Corina Anastasaki

Fusions between protein-coding genes are common oncogenic drivers, typically pairing a proto-oncogene with a partner that does not independently drive cancer. In all therapeutically actionable fusions, the proto-oncogene is the drug target, the contributions to oncogenicity of the fusion partner have largely been ignored. We studied the role of BRAF fusion partners and found that they are necessary for transformation. In the setting of KIAA1549::BRAF, the most common fusion protein across brain tumors, we found that KIAA1549 is necessary for oncogenicity of KIAA1549::BRAF and engenders a striking and specific dependency on the protein O-mannosyltransferase complex (POMT1/2). Specifically, we show that genetic silencing or pharmacologic inhibition of POMT1/2 reverses fusion-induced transformation, thereby representing a novel and MAPK-independent therapeutic target. Furthermore, POMT1/2 is required to glycosylate and enable maturation of the K::B fusion protein. These findings represent a proof-of-concept for targeting the partners in oncogenic fusions as a potential cancer therapeutic strategy.

Our labs have been studying neurofibromin function and phenotype for over a decade with the intent of generating targeted therapeutics for Neurofibromatosis type 1 (NF1). In the process, we have generated numerous human cell lines containing variants within the NF1 gene. Herein, we present data characterizing these cell lines and make them publicly available for use by researchers both within and outside the NF1 community. We describe lines that contain both well-characterized patient-specific variants either at their endogenous locus or as exogenous cDNAs, as well as variants of uncertain significance (VUS), engineered as heterozygous, homozygous, and compound heterozygous variants. Methods to generate each line and subsequent validation steps are detailed including targeted sequencing, Western blot analysis for neurofibromin expression and ERK activation. The utility of each line is dependent on the variant of interest, the parental cell line, and the mechanism of action relevant to possible therapeutic targeting.

Pilocytic astrocytomas (PAs), the most common pediatric brain tumor, grow slowly and depend heavily on their local brain microenvironment, hindering accurate humanized model development. To surmount this barrier, we developed mixed assembloids of PAs nested within human iPSC–cerebral organoids (PANCOs) that recapitulate PA histopathology and growth. Consistent with their cell-intrinsic ERK dependence, MEK inhibition reduces PANCO proliferation, whereas clozapine-N-oxide-activatable neural progenitors integrate into PANCOs, preferentially differentiate into glutamatergic neurons, and increase cell-extrinsic PA proliferation, which is blocked by glutamate receptor inhibition. These accurate in vitro pediatric PA avatars provide tractable platforms to discover and validate PA cell-intrinsic and cell-extrinsic growth dependencies.

Individuals with neurofibromatosis type 1 (NF1) are prone to the evolution of neurodevelopmental symptomatology including motor delays, learning disabilities, autism, and attention deficits. Caused by heterozygous germline mutations in theNF1gene, this monogenic condition offers unique opportunities to study the genetic etiologies for neurodevelopmental disorders and the mechanisms that underlie their formation. Although numerous small animal models have been generated to elucidate the causes of these alterations, there is little consensus on how to align preclinical observations with clinical outcomes, harmonize findings across species, and consolidate these insights to chart a cohesive path forward. Capitalizing on expertise from clinicians; human, animal, and cellular model research scientists; and bioinformatics researchers, the first Cognition and Behavior in NF1 (CABIN) meeting was convened at the Banbury Center of Cold Spring Harbor Laboratory in October 2024. This Perspective summarizes the state of our understanding and a proposed plan for future investigation and exploration to improve the quality of life of those with NF1.

Summary: The field of cancer neuroscience has begun to define the contributions of nerves to cancer initiation and progression; here, we highlight the future directions of basic and translational cancer neuroscience for malignancies arising outside of the central nervous system.

