Glioblastoma is the most common malignant brain tumor in adults, and radiotherapy (RT) is the most effective postoperative treatment for patients with glioblastoma. Nevertheless, glioblastoma recurrence after treatment is nearly universal, and cell state plasticity and intratumor heterogeneity underlie glioblastoma evolution and resistance to treatment. Here we integrate in vitro genome-wide CRISPR interference screens and in vivo perturb-seq in preclinical models with single-nucleus and spatial transcriptomic sequencing of human tumors to identify therapeutic vulnerabilities that overcome glioblastoma resistance to RT. Gene regulatory network modeling identifies DNA-PKcs as a RT-sensitizing target in glioblastoma cells in vitro and in vivo. Small molecule inhibition of DNA-PKcs plus RT improves survival and reprograms tumor cell states and immune microenvironment composition compared to DNA-PKcs inhibition or RT monotherapy. Bioinformatic and imaging analyses of patient-matched glioblastoma samples before and after DNA-PKcs inhibition and RT show that combination therapy drives inflammatory gene expression programs in tumor cells that recruits pro-inflammatory myeloid cells to the tumor microenvironment. Using this framework to inform rational sequential therapy in preclinical models, we show that immunomodulation in response to genomic stress after DNA-PKcs inhibition and RT primes glioblastoma for response to cGAS/STING activation. These data show that DNA-PKcs modulates tumor cell states and immune microenvironment cell types to drive resistance to RT in glioblastoma, and that targeting DNA-PKcs sensitizes glioblastomas to RT and cGAS/STING activation.

Tumor suppressor NF1 is recurrently mutated in glioblastoma, leading to aberrant activation of Ras/rapidly accelerated fibrosarcoma (RAF)/MEK signaling. However, how tumor heterogeneity shapes the molecular landscape and efficacy of targeted therapies remains unclear. Here, we combined bulk and single-cell genomics of human somatic NF1-mutant, isocitrate dehydrogenase (IDH) wild-type glioblastomas with functional studies in cell lines and mouse intracranial tumor models to identify mechanisms of tumor heterogeneity underlying clinical outcome and MEK inhibitor response. Targeted DNA sequencing identified CDKN2A/B homozygous deletion as a poor prognostic marker in somatic NF1-mutant, but not NF1 wild-type, glioblastoma. Single-nucleus RNA sequencing of human patient NF1-mutant glioblastomas demonstrated that mesenchymal-like (MES-like) tumor cells were enriched for MEK activation signatures. Single-cell RNA-sequencing of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib identified distinct responses among tumor subpopulations. MEK inhibition selectively depleted MES-like cells, and selumetinib-resistant MES-like cells upregulated Ras signaling while resistant non-MES cells expressed markers of glial differentiation. Finally, genome-wide CRISPR interference screens validated Ras/RAF/MEK signaling as a key mediator of selumetinib response. Repression of the RAF regulator SHOC2 sensitized glioblastomas to selumetinib in vitro and in vivo, suggesting a synergistic treatment strategy. Taken together, these results highlighted the heterogeneity of NF1-mutant glioblastomas and informed future combination therapies.

The NF1 tumor suppressor gene is recurrently mutated in human cancers and is associated with the neurofibromatosis type 1 (NF-1) cancer predisposition syndrome. NF1 encodes neurofibromin, a Ras guanosine triphosphate (GTPase) activating protein that negatively regulates Ras signaling. NF1 mutation accordingly leads to Ras misactivation and downstream activation of RAF/MEK/ERK signaling, leading to the approval of the MEK inhibitor selumetinib for NF-1 associated peripheral nervous system (PNS) tumors. However, how NF1 loss modifies response to selumetinib and the utility of targeting additional upstream inputs or downstream outputs of Ras these tumors remain unclear. Here, we perform RNA-sequencing, phosphoproteomic, pharmacologic, and proximal proteomic analysis across a panel of CRISPR interference immortalized peripheral nerve (iPN) cells to systematically dissect the function of neurofibromin loss. Small guide NF1 (sgNF1) repression is sufficient to increase Ras GTP levels and alter gene expression to promote cell proliferation and dedifferentiation, with sgNF1 iPNs showing decreased sensitivity to selumetinib due to altered feedback regulation to Ras/RAF/MEK/ERK. Upstream small guide PTPN11 (sgPTPN11) repression leads to the inverse gene expression signature, decreasing cell proliferation and promoting differentiation, and sgPTPN11 iPNs are more sensitive to selumetinib. However, upstream sonof sevenless 1 inhibition shows limited efficacy in iPNs due to compensation by SOS2. Finally, proximal proteomics reveals Kirsten rat sarcoma virus (KRAS), but not Harvey rat sarcoma virus (HRAS) or neuroblastoma Ras viral oncogene homolog (NRAS), associates with neurofibromin in iPN cells, and pan-KRAS inhibition is sufficient to block ERK activation and CDK1/2 activation in NF1 mutant cells, suggesting blocking KRAS may be a therapeutic approach for NF1 mutant PNS tumors.

