NF1 Loss Research Articles

BIOM-12. ALLIANCE A071401: GENOMICALLY GUIDED PHASE II TRIAL OF ABEMACICLIB IN PATIENTS WITH GRADE 2/3 MENINGIOMAS HARBORING SOMATIC NF2 OR CDK PATHWAY ALTERATIONS

Abstract BACKGROUND Patients with meningiomas that have progressed after surgery and/or radiation have limited treatments. Loss of NF2 and CDKN2A/B are common in higher-grade meningiomas and promote meningioma progression. We evaluated the efficacy of abemaciclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, as part of Alliance A071401, a genomically-driven phase II trial in recurrent or progressive meningiomas. METHODS Eligible patients (pts) with grade 2/3 tumors and NF2 mutations or CDK pathway alterations were treated with abemaciclib 200 mg orally twice daily. Two co-primary endpoints were used: progression-free survival at 6 months (PFS6) and response rate (RR); the trial would be declared positive if either endpoint was met. Twenty-four evaluable pts provided ≥85% power to detect a PFS6 ≥41.5% (vs. null 15%; alpha=0.02), and ≥89% power to detect RR ≥20% (vs. null 2.5%; alpha=0.021). RESULTS Of 83 pts screened while the abemaciclib arm was open between September 15, 2021 and October 3, 2022, 36 eligible pts received abemaciclib. The observed PFS6 rate in the first 24 evaluable pts was 54% (95% CI 33-75%), thus the study met PFS6 endpoint. Of all 35 evaluable pts, median PFS was 7.6 months (95% CI 3.6-17.1 months), estimated 6-month PFS was 55% (95% CI 40-75%) and estimated 6-month OS was 88% (95% CI 77-100%). No objective responses were observed. Of the 36 pts who started treatment, two had a grade 4 adverse event at least possibly related to treatment which included ALT elevation(1), AST elevation(1) and vomiting(1). Patients with NF2 alterations (n=24) had favorable PFS6 outcomes (58%, 95% CI 37-78%) compared to those with CDK (n=4, 25%, 95% CI 1-81%) and CDK+NF2 alterations (n=7, 29% 95% CI 4-71%), (Fisher’s Exact p=0.036). CONCLUSIONS Abemaciclib was well-tolerated and resulted in improved PFS6, meeting the trial’s co-primary endpoint. Abemaciclib warrants further investigation for the treatment of patients with progressive meningiomas.

TMOD-30. DISRUPTION OF NORMAL RADIAL GLIA DEVELOPMENT IN AN NF2-ALTERED NEUROEPITHELIAL STEM CELL MODEL

Abstract NF2-related schwannomatosis is a tumor predisposition syndrome caused by mutations in the NF2 gene and associated with spinal ependymomas (SP-EPN). SP-EPNs are suspected to originate from aberrations in the radial glia (RG) cell lineage. These tumors are only effectively treated through high-risk surgical resection, emphasizing the critical need for identification of targets for medical therapy. Yet, the role of NF2-dependent disruption in RG cell development is poorly understood. We hypothesize that this mutation may be halting normal RG cell differentiation, resulting in a RG-like stem cell that is driving SP-EPN formation. An NF2-knockout was generated in neuroepithelial stem (NES) cells using CRISPR/Cas9. Knockouts were validated using Western blot and Sanger sequencing. In vitro differentiation was induced with removal of growth factors. NF2-knockout phenotypes were assessed with real-time polymerase chain reaction and compared with wildtype NES cells. Preliminary data has shown that NF2-knockout cells express similar levels of early pan-neural and neural stem cell genes compared to wildtype after CRISPR editing. The NF2-knockout cells retain this stem cell-like gene expression following attempted differentiation, whereas wildtype cells take on a primarily neural phenotype. The knockout cells also form what appears to be pre-neoplastic spheres when allowed to differentiate. NF2-knockout NES cells fail to differentiate normally compared to wildtype NES cells. They retain early RG-like markers, including SOX2, BLBP, and GFAP. Furthermore, formation of spheres when growth factors were removed hints at NF2 loss being relevant to formation of pre-neoplastic growths. Future work will include investigating downstream effects of NF2 loss in this model.

Automatic construction of Petri net models for computational simulations of molecular interaction network

Petri nets are commonly applied in modeling biological systems. However, construction of a Petri net model for complex biological systems is often time consuming, and requires expertise in the research area, limiting their application. To address this challenge, we developed GINtoSPN, an R package that automates the conversion of multi-omics molecular interaction network extracted from the Global Integrative Network (GIN) into Petri nets in GraphML format. These GraphML files can be directly used for Signaling Petri Net (SPN) simulation. To demonstrate the utility of this tool, we built a Petri net model for neurofibromatosis type I. Simulation of NF1 gene knockout, compared to normal skin fibroblast cells, revealed persistent accumulation of Ras-GTPs as expected. Additionally, we identified several other genes substantially affected by the loss of NF1’s function, exhibiting individual-specific variability. These results highlight the effectiveness of GINtoSPN in streamlining the modeling and simulation of complex biological systems.

