, the FDA has approved the MEK inhibitor selumetinib for pediatric NF1 patients with symptomatic, inoperable plexiform neurofibromas. The clinical trial leading to this approval demonstrated that treatment with selumetinib results in tumor shrinkage and durable responses in this patient population. However, despite the encouraging anti-tumor activity observed in patients treated with selumetinib, a proportion of tumors remained unresponsive and/or developed acquired resistance to this treatment. Similar observations have been made in the oncology field, since RAS-driven tumors often become refractory to the treatment with MEK inhibitors. Due to the dynamic nature of the MAPK signaling regulation and the many feedback mechanisms that are relieved upon inhibition of the pathway, we hypothesize that different adaptive mechanisms are involved in the resistance to selumetinib. In this application, we propose the study of an emerging mechanism of adaptation to MEK inhibitors that we have recently discovered in a cell-based screening approach. Our project will focus on analyzing and characterizing at the molecular level this novel mechanism in cell lines derived from NF1 tumors, as well as cancer cell lines with somatic NF1 mutations. In addition, we will test whether counteracting this mechanism of adaptation to MEK inhibitors can be exploited as a potential combinatorial therapy to increase the efficacy of selumetinib in the clinic.

Clinical Impact. The therapeutic management of tumors arising in individuals with neurofibromatosis type 1, as well as cancers with somatic NF1 mutations, is part of the main goals of the DHART SPORE. The current standard of care treatment for plexiform neurofibromas includes the treatment with the MEK inhibitor Selumetinib; however, despite the efficacy of this therapy, there are many tumors that remain unresponsive or develop resistance to this drug. In this application, we will study the molecular mechanisms of adaptation to different MEK inhibitors in cellular and animal models of neurofibromatosis type 1-derived tumors and NF1 mutant cancers. We aim to characterize a potential novel mechanism of molecular adaptation to MEK inhibitors that can be used to predict resistance to these agents and/or overcome drug resistance

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.

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.

Staedtke V, Bai RY, Kim K, Darvas M, Davila ML, Riggins GJ, Rothman PB, Papadopoulos N, Kinzler KW, Vogelstein B, Zhou S. Disruption of a self-amplifying catecholamine loop reduces cytokine release syndrome. Nature. 2018 Dec;564(7735):273-277. doi: 10.1038/s41586-018-0774-y. Epub 2018 Dec 12. PMID: 30542164; PMCID: PMC6512810.

Gampala S, Shah F, Zhang C, Rhodes SD, Babb O, Grimard M, Wireman RS, Rad E, Calver B, Bai RY, Staedtke V, Hulsey EL, Saadatzadeh MR, Pollok KE, Tong Y, Smith AE, Clapp DW, Tee AR, Kelley MR, Fishel ML. Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours. Br J Cancer. 2021 Apr;124(9):1566-1580. doi: 10.1038/s41416-021-01270-8. Epub 2021 Mar 3. Erratum in: Br J Cancer. 2022 Aug 11;: PMID: 33658640; PMCID: PMC8076291.

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.

Bera AK, Lu J, Lu C, Li L, Gondi S, Yan W, Nelson A, Zhang G, Westover KD. GTP hydrolysis is modulated by Arg34 in the RASopathy-associated KRASP34R. Birth Defects Res. 2020 Jun;112(10):708-717. doi: 10.1002/bdr2.1647. Epub 2020 Mar 18. PMID: 32187889; PMCID: PMC7495839.

Bera AK, Lu J, Wales TE, Gondi S, Gurbani D, Nelson A, Engen JR, Westover KD. Structural basis of the atypical activation mechanism of KRASV14I. J Biol Chem. 2019 Sep 20;294(38):13964-13972. doi: 10.1074/jbc.RA119.009131. Epub 2019 Jul 24. PMID: 31341022; PMCID: PMC6755796.