Abstract Alterations in cis-regulatory elements, such as hypomethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter and activating mutations within the telomerase reverse transcriptase (TERT) promoter, are pervasive drivers of glioblastoma (GBM) tumorigenesis that are challenging to target. CRISPRoff is an engineered CRISPR/Cas9 system that can potently and heritably silence cis-regulatory elements through DNA methylation. Here, we develop an mRNA-based CRISPRoff platform to target the MGMT and TERT promoters in GBM, and we establish proof-of-principal in vivo targeting through lipid nanoparticles (LNP). Epigenetic editing in GBM cells and primary GBM organoids was performed using either electroporation or LNP encapsulation of CRISPRoff mRNA and sgRNAs. Target gene silencing and functional phenotypes were assessed by bisulfite sequencing, RT-qPCR, RNA-seq, western blot, cell viability/apoptosis, and telomere restriction fragment assays. In vivo efficacy was demonstrated using intracranial GBM xenografts, first through transplantation of cells with CRISPRoff delivered ex vivo, then with direct delivery of LNPs using convection enhanced delivery (CED) coupled with immunofluorescence/ immunohistochemistry. CRISPRoff targeting of MGMT promoter resulted in >99% reduction in MGMT expression in MGMT unmethylated primary GBM organoids, inducing up to 150-fold sensitization to temozolomide. Temozolomide sensitization was retained after intracrania transplantation, and silencing was durable in clonally isolated GBM cells continuously passaged for over 8 months after transient delivery of CRISPRoff. CRISPRoff targeting of the TERT promoter in primary GBM cultures harboring the G228A TERT promoter mutation silenced TERT expression by up to 99%, overcoming constitutive activation of TERT, and induced telomere shortening sufficient for complete replicative senescence. CED of mRNAs encapsulated by LNPs based on cKK-E12 or Lipid A9 resulted in tumor-selective uptake of mRNA transcripts, establishing a foundation for direct delivery of CRISPRoff in vivo bypassing the blood-brain-barrier. In summary, we establish a flexible and durable epigenetic editing system against multiple regulatory elements driving GBM, leveraging LNPs with potential for in vivo application.

Abstract PURPOSE Malignant peripheral nerve sheath tumors (MPNSTs) are the most common cause of death in neurofibromatosis type 1 (NF-1) and are resistant to radiation therapy (RT), yet the mechanisms underlying RT response are poorly understood. Here, we elucidate mechanisms of RT response in NF-1 associated peripheral nerve sheath tumors through genome-wide CRISPRi screens, genomic analysis of mouse models, and molecular analysis of patient-derived tumor specimens. METHODS Patient derived NF1 mutant plexiform neurofibroma (pNF) cells (NF9511b, NF95.6), and MPNST cells (ST88-14; JH2-002) were used to measure RT responses by cell counts and flow cytometry. Genome-wide CRISPRi screens were used to identify functional modifiers of RT response in ST88-14 and JH2-002 MPNST cells. Nf1-/-; Tp53-/- mouse MPNSTs were subcutaneously implanted in immunocompetent C57/B6 mice, irradiated (2Gyx5), and dissociated for single-cell RNA sequencing (scRNA-seq) using 10x Genomics. Human MPNST tumor specimens (n=45) were analyzed with targeted DNA mutation sequencing and methylation arrays for cell type deconvolution. RESULTS Radiation single-dose response curves revealed MPNST cells (IC50 6.85Gy) were radioresistant compared to pNF cells (IC50 3.13Gy). Genome wide CRISPRi screens in ST88-14 and JH2-002 MPNST cells converged on cell cycle, DNA repair, and immunomodulatory gene sets mediating RT sensitivity. Irradiation of subcutaneous mouse Nf1-/-; Tp53-/- MPNSTs significantly decreased tumor growth (p=0.01, t-test). scRNA-seq of 32,763 cells from 4 irradiated and 3 unirradiated mouse Nf1-/-; Tp53-/- MPNSTs identified 8 tumor clusters and 7 non-tumor clusters. Irradiation demonstrated a pro-inflammatory effect with increased T-cell presence (5.8% versus 3.3%, p=0.04, t-test) and greater activation of immunostimulatory genes (Cxcl10, Stat1, Irf7) in T cells. In human MPNSTs, CDKN2A/B deletion (p=0.04, log-rank test) and decreased immune cell composition (p=0.03, log-rank test) were associated with significantly worse overall survival in response to RT. CONCLUSION MPNST RT responses may depend on immunomodulatory mechanisms that could be leveraged to improve clinical RT response.