NF1 expression profiling in IDH-wildtype glioblastoma: genomic associations and survival outcomes

BackgroundNF1 inactivation is associated with sensitivity to MEK inhibitor targeted therapy in low-grade and some high-grade gliomas. NF1 loss may also be a harbinger of exploitable vulnerabilities in IDH-wildtype glioblastoma (GBM). Accurate and consistent detection of NF1 loss, however, is fraught given the large gene size, challenges with complete coverage and variant calling upon sequencing, and mechanisms of mRNA and protein regulation that result in early degradation in the absence of genomic alterations. Here, we seek to perform a composite analysis for NF1 loss accounting for genomic alterations and protein expression via immunohistochemistry. We also characterize the landscape of NF1 alterations in GBM.MethodsWe assembled a single-institution, retrospective cohort of 542 IDH-wildtype GBM with somatic next generation sequencing to investigate the frequency and nature of detected NF1 alterations. We selected 69 GBMs from which to build a tissue microarray (TMA) of 44 NF1-wildtype and 25 NF1-mutant cases. We performed NF1 immunohistochemistry using two different NF1 antibodies (NFC, Sigma-Aldrich; and iNF-07E, iNFixion Bioscience) and correlated results with clinical, genomic, and other immunohistochemical features.ResultsIn our retrospective cohort, we identified 88 IDH-wildtype GBM with NF1 alterations (16%). NF1 alterations were mutually exclusive with EGFR and MDM2 alterations (p-adj < 0.001, 0.05, respectively), but co-occurred with PIK3R1 alterations (Log2(OR) = − 1.6, p-adj = 0.03). Of the 63 scorable sporadic GBMs in the TMA, 14 harbored NF1 inactivating alterations and of those, 12 (86%) demonstrated minimal NF1 immunoreactivity by NFC antibody, compared to 8 (57%) by iNF-07E antibody. Among the 42 scorable NF1-wildtype GBM in the TMA, NF1 immunostaining was minimal in 18 (43%) by NFC antibody compared to 4 (10%) by iNF-07E antibody, potentially reflecting false positives or differential protein regulation. Minimal immunoreactivity by NFC antibody was associated with decreased median overall survival (8.5 vs. 16.4 months, p = 0.011). Cox proportional hazards model correcting for prognostic variables in this subset revealed HR 3.23 (95% CI 1.29–8.06, p = 0.01) associated with decreased NF1 expression by IHC.ConclusionNF1 immunostaining may serve as a sensitive surrogate marker of NF1 genomic inactivation and a valuable extension to next-generation sequencing for defining NF1 status. Minimal NF1 immunoreactivity is a poor prognostic marker, even in IDH-wildtype glioblastoma without apparent NF1 genomic alterations, but the underlying molecular mechanism requires further investigation.

MEK Inhibitors Lead to PDGFR Pathway Upregulation and Sensitize Tumors to RAF Dimer Inhibitors in NF1-Deficient Malignant Peripheral Nerve Sheath Tumor.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive subtype of soft-tissue sarcoma with a high propensity to metastasize and extremely limited treatment options. Loss of the RAS-GAP NF1 leads to sustained RAF/MEK/ERK signaling in MPNST. However, single-agent MEK inhibitors (MEKi) have failed to elicit a sustained inhibition of the MAPK signaling pathway in MPNST. We used pharmacological, biochemical, and genetic perturbations of the receptor tyrosine kinase and MAPK signaling pathway regulators to investigate the mechanisms of MEKi resistance and evaluated combination therapeutic strategies in various preclinical MPNST models in vitro and in vivo. Here, we report that MEKi treatment resistance in MPNST involves two adaptive pathways: direct transcriptional upregulation of the receptor tyrosine kinase PDGFRβ and MEKi-induced increase in RAF dimer formation and activation of downstream signaling. Although the pharmacologic combination of a MEKi with a PDGFRβ-specific inhibitor was more effective than treatment with the MEKi alone, the combination of the MEKi and RAF dimer inhibitors led to a robust inhibition of MAPK pathway signaling. This combination treatment was effective in vitro and in vivo, as demonstrated by the significant increase in drug synergism and its high effectiveness in decreasing MPNST viability. Our findings suggest that the combination of MEKis and PDGFR and/or RAF dimer inhibitors can overcome MEKi resistance and may serve as a novel targeted therapeutic strategy for patients with NF1-deficient MPNST, which in turn could impact future clinical investigations for this patient population.

Erratum for the Research Article "MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells" by S. Perrin et al.

Erratum for the Research Article "MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells" by S. Perrin et al.

Neurofibromin deficiency alters the patterning and prioritization of motor behaviors in a state-dependent manner.

