Development Programs

Project Abstract. : Juvenile myelomonocytic leukemia (JMML) is an aggressive hematological malignancy affecting young children, driven by mutations in genes such as NF1, CBL, KRAS, NRAS, or PTPN11. JMML is characterized by abnormal myeloid cell proliferation and a hyperinflammatory state resembling viral infections. Traditional chemotherapy is ineffective, and allogeneic hematopoietic stem cell transplantation (HSCT) is only curative in 50% of cases. The bone marrow microenvironment (BME) in JMML is thought to be altered due to hyperinflammation, contributing to relapse and poor outcomes post-HSCT. Understanding the role of the hyperinflammatory state in JMML is crucial for developing better therapies. Studies indicate that hyperinflammation in JMML involves hyperactive innate immune cells and hypersensitivity to GM-CSF, a cytokine stimulating myeloid cell production. The BME may also become conducive to leukemia cell survival and expansion. Inflammasomes, which are protein complexes involved in host defense and cellular stress response, play a role in myeloid cell inflammation. Two important genes in this process are NLR family pyrin domain containing 3 (NLRP3) and prostaglandin-endoperoxide synthase 2 (PTGS2), associated with inflammasome signaling and prostaglandin synthesis, respectively. However, their specific contributions to JMML pathogenesis remain unclear. Our preliminary RNA-seq analysis of JMML patients showed elevated NLRP3 and PTGS2 expression compared to healthy controls, and their high expression correlated with poor overall survival in leukemia. Utilizing a mouse model of JMML bearing the most common JMML mutation, PTPN11 (Ptpn11E76K/+; seen in 35% of patients), which encodes the protein tyrosine phosphatase, Shp2 (Shp2E76K/+), we have initial evidence suggesting a potential role of increased Nlrp3 expression in JMML pathogenesis. Based on these findings, we hypothesize that overexpression of NLRP3 and PTGS2 contributes to JMML pathology. Targeting the NLRP3 inflammasome, either alone or in combination with other therapies, presents a promising approach for treating JMML. By understanding the molecular mechanisms underlying JMML-associated hyperinflammation and its impact on disease progression, we aim to identify novel therapeutic strategies for improved outcomes in JMML patients.

Clinical Impact. The proposed research is highly relevant to the DHART SPORE program, particularly focusing on PTPN11-mutant JMML. PTPN11, along with NF1, is involved in the Ras signaling pathway, which is critical in both normal cellular processes and in the development of various cancers, including JMML. While NF1 mutations directly impair Ras regulation by promoting its hyperactivity, mutations in PTPN11 also lead to Ras hyperactivation through distinct mechanisms. Notably, approximately 35% of JMML cases exhibit somatic PTPN11 mutations, underscoring their significance in disease pathogenesis. Therefore, understanding the role of PTPN11 in Ras pathway dysregulation is crucial for elucidating the pathogenesis of JMML. By investigating how PTPN11 mutations contribute to JMML development, particularly focusing on the hyperinflammatory state mediated by factors like the NLRP3 inflammasome and PTGS2, this research aims to uncover underlying mechanisms that are implicated in the disease progression. Moreover, exploring novel therapeutic strategies targeting NLRP3/PTGS2 pathways aligns directly with the DHART SPORE program's objective of developing effective treatments for Ras-driven tumors. The findings from this study have the potential not only to advance our understanding of JMML but also to inform broader strategies for treating cancers associated with Ras pathway dysregulation, including those involving PTPN11-mutations.