Nervous system tumors, particularly brain tumors, represent the most common tumors in children and one of the most lethal tumors in adults. Despite decades of research, there are few effective therapies for these cancers. Although human nervous system tumor cells and genetically engineered mouse models have served as excellent platforms for drug discovery and preclinical testing, they have limitations with respect to accurately recapitulating important aspects of the pathobiology of spontaneously arising human tumors. For this reason, attention has turned to the deployment of human stem cell engineering involving human embryonic or induced pluripotent stem cells, in which genetic alterations associated with nervous system cancers can be introduced. These stem cells can be used to create self-assembling three-dimensional cerebral organoids that preserve key features of the developing human brain. Moreover, stem cell-engineered lines are amenable to xenotransplantation into mice as a platform to investigate the tumor cell of origin, discover cancer evolutionary trajectories and identify therapeutic vulnerabilities. In this article, we review the current state of human stem cell models of nervous system tumors, discuss their advantages and disadvantages, and provide consensus recommendations for future research.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by germline mutations in the neurofibromin 1 (NF1) gene. Children with NF1 are prone to the development of multiple nervous system abnormalities, including autism and brain tumors, which could reflect the effect of NF1 mutation on microglia function. Using heterozygous Nf1-mutant mice, we previously demonstrated that impaired purinergic signaling underlies deficits in microglia process extension and phagocytosis in situ. To determine whether these abnormalities are also observed in human microglia in the setting of NF1, we leveraged an engineered isogenic series of human induced pluripotent stem cells to generate human microglia-like (hiMGL) cells heterozygous for three different NF1 gene mutations found in patients with NF1. Whereas all NF1-mutant and isogenic control hiMGL cells expressed classical microglia markers and exhibited similar transcriptomes and cytokine/chemokine release profiles, only NF1-mutant hiMGL cells had defects in P2X receptor activation, phagocytosis and motility. Taken together, these findings indicate that heterozygous NF1 mutations impair a subset of the functional properties of human microglia, which could contribute to the neurological abnormalities seen in children with NF1.

While neuronal activity has previously been implicated in malignant glioma growth control, we recently demonstrated that neuron excitability can also regulate low-grade (optic) glioma initiation and progression. In this prior study, we showed that NF1 germline mutation-dependent neuronal excitability sustains optic glioma growth through HCN channel-mediated midkine activation of a T cell-microglia stromal circuit. Relevant to clinical translation, lamotrigine modulation of HCN channel function reduced neuronal hyperexcitability, midkine production and optic glioma (OPG) growth. To establish an obligate role for neuronal activity and midkine (Mdk) production in OPG growth, we leveraged a series of unique genetically engineered mouse models. First, we demonstrate that midkine is necessary for gliomagenesis, as Nf1-OPG; Mdk-/- mice do not develop tumors and have restored retinal pathology, as evidenced by increased retinal ganglion cell (RGC) numbers and retinal nerve fiber layer thickness. Second, we show that midkine production and neuronal hyperactivity are interdependent, such that only tumor-associated germline Nf1 mutations result in increased RGC excitability and midkine production, whereas those mutations that do not cause optic gliomas lack RGC hyperexcitability or elevated midkine expression. Third, lamotrigine treatment both during and after Nf1-OPG development attenuates midkine levels and tumor proliferation. Moreover, this anti-tumoral effect is durable for months after the cessation of treatment. Fourth, oral treatment of Nf1-OPG-bearing mice with clinically relevant doses of lamotrigine (2.5-7.5mg/kg/day) during OPG development blocks tumor formation. Together, these data establish midkine and neuronal hyperexcitability as key drivers of Nf1 optic glioma growth, relevant to planned clinical trials of this anti-seizure medication for children with NF1-OPG.

Oligodendrocyte precursor cells (OPCs) are progenitor cells that give rise to oligodendrocytes in the central nervous system throughout the lifespan. Transcriptomic and lineage tracing studies suggest that OPCs may be the putative cell of origin for many gliomas in children and adults. While children with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome typically develop optic pathway and brainstem gliomas, as many as 50% of children develop gliomas in other brain regions using radiologic criteria. Relevant to these gliomas, discrete patches of hyperdense OPCs were found in the brains of Nf1+/- mice. The OPCs within the lesions display a transient increase in proliferation and hypercellularity. When the brains of mice carrying different heterozygous NF1 patient derived germline Nf1 gene mutations were examined, hyperdense OPC lesions were only seen in mice whose Nf1 mutations also predispose to optic gliomas. Mechanistically, monoallelic KRAS hyperactivation also results in similar OPC hyperdense lesions. Moreover, OPC-specific Nf1-heterozygous mice display similar lesions, while OPC-specific Nf1-knockout results in generalized OPC hyperdensity throughout the entire brain. Taken together, these data suggest the existence of a preneoplastic state conferred by Nf1 mutation in OPCs and establish a tractable model system to explore the transition to glioma in children with NF1.