Genetic disorders such as neurofibromatosis type 1 increase vulnerability to cognitive and behavioral disorders, such as autism spectrum disorder and attention-deficit/hyperactivity disorder. Neurofibromatosis type 1 results from loss-of-function mutations in the neurofibromin gene and subsequent reduction in the neurofibromin protein (Nf1). While the mechanisms have yet to be fully elucidated, loss of Nf1 may alter neuronal circuit activity leading to changes in behavior and susceptibility to cognitive and behavioral comorbidities. Here we show that mutations decreasing Nf1 expression alter motor behaviors, impacting the patterning, prioritization, and behavioral state dependence in a Drosophila model of neurofibromatosis type 1. Loss of Nf1 increases spontaneous grooming in a nonlinear spatial and temporal pattern, differentially increasing grooming of certain body parts, including the abdomen, head, and wings. This increase in grooming could be overridden by hunger in food-deprived foraging animals, demonstrating that the Nf1 effect is plastic and internal state-dependent. Stimulus-evoked grooming patterns were altered as well, with nf1 mutants exhibiting reductions in wing grooming when coated with dust, suggesting that hierarchical recruitment of grooming command circuits was altered. Yet loss of Nf1 in sensory neurons and/or grooming command neurons did not alter grooming frequency, suggesting that Nf1 affects grooming via higher-order circuit alterations. Changes in grooming coincided with alterations in walking. Flies lacking Nf1 walked with increased forward velocity on a spherical treadmill, yet there was no detectable change in leg kinematics or gait. Thus, loss of Nf1 alters motor function without affecting overall motor coordination, in contrast to other genetic disorders that impair coordination. Overall, these results demonstrate that loss of Nf1 alters the patterning and prioritization of repetitive behaviors, in a state-dependent manner, without affecting motor coordination.

Pediatric Hemispheric High-Grade Gliomas and H3.3-G34 Mutation: A Review of the Literature on Biological Features and New Therapeutic Strategies.

Pediatric high-grade glioma (pHGG) encompasses a wide range of gliomas with different genomic, epigenomic, and transcriptomic features. Almost 50% of pHGGs present a mutation in genes coding for histone 3, including the subtype harboring the H3.3-G34 mutation. In this context, histone mutations are frequently associated with mutations in TP53 and ATRX, along with PDGFRA and NOTCH2NL amplifications. Moreover, the H3.3-G34 histone mutation induces epigenetic changes in immune-related genes and exerts modulatory functions on the microenvironment. Also, the functionality of the blood-brain barrier (BBB) has an impact on treatment response. The prognosis remains poor with conventional treatments, thus eliciting the investigation of additional and alternative therapies. Promising molecular targets include PDGFRA amplification, BRAF mutation, EGFR amplification, NF1 loss, and IDH mutation. Considering that pHGGs harboring the H3.3-G34R mutation appear to be more susceptible to immunotherapies (ITs), different options have been recently explored, including immune checkpoint inhibitors, antibody mediated IT, and Car-T cells. This review aims to summarize the knowledge concerning cancer biology and cancer-immune cell interaction in this set of pediatric gliomas, with a focus on possible therapeutic options.

First person – Stephanie Bouley

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Stephanie Bouley is first author on ‘ Chemical genetic screens reveal defective lysosomal trafficking as synthetic lethal with NF1 loss', published in JCS. Stephanie conducted the research described in this article while a PhD candidate in Yolanda Sanchez's lab at Geisel School of Medicine at Dartmouth College, Hanover, USA. She is now a post-doctoral research fellow in the lab of James A. Walker at Massachusetts General Hospital Center for Genomic Medicine, Boston, MA USA, where she is interested in identifying novel therapeutic targets for treating neurofibromatosis type 1-associated tumors.

Chemical genetic screens reveal defective lysosomal trafficking as synthetic lethal with NF1 loss.

Neurofibromatosis type 1, a genetic disorder caused by pathogenic germline variations in NF1, predisposes individuals to the development of tumors, including cutaneous and plexiform neurofibromas (CNs and PNs), optic gliomas, astrocytomas, juvenile myelomonocytic leukemia, high-grade gliomas and malignant peripheral nerve sheath tumors (MPNSTs), which are chemotherapy- and radiation-resistant sarcomas with poor survival. Loss of NF1 also occurs in sporadic tumors, such as glioblastoma (GBM), melanoma, breast, ovarian and lung cancers. We performed a high-throughput screen for compounds that were synthetic lethal with NF1 loss, which identified several leads, including the small molecule Y102. Treatment of cells with Y102 perturbed autophagy, mitophagy and lysosome positioning in NF1-deficient cells. A dual proteomics approach identified BLOC-one-related complex (BORC), which is required for lysosome positioning and trafficking, as a potential target of Y102. Knockdown of a BORC subunit using siRNA recapitulated the phenotypes observed with Y102 treatment. Our findings demonstrate that BORC might be a promising therapeutic target for NF1-deficient tumors.