Project Abstract. Patients with neurofibromatosis type 1 (NF-1), caused by loss of the tumor suppressor gene NF1 encoding neurofibromin, develop both benign plexiform neurofibromas (pNF) and malignant peripheral nerve sheath tumors (MPNSTs). Although the MEK inhibitor selumetinib is FDA approved for pNFs in patients with NF-1, partial responses are common, and MPNSTs are resistant to MEK inhibition. Given pNFs cause significant morbidity and MPNSTs are the most common cause of death in adults with NF-1, there is an urgent, unmet clinical need for novel therapies. My preliminary data demonstrates repression of NF2, encoding the tumor suppressor Merlin, is sufficient for selumetinib resistance in MPNST models and modulates multiple disparate pathways in pNFs and MPNSTs with selective activation of Hippo signaling in MPNSTs alone. The central focus of this proposal is to understand how NF2 loss underlies resistance in NF1 deficient tumors. In particular, the key knowledge gap addressed by this proposal is to shed light on the critical signaling intermediates, biochemical mechanisms, and druggable dependencies underlying cooperation between NF1 and NF2 loss. In order to address this gap, we propose to (i) define if Hippo signaling following NF2 loss is a druggable dependency in MPNSTs, and (ii) determine if Merlin reconstitution blocks MPNST growth, The technical innovation lies in the integration of validated genetic CRISPRi and inducible overexpression systems with cutting edge pharmacologic agents to define the functional and biochemical network connecting neurofibromin and Merlin. The biological innovation is the elucidation of a functional link between the NF1 and NF2 tumor suppressors, which cause overlapping cancer syndromes defined by peripheral nervous system tumors. In sum, we will leverage a multidisciplinary approach combining CRISPR interference, pharmacology, and biochemistry to understand Merlin function in pNF and MPNST model systems and identify new therapeutic approaches for patients.

Clinical Impact. Peripheral nervous system tumors such as plexiform neurofibromas (pNFs) and malignant peripheral nerve sheath tumors (MPNSTs) are significant sources of morbidity and mortality in persons with neurofibromatosis type 1 (NF1).The goal of this project is to identify novel therapeutic approaches beyond the MEK inhibitor selumetinib, which is the only FDA approved molecular therapy for pNFs. This proposal builds on my prior work integrating human pNF and MPNST samples with biochemical and functional genomic studies in cell lines and mice to nominate NF2 loss as a potential driver of MEK inhibitor resistance. However, the mechanisms underlying this functional relationship are unclear, and by leveraging the biochemical and pharmacologic expertise of my mentor Dr. Frank McCormick, we aim to understand the signaling alterations and druggable dependencies following NF2 loss in NF1 mutant peripheral nervous system tumors with the goal of identifying new treatments for pNF and MPNSTs. The goal of this renewal application is to specifically focus on (i) Hippo signalling and (ii) NF2 reconstitution as therapeutic approaches for MPNSTs leveraging our efforts to date. Thus, our proposal is well aligned with the DHART SPORE’s emphasis on tumors arising in persons with NF1.

Project Abstract. : Neurofibromatosis type I patients frequently develop low grade gliomas that can progress to malignancy, especially when located outside of the optic pathway. Even though the frequency of High-Grade Glioma (HGG) in the NF1 population is low, they have a disproportional effect on the morbidity of NF1 patients. The prognosis of NF1 associated HGG remains dismal as the search for an effective therapy continues. Hence, there is a high unmet need to accelerate preclinical model building, so we can use these models to better understand what drives tumor formation and progression and design informed clinical trials. The NF1 heterozygous microenvironment is essential for the formation and progression of multiple NF1 associated tumors, including plexiform neurofibromas and optic pathway gliomas. The role of the NF1 heterozygous tumor microenvironment (TME) in NF1 high grade gliomas, however, remains elusive. In our lab we have 2 mouse NF1 HGG cell lines, derived from the NPcis genetically engineered mouse model, that readily form tumors when injected into the brains of syngeneic mice. Using single cell RNAseq, we will compare the differences between the Nf1 wildtype and heterozygous HGG TME. Additionally, with this RNAseq we will elucidate if the tumor location (hemisphere or cerebellum) affects the TME. Finally, the overall survival of Nf1 wildtype or heterozygous mice injected with our NF1 HGG lines will be determined. By analyzing these results in great detail, we will not only shed light on the potential contribution of the NF1 heterozygous TME in glioblastoma formation, but also better understand how NF1 associated HGG differ from sporadic HGG with a somatic NF1 mutation. This knowledge will help us target cell populations that are potentially key in tumor formation and progression.

Clinical Impact. This proposal investigates the role of the NF1 heterozygous tumor microenvironment in the development and progression of Neurofibromatosis Type I associated High-Grade Gliomas, one focus area of the DHART SPORE. Currently, it is unclear how or if the glioblastoma TME differs between non-NF1 patients and NF1 patients, and if this altered TME contributes to tumor formation. By fully understanding the how the TME differs between NF1 wildtype and heterozygous mice, we will be able to make informed decisions on what mouse strain to use for preclinical trials. Additionally, this knowledge will allow us to design specific therapies that target the tumor immune TME to hopefully enhance patient therapeutic outcomes.