Schwann cell tumors are the most common cancers of the peripheral nervous system and can arise in patients with neurofibromatosis type-1 (NF-1) or neurofibromatosis type-2 (NF-2). Functional interactions between NF1 and NF2 and broader mechanisms underlying malignant transformation of the Schwann lineage are unclear. Here we integrate bulk and single-cell genomics, biochemistry, and pharmacology across human samples, cell lines, and mouse allografts to identify cellular de-differentiation mechanisms driving malignant transformation and treatment resistance. We find DNA methylation groups of Schwann cell tumors can be distinguished by differentiation programs that correlate with response to the MEK inhibitor selumetinib. Functional genomic screening in NF1-mutant tumor cells reveals NF2 loss and PAK activation underlie selumetinib resistance, and we find that concurrent MEK and PAK inhibition is effective in vivo. These data support a de-differentiation paradigm underlying malignant transformation and treatment resistance of Schwann cell tumors and elucidate a functional link between NF1 and NF2.

Abstract BACKGROUND Glioblastoma (GBM) is a heterogenous disease comprised of malignant cell states and an immunosuppressive microenvironment that underlie radiotherapy (RT) resistance. Here we integrate genome wide and single cell in vivo functional genomic screens of preclinical models with single nuclei and spatial transcriptomics of human tumors to identify therapeutic vulnerabilities driving GBM radioresistance. MATERIAL AND METHODS Perturb-seq of intracranial GBM models was performed using human (GBM43) or mouse (GL261, SB28) GBM cells expressing CRISPR interference (CRISPRi) machinery, with sgRNA libraries delivered to intracranial tumors using convection enhanced delivery. Genome wide dual sgRNA CRISPRi screens were performed in GBM cells (GBM43, LN18, GL261, SB28) ± RT. Functional genomic analyses across preclinical models were integrated with single nuclei and spatial transcriptomics from human GBMs. Validation of genetic phenotypes was performed using the DNA-PK inhibitor peposertib to treat GBM cells and orthotopic mouse models (SB28, GBM43, GBM6), which were analyzed using IF and single cell RNA sequencing. RESULTS Genome wide CRISPRi screens identified 114 radiation sensitizing targets. In vivo perturb-seq of radiation sensitizing targets reprogrammed 25 gene modules that spanned homeostatic, metabolic, and inflammatory pathways and nominated DNA-PK as a pleotropic radiation sensitizer that regulates interactions between malignant cells and the myeloid microenvironment. Genetic and pharmacologic inhibition of DNA-PK revealed radiotherapy dependent reprograming of cell states through cell intrinsic mechanisms (e.g. cell cycle, proteasome) and cell extrinsic pathways (e.g. TNFα, IFN). Peposertib sensitized GBMs to radiotherapy and extended survival in mice harboring intracranial xenografts. DNA-PK inhibition induced cGAS/STING and IFN signaling in malignant cells (n = 58,161) that were associated with pro-inflammatory polarization of infiltrating myeloid cells (n = 22,067). These alterations were synergistic with RT but were not induced by RT alone. Spatial transcriptomics of 8 matched primary-recurrent GBMs (20,793 transcriptomes) from patients treated with RT and peposertib, and single nuclei transcriptomics from 100 GBMs (76 matched primary-recurrent tumors, 4 after treatment with RT and peposertib, 356,997 nuclei) demonstrated malignant cell differentiation and enrichment of pro-inflammatory macrophages in the tumor microenvironment after inhibition of DNA-PK. CONCLUSION Integration of genome wide CRISPRi screens and in vivo perturb-seq in preclinical models with single nuclei and spatial transcriptomic analyses of human tumors enables discovery and mechanistic validation of therapeutic targets in GBM. These results reveal DNA-PK as a target for radiation sensitization of tumor and myeloid microenvironment cells in GBM.