NFS-09.NF1 MUTATION IN OLIGODENDROCYTE PRECURSOR CELLS LEADS TO PRENEOPLASTIC LESION FORMATION IN THE BRAIN

Abstract Oligodendrocyte precursor cells (OPCs) are progenitor cells that give rise to central nervous system oligodendrocytes throughout life. Transcriptomic and lineage tracing studies suggest that OPCs may be the putative cell of origin for many glioma histologic subtypes. While children with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome are prone to low-grade gliomas following bi-allelic NF1 loss, the cellular and molecular etiologies underlying tumorigenesis remain incompletely elucidated. Using a unique collection of genetically engineered Nf1 mouse strains, we identified discrete hyperdense lesions composed of OPCs. The OPCs within the lesions display a transient increase in proliferation and hypercellularity (hyperplasia). This phenotype was not observed following heterozygous mutation in other tumor suppressors (Trp53, Pten, or Rb1). OPC-specific heterozygous Nf1-mutant mice harbored similar lesions, suggesting an OPC-intrinsic phenotype. OPC-specific Nf1 loss resulted in increased OPC density throughout the entire brain, suggesting that the lesions in heterozygous Nf1-mutant mice represent spontaneous OPC Nf1 loss-of-heterozygosity events. Mechanistically, monoallelic KRAS hyperactivation phenocopies OPC hyperdensities in the brain of Nf1+/- mice; however, KRAS hyperactivity is not necessary for lesion formation or expansion. When the brains of mice engineered with heterozygous NF1 patient-derived Nf1 mutations were examined, the development of hyperdense OPC lesions reflected RAS-independent dysregulated PI3K-AKT signaling. Collectively, our study reveals a potential mechanism driving the development of preneoplastic lesions in the brains of individuals with NF1.

NFS-10. ROLE OF CDKN2A CO-DELETION IN DRIVING TUMOR INVASIVENESS IN NF1 MUTANT GLIOMA

Abstract BACKGROUND Neurofibromatosis type 1, caused by mutations in the NF1 gene, is a common autosomal dominant genetic disorder associated with an increased risk of glioma tumor development. A high occurrence of CDKN2a codeletion in NF1-mutant high-grade gliomas (HGG) compared to low-grade gliomas (LGG) implies a potential role of CDKN2a loss in the transformation of NF1-mutant LGG to HGG. In our study, we aimed to elucidate the role of NF1 and CDKN2a in tumor transformation using a panel of glioma cell line models with varying NF1 and CDKN2a expression. METHOD A diverse panel of glioma cell lines, encompassing LGG (1667-ON, RES259, RES186, JHH-NF1-PA1) and HGG (GBM511, GBM110, SF8628, U87-MG, TM-31, 7316-2189, 7316-3058, 7316-4917), was cultured. Their growth rates and invasiveness were further evaluated using cell proliferation and Boyden chamber invasiveness assays. RESULTS LGG cell lines exhibited relatively lower growth rates and invasiveness compared to their high-grade counterparts. Intriguingly, RES259, a LGG cell line harboring both NF1 and CDKN2a variants, demonstrated the highest growth rate and invasiveness among LGG cell lines, surpassing even those with intact NF1 and CDKN2a, such as JHH-NF1-PA1 and RES186. To further elucidate the roles of NF1 and CDKN2a, CRISPR-Cas9 technology was employed to knockout these genes individually and in various combinations in RES186. Our findings unveiled that sole NF1 knockout had negligible impact on RES186. Conversely, CDKN2a knockout increased both growth rate and invasiveness. Strikingly, the codeletion of NF1 and CDKN2a in RES186 significantly exacerbated its growth rate and invasiveness. These results underscore the intricate interplay of NF1 and CDKN2a in governing the growth and invasiveness dynamics of glioma cells. CONCLUSION In conclusion, our preliminary study suggests that the concurrent loss of NF1 and CDKN2a promotes enhanced growth and invasiveness in gliomas, shedding light on the collaborative role of these genetic alterations in tumor transformation.

Molecular-targeted therapy for childhood low-grade glial and glioneuronal tumors.

Since the discovery of the association between BRAF mutations and fusions in the development of childhood low-grade gliomas and the subsequent recognition that most childhood low-grade glial and glioneuronal tumors have aberrant signaling through the RAS/RAF/MAP kinase pathway, there has been a dramatic change in how these tumors are conceptualized. Many of the fusions and mutations present in these tumors are associated with molecular targets, which have agents in development or already in clinical use. Various agents, including MEK inhibitors, BRAF inhibitors, MTOR inhibitors and, in small subsets of patients NTRK inhibitors, have been used successfully to treat children with recurrent disease, after failure of conventional approaches such as surgery or chemotherapy. The relative benefits of chemotherapy as compared to molecular-targeted therapy for children with newly diagnosed gliomas and neuroglial tumors are under study. Already the combination of an MEK inhibitor and a BRAF inhibitor has been shown superior to conventional chemotherapy (carboplatin and vincristine) in newly diagnosed children with BRAF-V600E mutated low-grade gliomas and neuroglial tumors. However, the long-term effects of such molecular-targeted treatment are unknown. The potential use of molecular-targeted therapy in early treatment has made it mandatory that the molecular make-up of the majority of low-grade glial and glioneuronal tumors is known before initiation of therapy. The primary exception to this rule is in children with neurofibromatosis type 1 who, by definition, have NF1 loss; however, even in this population, gliomas arising in late childhood and adolescence or those not responding to conventional treatment may be candidates for biopsy, especially before entry on molecular-targeted therapy trials.