Project Abstract. : Inactivation of the tumor suppressor NF1, due to loss of function (LoF) mutations, represents a subtype of melanoma, for which currently no effective targeted therapies are clinically available. NF1-LoF mutations abrogate negative feedback on RAS, thereby causing hyperactivation of multiple downstream signaling pathways. NF1-LoF melanoma cells, however, show only fractional responses to individual inhibitors of pro-growth pathways downstream of RAS, including clinically available MEK, ERK, or PI3K/mTOR inhibitors. To overcome this challenge, we recently performed a targeted compound screen to search for actionable kinases upstream, parallel to, or downstream of MEK/ERK and PI3K/mTOR pathways, whose inhibition individually or in combination with one another might be lethal to NF1-LoF melanoma cells. Through the integration of systems pharmacology approaches, including kinase inhibitor screening, kinome inhibition assays, transcriptomic profiling and bioinformatics analysis, single-cell analysis, and genetic experiments, we identified SYK, a non-receptor tyrosine kinase and mediator of oxidative metabolism, as a new molecular target that is required for NF1-LoF melanoma cell growth and survival. Building upon these data, in this project, we aim to evaluate the in vivo efficacy of the combination of two clinical-grade small molecule inhibitors, MEK inhibitor trametinib and SYK inhibitor fostamatinib, in NF1-LoF melanoma tumors in vivo (Aim 1). Given the established importance of SYK in metabolic homeostasis (including mitochondrial function, respiration and oxidative metabolism of fatty acids), and recent reports about unique metabolic dependencies of NF1-LoF cells, we also aim to take a systematic approach to mechanistic understanding of the connection between SYK dependency and metabolic reprogramming in NF1-LoF melanomas. This will be accomplished via integrative analysis across two metabolic screening platforms in the presence of loss- and gain-of function SYK perturbations. We will validate the identified metabolic needs and actionable vulnerabilities mediated by SYK in NF1-LoF melanoma cells in additional cell lines and in vivo (Aim 2). Overall, determination of the selective efficacy of SYK inhibition and its associated metabolic vulnerabilities is likely to offer a solid path toward the development, pre-clinical testing, and ultimately clinical evaluation of new therapies for NF1-LoF melanomas.

Clinical Impact. The proposed studies in this project are highly relevant to the goals of DHART SPORE, because they address an unmet clinical need by focusing on effective therapeutic targets and treatment strategies for a significant subtype of melanoma driven by NF1 loss of function (NF1LᵒF). There are currently no targeted therapies available for NF1LᵒF melanoma patients (i.e., ~13% of all melanoma patients), especially those that acquire resistance to immunotherapies. Importantly, our pilot project not only provides a path to evaluate a novel mechanism-based therapy in validated preclinical NF1LᵒF melanoma models (Aim 1), but also takes a systematic approach to exploit the relatively understudied signaling metabolic axis in NF1LᵒF melanoma as a novel therapeutic opportunity (Aim 2). Our findings, therefore, will have the potential of applying important fundamental and preclinical discoveries to the near-term development of clinical studies.

Project Abstract. : Understanding the epidemiology of disease is an important contributor to health of individuals and populations. Epidemiology can inform the anticipatory guidance provided to patients and patient families, enable prevention of disease (both by influencing individual behavior and by guiding public health policies), and identify avenues for research that may ultimately lead to improved treatments. This proposal seeks to advance our understanding of the epidemiology of Juvenile Myelomonocytic Leukemia (JMML), a cancer that occurs with increased frequency in children with Neurofibromatosis 1 (NF-1) and related disorders. Prior epidemiologic studies identified that JMML has an incidence of approximately 1.2 per million in the United States. Patients with NF-1 are at an approximately 200-fold increased risk of JMML and patients with related disorders (for example, Noonan Syndrome) are also at substantially increased risk. JMML develops in more boys than girls (above 2:1 male:female sex bias). This sex bias is especially marked in children with NF-1 (~80% of JMML in children with NF-1 are boys). Although studies have examined whether a child’s sex influences outcome in JMML (which it presently does not), the biological basis of male sex bias in JMML has not been reported (to my knowledge, based on literature review). - The current proposal seeks to address this gap in our knowledge. Hypothesis: Integrated Omics analyses to delineate sex differences will identify mechanisms that contribute to the sex bias of JMML. This proposal begins to address this hypothesis through study of pre-leukemic myelopoiesis in NF-1 deficient mouse myeloid cells and through analysis of existing data generated from human JMML patient samples. - In Aim 1 we will delineate sex differences of NF-1 deficient myelopoiesis by identifying sex differences in the methylome, areas of open chromatin, and gene expression in Nf1 deficient mouse myeloid cells. - In Aim 2 we will delineate sex differences in human JMML by analysis of OMICs data in the UCSF JMML REDCap database (which currently includes 426 JMML patients). - In our Integrative Aim, we will perform pathway analyses of mouse and human sex differences, enabling identification of higher order homologous changes across species and across disease stages.