Abstract Poly-aneuploid cancer cells (PACCs) or as also known as “giant cells”, are a treatment resistant stem cell-like cancer phenotype. PACCs have been identified in numerous cancer types, and their presence appears to play a significant role in chemoresistance and poor patient outcomes. PACCs employ a multitude of polyploidization programs to enter this transient state in response to stress. After removal of stress, PACCs give rise to non-PACC progeny through potential mechanisms of neosis or depolyploidization that maintain chemotherapeutic resistance. Leiomyosarcoma (LMS) is a difficult to treat sarcoma that grows in smooth muscle that often develops chemoresistance. Here we report the first description of PACCs in LMS cell culture, LMS xenografts, and LMS patient tumor microarrays. LMS cells treated (doxorubicin, docetaxel, and gemcitabine) in vitro and in vivo lead to the development of mononucleated and multinucleated polypoid cells as the predominant phenotype via polyploidization. Polyploidization mechanisms identified included endocycling, endomitosis, and cell-cell fusion. In vitro, LMS PACCs had a significant increase in size and DNA content as well as an increase in resistance to chemotherapy as compared to parents. LMS PACCs also repopulated the culture with non-PACC LMS progeny cells. The PACC progeny cells maintained a significantly decreased (p<0.0001) response to chemotherapy compared to parent cells. PACCs were also identified in clinical samples from patients with LMS. Twenty-nine percent of samples (N=289) contained abnormally large tumor cells and fifty-eight percent contained abnormal nuclei. A positive correlation was observed in patient tumor samples between PACCs in metastatic tumors and recurrence (p=0.0277) and survival (p=0.0009) than those patient tumors without PACCs. The high rate of functional loss of TP53 in LMS (>90%) may contribute to the ability of PACCs to form abnormal, multiple nuclei. We therefore transfected LMS PACCs to express TP53 and observed an increased apoptotic response. Additionally, combining functional TP53 expression with low doses of doxorubicin increases the apoptotic response of LMS cells, potentially by targeting PACCs. LMS PACCs may contribute to the lack of response in patients with LMS. Identifying therapeutic approaches that prevent or target LMS PACCs, including TP53 replacement, may improve response to chemotherapy and improve outcomes for patients. Citation Format: Ryan Kennington, Gareth Mitchell, Emily Payne, Niraja Bhachech, Jonathan A. Fletcher, Ting Liu, Matt van de Rijn, Sarah Amend, Kenneth J. Pienta, Joshua D. Schiffman, Lisa M. Abegglen. Leiomyosarcoma poly-aneuploid cancer cells form in response to chemotherapy, contribute to chemoresistance, and lead to higher rates of metastatic recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1991.

Abstract The advent of single cell techniques has advanced our understanding of glioblastoma (GBM) evolution and cell lineage relationships. However, study of mutational co-occurrence and clonal phylogeny has been hampered by limitations in single cell genotyping. Here, we perform semi-automated, rapid single nuclear dissociation followed by targeted single nucleus DNA sequencing (snDNA-seq) of eleven retrospectively identified somatic NF1 mutant IDH-wildtype glioblastomas. Bulk DNA sequencing was performed as part of routine clinical care using a CLIA-certified targeted sequencing panel. For snDNA-seq, tissue cores with greater than 30% tumor were dissociated on a S2 genomics S100 Singulator followed by snDNA-seq using a targeted 361 amplicon panel (Tapestri, Mission Bio, USA) to sequence for recurrently observed single nucleotide substitutions and copy number alterations. A mean of 3,661 cells were recovered per sample with a mean of 96 reads per cell per amplicon and 87% mean panel uniformity. SnDNA-seq validated point mutations and copy number alterations observed in bulk DNA-sequencing and revealed novel alterations and subclonal copy number alterations not detected on bulk analysis. Within individual samples, snDNA-seq based phylogenetic reconstruction identified mutually exclusive patterns of mutation between NF1 and PI3K signaling alterations, suggesting these events may occur in distinct tumor subclones. With regard to copy number alterations (CNAs), snDNA-seq demonstrated intra-tumoral heterogeneity for chromosome 9p loss, chromosome 7 gain, and chromosome 10 monosomy. Subclonal CNA clones not detected by bulk sequencing were identified in 5 samples. Analysis of a matched primary-recurrent tumor pair revealed expansion of a specific mutational clone (loss of 9p, 10p, gains of 1p, 7, 15, and 19q) at recurrence. SnDNA-seq recapitulated bulk DNA sequencing alterations and identified distinct patterns of mutational co-occurrence, CNAs, and tumor evolution over time. Reconstructing GBM phylogeny at single cell resolution has potential translational implications for understanding tumor evolution and treatment resistance.