Abstract A013: NF1 loss is syntetic lethal with Trastuzumab emtansine

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). Most research has focused on target expression, but key predictors of payload efficacy have not been indeitifed. NF1 is a tumor suppressor classically considered as an inhibitor of RAS signaling, and often mutated in metastatic HER2+ breast cancer (BC). We screened multiple approved drugs for differential sensitivityin CRISPR-engineeredf NF1 KO cells. HER2-targeted agents (small molecules or antibodies) were found to be less effective upon NF1 loss; surprisingly, we identified increased sensitivity to the approved ADC T-DM1, but not to Trastuzumab Deruxtecan (T-Dxd). We then elucidated the underlying molecular cause employing in vivo, in vitro and in vitro reductionist systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3- GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization and completely rescued DM1-induced inhibition. NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a dose-dependent significant increase in MT dynamics (fraction of elongating MTs, elongation speed, catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intratubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair. In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intratubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. These results prompt the use of NF1 as a biomarker to select patients for ADC treatment. Funding: FIEO fellowship 2023, AIRC (n25791), Italian MoH-Ricerca Corrente di Rete (ACCORD) 2022, Next Generation EU – PNRR M6C2 – PNRR-MAD-2022-12376934 Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Marcus Braun, Zdenek Lansky, Luigi Scietti, Luca Mazzarella. NF1 loss is syntetic lethal with Trastuzumab emtansine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A013.

Abstract PR012: KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer

Abstract The estrogen receptor (ER) is expressed in ∼70% of breast cancers where it functions as a critical transcription factor that regulates cell growth and tumor progression. ER inhibition is a mainstay treatment for ER-positive (ER+) breast cancer, but despite the clinical benefit of targeting ER signaling, breast cancer remains the second leading cause of cancer-related death in women, with ∼50% of deaths resulting from ER+ tumors. While recurrent and metastatic ER+ tumors are typically resistant to endocrine therapies they continue to rely on ER signaling suggesting that disruption of other chromatin associated complexes that mediate ER-driven gene expression may present an alternative therapeutic strategy. One chromatin modifier important in regulating ER function is the histone acetyltransferase KAT6A. KAT6A is amplified and/or overexpressed in ∼15% of ER+ tumors and a novel KAT6A/B inhibitor has entered clinical trials for advanced breast cancer. However, as with most single agent therapies, compensatory mechanisms may attenuate the effects of KAT6A/B inhibition on ER-driven gene expression. We hypothesized that the dual targeting of distinct chromatin complexes would enhance the effects of KAT6A/B inhibition, by altering chromatin state and transcriptional output. To that end we performed an epigenetic focused CRISPR-Cas9-based functional genetic screen to identify chromatin-associated proteins that enhance KAT6A/B inhibition. CRISPR guides targeting MEN1 (encoding Menin) were among our top hits as sensitizers to treatment with the KAT6A/B inhibitor PF-9363. We confirmed that co-treatment of PF-9363, and the Menin inhibitor, SNDX-5613, had strong synergistic anti-proliferative effects in a panel of ER+ breast cancer cell lines. Mechanistically, our data revealed that KAT6A/B and Menin cooperatively regulate ER-driven gene expression and chromatin accessibility, both through direct effects on ESR1 expression as well as through effects at ER target genes. Specifically, we found that KAT6A and Menin-KMT2A complexes co-localized at promoters of ER target genes and treatment with KAT6A/B or Menin inhibitors displaced KAT6A and Menin/KMT2A from ER target genes, respectively. Yet only the combination effectively displaced both complexes from chromatin, which produced more dramatic changes in ER-directed gene expression than either small molecule alone. Combined displacement of KAT6A and Menin/KMT2A at ER-regulated genes induced loss of an active chromatin landscape characterized by reduction of activating histone modifications and loss of RNA Polymerase II binding to ER target loci. Importantly, the combination of KAT6A/B and Menin inhibition was effective in a panel of ER+ patient-derived organoid models as well as in various models of endocrine resistance, including models with ESR1 mutations, NF1 loss, and FOXA1 mutations. Both KAT6A/B and Menin inhibitors are in Phase I/II clinical trials and have shown manageable toxicity profiles to date. This combination represents a novel potential therapeutic combination for ER+ breast cancer. Citation Format: Sarah Naomi Olsen, Bryn Anderson, Charles Hatton, Rinath Jeselsohn, Myles Brown, Eneda Toska, Scott Armstrong. KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR012.