Clinical Impact. The Developmental and Hyperactive Ras Tumor (DHART) SPORE “has the goal of improving the diagnosis and management of tumors arising in persons with neurofibromatosis type 1 (NF1) and other inherited ‘Rasopathy’ syndromes through basic, translational, and clinical research.” The SPORE Developmental Research Program supports “rigorous translational research in the areas of population science, therapeutics, and mechanisms of disease and by facilitating collaborative interactions between DRP-funded scientists and other SPORE investigators.” This proposal includes basic research on the nature of sex differences in NF-1 deficient mice as well as translational research utilizing human data derived from samples of patients with Juvenile Myelomonocytic Leukemia (JMML). The proposal directly addresses a population science question (sex bias in JMML) and has the potential to identify mechanisms that contribute to disease and/or to inform future therapeutics. The proposal has been developed in collaboration with DHART SPORE investigator Elliot Stieglitz and DHART OMICs core co-investigator Steve Angus. Should this work be funded the resulting work will require collaboration of the project PI (Scott Kogan) with Project 3 investigators Elliot Stieglitz, Benjamin Braun, and Kevin Shannon as well as the use of DHART OMICs Core resources.

Project Abstract. : Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcoma and the leading cause of mortality in individuals with Neurofibromatosis type 1 (NF1). MPNST are difficult to treat due to their relative insensitivity to chemotherapy or radiotherapy. Currently, no therapeutic agents have proven efficacy in clinical trials for patients with MPNST, and patient outcomes remain poor, particularly for those with locally invasive and metastatic disease. Thus, novel therapeutic approaches are urgently needed. MPNST tumorigenesis is driven by the loss of a tumor suppressor protein, neurofibromin, which subsequently causes hyperactivation of RAS effector pathways. Therefore, targeting downstream RAS signaling pathways is a logical approach for patients with MPNST. There are currently several novel small molecule targeted agents that inhibit various nodes in RAS activation pathways. These agents are in preclinical and early phase clinical development for other RAS-dependent cancers, and it is likely that they may also have anti-tumor efficacy in MPNST. Concurrently, emerging evidence suggests that molecularly targeted therapies have immunomodulatory effects, indicating a potential role for immunotherapy in providing more durable responses. Therefore, we aim to determine the interaction between molecularly targeted therapies and tumor immunobiology. We hypothesize that RAS signaling pathway inhibitors will exhibit anti-tumor effects and that the resulting signaling changes will influence the composition and function of tumor-infiltrating immune cells, thereby releasing an anti-tumor immune response. We will use a feasible and established immunocompetent, syngeneic mouse model to test a panel of RAS pathway-targeted agents that currently have tolerable toxicities and adequate pharmacodynamic activity in early phase clinical trials for RAS-dependent cancers. We will administer SHP2, SOS1, RAS(ON), pan-RAF, and CDK4/6 inhibitors as monotherapies to the tumor-bearing mice and measure tumor response, survival, and toxicity. Next, we will process the differentially treated tumors, isolate the infiltrating immune cells, and analyze their composition and function using multiparameter flow cytometric analysis (MFC). Successfully deconvoluting the tumor microenvironment following treatment with targeted agents is key to advancing the application of novel combination therapies that will provide long-term, durable responses in patients with MPNST.