Project Abstract. Neurofibromatosis type 1 (NF1) is a cancer-predisposition syndrome characterized by pleiotropic secondary manifestation, including malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are a leading cause of death in individuals with NF1. Despite attempts at surgical resection, few therapeutic options are available to treat MPNSTs, highlighting the critical need for new therapeutic opportunities. With prior DRP support from the DHART SPORE (2019-2020), we demonstrated that aberrant MET splicing (METDJMD) was enriched in a subset of NF1 patient MPNSTs. Expression of the METDJMD allele leads to splice-exclusion of the MET juxtamembrane domain (JMD), resulting in ligand-dependent gain-of-function. Expression of the METDJMD allele has been studied extensively in lung cancer, including metastatic disease, and leads to a “MET addiction” phenotype in the tumor. Thus, tumors that express the METDJMD allele are exquisitely sensitive to MET inhibitors, which was recently demonstrated in multiple phase 2 lung cancer clinical trials. Moreover, we tested the hypothesis that expression of the MetDJMD allele in a mouse model of spontaneous MPNST formation (cisNP mice) would alter the development of MPNSTs in vivo. Indeed, we showed that tumor formation and lethality were significantly accelerated in cisNP;Met+/DJMD mice compared to cisNP;Met+/+ mice (hazard ratio=10.36; p<0.0001). Building on this exciting preliminary data, the study proposed here will investigate the molecular basis for the accelerated tumor formation in these mice (Aim 1) and will test whether MetDJMD-expressing tumors are sensitive to MET inhibitors in vivo (Aim 2). First, we will utilize the DHART Omics core to perform comprehensive kinome, transcriptome, and mutation analyses of existing S100+ MPNSTs from cisNP;Met+/+ and cisNP;Met+/DJMD mice (i.e. collected from our 2019 DRP study). Second, we will test the potential for the MET inhibitor Capmatinib to treat MetDJMD-expressing MPNSTs. Our laboratory is committed to investigating a potential therapeutic role for MET inhibitors in this novel sub-class of NF1 MPNSTs. At the completion of this study, we will be positioned to begin testing patient-derived samples using in vitro and in vivo (i.e. xenograft) approaches (see support letter from Dr. Angela Hirbe).

Clinical Impact. MPNSTs are the primary cause of death in individuals with NF1, and new therapeutic strategies are urgently needed. The study proposed here builds on the success of our previous DRP award (2019) in which we 1) identified a molecularly-defined subset of MPNSTs predicted to be sensitive to MET inhibitor therapy, and 2) successfully demonstrated that expression of the MetDJMD allele leads to advanced disease progression and significantly reduced survival in mice. Here, we will leverage the infrastructure of the DHART Omics core facility to determine the molecular basis for the advanced MPNST progression in cisNP;Met+/DJMD mice. As well, we will test whether expression of the MetDJMD allele sensitizes MPNSTs to the MET inhibitor Capmatenib, as was recently demonstrated in clinical trials for human METDJMD-expressing non-small cell lung cancer. Taken together, we will both leverage the infrastructure of the DHART SPORE and build on our previous DHART SPORE DRP award to implicate a novel therapeutic strategy for a molecularly-defined subset of NF1 MPNSTs.

Project Abstract. This application seeks to evaluate a series of BRAF mutations found in RASopathy syndromes. BRAF, a protein central to RAS/MAPK signaling, is normally negatively regulated by forming a complex with 14-3-3. Recent structures of BRAF/MEK/14-3-3 complexes place these RASopathy mutations at the 14-3-3:BRAF interface and are predicted to disrupt the interaction. This suggests that these mutations work by impairing negative regulatory mechanisms downstream of RAS. In this proposal we will evaluate if the RASopathy-associated BRAF mutations in question do, in fact, impair 14-3-3:BRAF interactions in vitro and in cells. We will also evaluate if these mutations function in a RAS-independent manner, as might be expected for a component downstream of RAS in the MAPK pathway. If they do, patients with these mutations may be treatable with RAF inhibitors, similar to other RAS-independent activating BRAF mutations such as BRAFV600E, an established therapeutic target in cancer patients.