Combined NF2/PTEN mutations as predictors of meningioma aggressiveness.

e14044 Background: In meningiomas, loss of the NF2 tumor suppressor gene function leads to dysregulation of cell proliferation and tumor formation. PTEN, another tumor suppressor gene correlated with worse prognosis in tumors like gliomas, has also been reported in meningiomas, but the impact of its mutation alone or in conjunction with NF2 loss on outcomes in these tumors is unexplored. We aimed to evaluate whether dual NF2/PTEN mutations comprise a more aggressive meningioma phenotype compared to tumors with only NF2 mutations. Identifying this high-risk genetic profile could allow for preventative and individualized treatment of these aggressive meningiomas. Methods: We reviewed patients who underwent meningioma resection at our institution from January 2018 through October 2022. Demographic information, tumor characteristics, pre- and post-operative clinical characteristics, and outcomes were collected. Meningioma specimens were analyzed by immunohistochemistry for WHO grading and Ki67 proliferation index and by sequencing for genomic alterations. We performed multivariable logistic regression analyses to identify clinical outcomes associated with proliferation index and genetic mutations. Results: We identified NF2 mutations in 24 meningiomas and dual NF2/PTEN mutations in 7 meningiomas. Mutation rates did not significantly differ based on demographic factors including sex, age, race, socioeconomic status, or prior cranial radiation status (p&gt;0.05). The majority of tumors with these mutations (93%) were in non-skull base regions (i.e. convexity, parafalcine). Tumors with both NF2 and PTEN mutations were more likely to be WHO grade III tumors than those with the NF2 mutation alone (29% versus 9%). Tumors with dual mutations had higher likelihood of having Ki67 proliferation indices ≥ 10% (OR 5.98, p&lt;0.01). While higher Ki67 index was significantly associated with features of more aggressive tumors including higher WHO grade and recurrence rates, dual mutation in NF2 and PTEN is associated with higher rate of recurrence (OR 13.13, p&lt;0.001) and mortality (OR 5.14, p&lt;0.05) compared to NF2 irrespective of Ki67 index. Notably, patients with mutations in NF2 alone or both NF2/PTEN did not differ in preoperative Karnofsky performance score, Simpson resection grade, or presence of post operative residual tumor. Conclusions: Our results demonstrated that concomitant NF2 and PTEN mutations in meningiomas were associated with higher WHO grades, tumor proliferation indices, recurrence rates, and mortality rates. This finding suggests that NF2/PTEN mutated meningiomas represent a distinct subset of tumors that demonstrate a more aggressive clinical and histopathological phenotype that may require more aggressive treatment and closer surveillance. Further investigation is warranted to confirm the prognostic relevance of NF2/PTEN mutations in meningioma and elucidate their underlying molecular mechanisms.

Nf1 mutation disrupts activity-dependent oligodendroglial plasticity and motor learning in mice

Neurogenetic disorders, such as neurofibromatosis type 1 (NF1), can cause cognitive and motor impairments, traditionally attributed to intrinsic neuronal defects such as disruption of synaptic function. Activity-regulated oligodendroglial plasticity also contributes to cognitive and motor functions by tuning neural circuit dynamics. However, the relevance of oligodendroglial plasticity to neurological dysfunction in NF1 is unclear. Here we explore the contribution of oligodendrocyte progenitor cells (OPCs) to pathological features of the NF1 syndrome in mice. Both male and female littermates (4–24 weeks of age) were used equally in this study. We demonstrate that mice with global or OPC-specific Nf1 heterozygosity exhibit defects in activity-dependent oligodendrogenesis and harbor focal OPC hyperdensities with disrupted homeostatic OPC territorial boundaries. These OPC hyperdensities develop in a cell-intrinsic Nf1 mutation-specific manner due to differential PI3K/AKT activation. OPC-specific Nf1 loss impairs oligodendroglial differentiation and abrogates the normal oligodendroglial response to neuronal activity, leading to impaired motor learning performance. Collectively, these findings show that Nf1 mutation delays oligodendroglial development and disrupts activity-dependent OPC function essential for normal motor learning in mice.

Columnar Cell Thyroid Carcinoma: A Heterogeneous Entity Demonstrating Overlap Between Papillary Thyroid Carcinoma and Follicular Neoplasms.