Clinical Impact. The leading cause of mortality in individuals with NF1 is the development of malignant peripheral nerve sheath tumors (MPNST). Currently, the only definitive treatment for localized disease is oncologic surgery with wide margins. However, this is often unfeasible due to tumor location, the morbidities associated with the procedure, or the presence of metastases. Patient outcomes have not improved with chemotherapy or radiotherapy. Therefore, developing effective therapeutics for MPNST will fill an unmet clinical need. We aim to establish preclinical data on the immunomodulatory effects of molecularly targeted therapies and identify potential biomarkers in patients whose tumors may respond to the addition of immunotherapy. Our results will provide deeper insights into the relationship between MPNST immunobiology and small-molecule inhibitor therapeutics, informing the design of future clinical trials using novel combination therapies. Moreover, we propose to use clinical therapeutics that already have clinical trials data in patients with RAS-driven cancers. With promising pre-clinical data in models of MPNST, we could accelerate their advancement to clinical trials for this patient population. The successful development of novel therapeutics and targets for MPNST also has potential applications for other chemotherapy-refractory soft-tissue sarcomas and NF1-deficient malignancies.

Project Abstract. : Neurofibromatosis type 1 (NF1) is one of the most common “RASopathies” and is associated with more than double the general population’s risk for multiple cancer types.1-4 Plexiform neurofibromas (PNF) are benign peripheral nerve sheath tumors of Schwann cell origin that occur in nearly 50% of individuals with NF1.5 Approximately 10% of PNF progress to malignant peripheral nerve sheath tumors (MPNST), a highly aggressive family of sarcoma that is resistant to both chemotherapy and radiation.6-8 Complete resection is the only effective treatment for MPNST, however due to their location and prevalence for infiltration of surrounding tissue, MPNST are frequently unresectable.6,7 Patients with unresectable tumors or who present with metastatic disease have 5-year survival estimates as low as 5%. Thus, early detection, accurate identification of the PNF-to-MPNST transition, and development of effective therapies for unresectable MPNST are critical needs for patients with NF1. The GD2 ganglioside is a cell surface glycolipid expressed on multiple types of solid tumors, including neuroblastoma, melanoma, osteosarcoma, and some soft-tissue sarcomas. We recent assessed GD2 expression across several families of sarcomas, including MPNST. Strikingly, we observed high expression (>80% GD2-positive cells by flow cytometry) in 5/5 MPNST cell lines. In contrast, GD2 was low (<15%) in all three PNF cell lines examined. Thus, GD2 expression could serve as a biomarker to differentiate between PNF and MPNST. In addition, uniformly high GD2 expression in MPNST supports the potential utility of GD2-targeted therapies for patients with metastatic or unresectable disease. Dinutuximab (Unituxin®) is a GD2-specific antibody that has shown clinical success for immunotherapy in recurrent and metastatic neuroblastoma. We have demonstrated that PET radiotracers based on 64Cu- and 89Zr-radiolabeled dinutuximab are highly sensitive for in vivo measurement of GD2 expression in tumor-bearing mouse models. We hypothesize that PET imaging with 89Zr-dinutuximab can differentiate benign PNF from MPNST, enabling acurate diagnosis and potentiating the application of GD2-targeted MPNST therapies. Thus, the aims of this project are to: a) assess GD2 expression as a biomarker of the PNF-to-MPNST malignant transformation, b) evaluate the efficacy of 89Zr-dinutuximab PET for the in vivo detection and diagnosis of NF1-associated MPNST in mouse models.

Clinical Impact. The expressed goal of the DHART SPORE program is “improving the diagnosis and management of tumors arising in persons with NF1”. This project represents, to our knowledge, the first recognition of high GD2 expression as a distinguishing characteristic of MPNST. The low GD2 expression we demonstrated on PNF cell lines together with plexiform and atypical neurofibroma expression data from public databases that report uniformly low expression of B4GALNT1, which encodes a GD2 synthase, support the hypothesis that GD2 is a unique biomarker of malignant transition in MPNST and a highly selective potential target for therapy. Thus, this work will be the first investigation of GD2 as a target for “improving diagnosis and management” in MPNST. This interdisciplinary collaboration leverages the 89Zr-dinutuximab radiotracer developed by the PI, a unique genetically engineered NF1 mouse model developed in the Clapp lab and patient tissue specimens provided by the DHART SPORE Biospecimen/Pathology Core. If successful, this study will provide the first in vivo demonstration of GD2 expression in MPNST. This will in turn enable rapid translation of this new biomarker to clinical imaging and therapeutic trials and could open a completely new avenue of critically needed therapeutic development for this deadly disease.