Clinical Impact. This work concerns germline mutations in BRAF that occur in persons with RASopathy syndromes. The proposed work will enable a new mechanistic understanding of how these mutations activate MAPK signaling, leading to RASopathy phenotypes. This work also has the potential to guide management of patients with these mutations, if we can show these mutations act as predictive biomarkers. Successful completion of the aims would provide actionable biomarkers for treatment of a subset of RASopathy patients with BRAF CRD mutations using certain BRAF-directed therapies.

Project Abstract. Neurofibromatosis 1 (NF1) is a neurogenetic disorder that can affect many organs in patients. By a wide margin, the most common complication is the appearance of cutaneous neurofibromas (cNF). These tumors, although benign, often number in the hundreds and are greatly disfiguring. Many NF1 patients consider cNF the most significant life burden of the disorder. Current management of the tumors is eradication often with significant morbidity. Typically, cNF begin to appear around puberty and continue to grow in size and number as the patient ages leading to a diminished quality of life. The investigators believe that early intervention, while the tumors are small and imperceptible, will have a major impact on the patients’ quality of life. To make early intervention possible developments in imaging and therapy are required – these are encompassed by Aims 1 and 2.

First, is the identification of a biomarker to locate and evaluate tumors at an early stage. Our preliminary data indicates that optical scattering is a novel cNF tumor biomarker. We propose to employ Spatial Frequency Domain Imaging (SFDI) and Optical Coherence Tomography (OCT) to measure optical scattering. This approach will enable us to identify and locate the tumors for subsequent treatment. Histological verification will validate optical scattering as a tumor biomarker and the applicability of SFDI and OCT.

Second, is development of a treatment of early-stage tumors. Local hyperthermia is proposed as the mechanism of action and two possible laser treatment implementations are proposed based on known characteristics of the tumors tested. One implementation makes use of dense tumor vascularity. In this case, selective heating of the tumor can be achieved with the use of a vascular selective laser. Local hyperthermia by controlling the laser heating source is a second implementation. Here the laser wavelength, beam diameter and power are adjusted to heat the tumor volume with negligible or no impact on surrounding tissues. Lasers in the wavelength range 1600-1800nm are proposed. Laser dosimetry guided by SFDI and OCT using both laser treatment implementations will be carried out on volunteer NF1 patients. Tissue response and evidence of damage or apoptosis will be investigated histologically.

Clinical Impact. The image guided treatment of cutaneous neurofibromas (cNF) addresses the manifestation of neurofibromatosis type 1 (NF1) regarded by most patients as the most significant burden of the disorder. Early treatment intervention will have a major improvement on patients’ quality of life. The proposed program is translational in nature. Preliminary basic optical data obtained at the Beckman Laser Institute and Medical Clinic have demonstrated optical scattering properties of cNF are different from those of normal tissue. These scattering property differences will be exploited as tumor biomarkers. Verification of these early data by histological evaluation will enable the first treatments of cNF by localized hyperthermia to be carried out under this proposal. These treatments will demonstrate the clinical potential for a treatment of early stage cNF that can be continued throughout the patients’ life. The management of these tumors will radically change current practice - eradication of large cNF - to treatment of small mostly imperceptible tumors and avoiding disfiguring cNF altogether.

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.

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.

Wang J, Pollard K, Calizo A, Pratilas CA. Activation of Receptor Tyrosine Kinases Mediates Acquired Resistance to MEK Inhibition in Malignant PeripheNerve Sheath Tumors. Cancer Res. 2021 Feb 1;81(3):747-762. doi: 10.1158/0008-5472.CAN-20-1992. Epub 2020 Nov 17. PMID: 33203698; PMCID: PMC7854512.

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.

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.