Columnar cell papillary thyroid carcinoma (CC-PTC) is a morphologic subtype of papillary thyroid carcinoma with a variable prognosis. It is characterized by neoplastic thyroid follicular-derived cells with pseudostratified columnar morphology arranged in papillary or follicular structures with supranuclear or subnuclear vacuoles. The molecular profile of this subtype has only recently come under scrutiny, with mixed results. The aim of this study is to further explore the morphologic, immunohistochemical, and genetic profile of CC-PTC, as well as to correlate these features with clinical outcomes. CC-PTC cases were identified from 3 institutions. Immunohistochemistry (ER, CDX2) and molecular testing (DNA and RNA sequencing) were performed. Clinicopathologic parameters and patient outcomes were recorded. Twelve cases (2006-2023) were identified, all in adults (age 45-91). Two presented with disease outside the thyroid gland (neck and mediastinum) and two presented with distant metastasis. Four were high-grade differentiated thyroid carcinomas (necrosis or mitoses), one of which died of disease. Four were noninvasive or minimally invasive, one of which locally recurred. Three patients had lymph node metastases. ER and CDX2 were positive in 73% and 50%, respectively. Pathogenic mutations were found in TERT promoter (n = 3), RAS (n = 2), ATM, NOTCH1, APC, and ESR1, along with cases bearing AGK::BRAF fusion (n = 1), BRAF VE1 expression (n = 1), and NF2 loss (n = 1). This study represents the largest molecularly defined cohort of non-oncocytic thyroid carcinomas with columnar cell morphology. These tumors represent a genetically and behaviorally heterogeneous group of neoplasms, some of which have RAS-like or follicular neoplasm-like genetics, some of which have BRAF-p.V600E-like or classic papillary thyroid carcinoma-like genetics, and some of which remain unclear. Noninvasive or minimally invasive tumors showed an indolent course compared to those with angioinvasion, gross extrathyroidal growth, or high-grade morphology. Consideration could be given to reclassification of this neoplasm outside of the subtyping of papillary thyroid carcinoma in light of its genetic diversity, distinct morphology, and clinical behavior more closely aligned with follicular thyroid neoplasms.

Abstract PO1-23-09: A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC)

Abstract Background: CDK4/6i are part of the standard management of HR+ MBC. The MAPK pathway has been implicated in mediating resistance to CDK4/6 blockade (Wander 2020). A variety of molecular alterations dysregulate MAPK signaling, including NF1 mutations (NF1-MUT) (Wallace 2012), though NF1 has not been implicated in CDK4/6i resistance. To assess this, we analyzed retrospective clinical data from multiple institutions, real-world data (RWD) from the GuardantINFORM database, and preclinical models of HR+ breast tumors. The primary objective of this study was to characterize clinical outcomes of CDK4/6i in patients (pts) with NF1-MUT MBC, and to validate those findings in laboratory models. Methods: Pts with pathogenic (p)NF1-MUT were identified via sequencing of circulating tumor DNA (ctDNA) or tumor tissue. Pathogenic was defined as nonsense, insertion/deletion, frameshift, or oncogenic missense mutations. Progression free survival (PFS) on CDK4/6i was analyzed, and intrinsic resistance was defined as progression &amp;lt; 6 months on 1st line (1L) CDK4/6i or &amp;lt; 3 mo on 2nd line and beyond (2L+). Acquired resistance was defined as PFS &amp;gt;6 mo (1L) or &amp;gt;3 mo (2L+) with a NF1 alteration detected post-progression. RWD from the GuardantINFORM dataset had Guardant360 (G360) ctDNA testing linked with claims data on &amp;gt;37,000 pts with advanced or MBC tested between June 2014 and March 2023. Pts on CDK4/6i within 90 days pre-G360 were stratified as NF1-MUT (n = 28) vs. NF1-non-mutant (NF1-nonMUT) (n = 1133) and analyzed for real-world time-to-next-treatment (rwTTNT) and real-world overall survival (rwOS). Propensity score weighting for age, sex, line of therapy, and year of G360 were used to create a balanced control group. To determine whether NF1 loss-of-function alterations were causal of CDK4/6i resistance, we used CRISPR/Cas9 to delete NF1 in MCF7 breast cancer cells. MCF7 wild-type (WT) and two NF1-knockout (KO) clones were treated with palbociclib ± estrogen deprivation, followed by cell viability assays. MAPK and PI3K/AKT pathway activation were measured by immunoblot analysis. Results: Across 4 institutions, 40 pts with HR+, HER2- MBC expressing pNF1-MUT and prior CDK4/6i were included. The mean age at MBC diagnosis was 56 years, and the majority of pts received no prior treatment in the metastatic setting before CDK4/6i (range 0 - 5 prior lines). Intrinsic or acquired resistance to CDK4/6i was seen in 29/40 (73%) tumors harboring pNF1-MUT. We analyzed pts with baseline pNF1-MUT who received 1L CDK4/6i plus endocrine therapy (n = 13) and found that the median PFS was 7.7 months (range 2 - 18 mo). We next examined GuardantINFORM RWD and discovered a significant difference in rwTTNT and rwOS on CDK4/6i for NF1-MUT (n = 28) vs. weighted NF1-nonMUT (n = 28) tumors. Mean age (62 years) and level of pretreatment (majority 2L+) were similar between the groups. Compared to NF1-nonMUT, NF1-MUT pts had a shorter median rwTTNT on CDK4/6i regimens (4.2 vs. 12.4 mo, weighted log-rank p&amp;lt; 0.0001) and worse rwOS (15.8 vs 45.2 mo, weighted log-rank p=0.016). In MCF7 cells, NF1-KO exhibited a palbociclib IC50 11- to 13-fold higher than NF1-WT controls. In estrogen-deprived media, treatment with 250 nM palbociclib for 6 days reduced MCF7 NF1-WT cell viability 75% but only 5-20% in NF1-KO cells. As expected, phosphorylation of ERK and AKT were strongly induced in NF1-KO cells relative to NF1-WT. Conclusions: Using multicenter clinical and real-world evidence, we demonstrate that NF1-MUT is associated with inferior duration and worse outcomes on CDK4/6i in MBC. Pts with baseline pNF1-MUT on 1L CDK4/6i and endocrine therapy had a median PFS below typical responses expected in clinical practice. A link between NF1 loss and CDK4/6i resistance was supported by in vitro experiments in HR+ breast cancer cells. NF1 deletion was accompanied by activation of ERK and AKT, therefore, future directions include investigating alternative or combination treatment strategies targeting the MAPK and/or PI3K/AKT pathways in these tumors. *MRL and RCP contributed equally. ABH and SAW are co-senior authors. Citation Format: Maxwell Lloyd, M Rosario Chica-Parrado, Todd Knepper, Chang-Ching Lin, Emily Podany, Christine Walko, Fabiana Napolitano, Caroline Weipert, Jiemin Liao, Arielle Medford, Lianne Ryan, Jennifer Keenan, Nicole Zhang, Shiyuan Liu, Gerburg Wulf, Katherine Clifton, Cynthia Ma, Hyo Han, Leif Ellisen, Aditya Bardia, Carlos Arteaga, Ariella Hanker, Seth Wander. A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-09.

Abstract GS01-08: CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ breast cancer

Abstract CDK4/6 control a key checkpoint by phosphorylating Rb, leading to Rb degradation and subsequent S-phase entry. CDK4/6 inhibition, together with endocrine therapy, is standard of care for advanced ER+ breast cancer; however, CDK4/6 inhibition typically is cytostatic. This study aims to identify breast cancer patients who may better respond to treatments targeting CDK4/6. NF1/neurofibromin is a key tumor suppressor that we have shown can represses not only RAS as a GAP but also ER as a transcriptional co-repressor. Dual activation of RAS and ER promotes endocrine therapy resistance. We computed kinase activity scores from phosphor-proteomic data in the CPTAC breast cancer cohort and found that both CDK4/6 activity scores and Rb-pS780 levels negatively correlated with NF1 protein levels. Furthermore, NF1 mutations co-occur with mutations in CDK4/6 and CCND1-3 in breast tumors. These results suggest a functional dependency between NF1 loss and activation of the CDK4/6-Rb pathway. To delineate how NF1 and CDK4 interact molecularly, we depleted NF1 in ER+ breast cancer cells and found an increase in Rb-pS780 and in phosphorylation in CDK4’s activation loop (CDK4-pT172). Cyclin-D binding is required for CDK4 activation. CCND1 expression is under the direct control by ER, and upon NF1-depletion, Cyclin D1 expression was increased, partly due to enhanced ER recruitment to an ERE in CCND1. Cyclin-D-bound CDK4 must be phosphorylated at T172 to be fully active; however, what kinase is responsible for this is largely unknown. We have evidence that CDK4-T172 phosphorylation is RAF, but not ERK, dependent. When the NF1-depleted ER+ cells were seeded in the presence of fulvestrant together with either palbociclib or abemaciclib, apoptosis readily occurred. Remarkably, fulvestrant plus palbociclib efficiently caused durable tumor regression in two ER+ PDX models, in which NF1 is undetectable by IHC. In contrast, no tumor regression was seen in a PDX model with detectable NF1. Finally, greater growth inhibition and apoptosis were detected in NF1-low tumors in the neoadjuvant NeoPalAna trial when palbociclib was added after anastrozole. These data support a model whereby ER and RAS signaling converge upon CDK4/6, and CDK4/6 activation is a key survival mechanism when ER signaling is attenuated by treatment in NF1-depleted ER+ breast cancer cells. This apparent addiction for CDK4/6 activity makes NF1-depleted ER+ breast tumors vulnerable to CDK4/6 inhibition, thus creating a potential therapeutic opportunity to match CDK4/6 inhibition with patients who can benefit the most. Citation Format: Ze-Yi Zheng, Anran Chen, Eric Jaehnig, Meenakshi Anurag, Jonathan Lei, Chenwei Wang, Long Feng, Purba Singh, Hilda Kennedy, Jin Cao, Ghazal Yadav, Jill Tsai, Xi Chen, Charles Foulds, Bora Lim, Matthew Ellis, Bing Zhang, Eric Chang. CDK4/6 inhibition is a potential vulnerability in NF1-depleted ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS01-08.