NF1 is recurrently mutated in glioblastoma yet the molecular landscape and efficacy of targeted therapies remain unclear. Here, we combine bulk and single cell genomics of human somatic NF1 mutant, IDH-wildtype glioblastomas with functional genomic analysis of cell lines and mouse intracranial tumor models to identify molecular subgroups within NF1 mutant glioblastomas and mechanisms underlying MEK inhibitor response. Targeted DNA sequencing showed homozygous deletion of the cell cycle regulator CDKN2A/B is a poor prognostic marker in somatic NF1 mutant, but not NF1 wildtype, tumors. DNA methylation array profiling revealed three epigenetic groups highlighted by distinct clinical features, co-mutation patterns, and reference methylation classifier identities. Genome-wide CRISPRi screens in glioblastoma cells revealed cell cycle regulators are conserved mediators of cell growth while response to the MEK inhibitor selumetinib converges on Ras/RAF/MEK pathway activation. Repression of the RAF regulator SHOC2 sensitizes glioblastomas to selumetinib in vitro and in vivo in mouse intracranial glioblastoma models. Single cell RNA-sequencing of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib reveals distinct responses between mesenchymal-like (MES-like) and non MES-like subpopulations suggesting non-MES like cells are intrinsically resistant to MEK inhibition. Finally, single nuclear RNA-sequencing (snRNA-seq) of human NF1 mutant, CDKN2A/B deleted glioblastomas reveals MES-like tumor cells are associated with selumetinib sensitivity signatures while non MES-like cells exhibit increased cell cycle progression and lack selumetinib sensitivity, further supporting the notion that MEK inhibition specifically targets MES-like tumor cell subpopulations. Taken together, our data underscores the heterogeneity between and within somatic NF1 mutant glioblastomas and delineates mechanisms of MEK inhibitor response across distinct tumor subpopulations, guiding the development of future therapeutic strategies that may synergize with MEK inhibition for NF1 mutant tumors. The tumor suppressor NF1 is mutated in 15% of glioblastomas,[1][1]–[3][2] the most common malignant brain tumor with poor outcomes and few effective treatments.[4][3] NF1 is a GTPase activating protein (GAP) that negatively regulates Ras, and thus, NF1 loss leads to induction of Ras/RAF/MEK/ERK signaling, driving tumorigenesis and comprising a targetable molecular cascade.[5][4],[6][5] Genomic analysis of glioblastoma demonstrates NF1 mutation is associated with a mesenchymal-like (MES-like) transcriptomic tumor cell subpopulation and altered tumor microenvironment.[7][6],[8][7] More broadly, DNA methylation analysis reveals multiple epigenetic subgroups with overlapping relationships to transcriptomic subtype and DNA alterations, underscoring the complex relationship between genetic drivers and molecular signatures.[9][8] While the updated 2021 Central Nervous System WHO tumor classification incorporates an ever increasing amount of molecular criteria for diffuse astrocytic tumors,[10][9] the existence and clinical significance of molecular subgroups within somatic NF1 mutant, IDH-wildtype glioblastomas based on genetic co-mutations, epigenetic profile, or transcriptomic signatures remain unclear. Preclinical data support the utility of MEK inhibition in NF1 mutant gliomas,[11][10],[12][11] and the MEK inhibitor selumetinib is FDA approved for tumors arising in patients with syndromic neurofibromatosis type 1 (NF-1) harboring a germline NF1 mutation.[13][12],[14][13] In NF-1 associated gliomas, MEK inhibition demonstrates efficacy in a limited case series,[15][14] and combined BRAF/MEK inhibition shows efficacy in BRAF p.V600E mutant gliomas,[16][15] further supporting the translational potential of Ras/Raf/MEK/ERK blockade within genetically defined glioma subtypes. Nevertheless, treatment resistance to molecular monotherapy remains a challenge,[17][16]–[20][17] and the mechanisms underlying MEK inhibitor resistance in NF1 mutant glioma are unknown. Here, we integrate targeted DNA sequencing, DNA methylation profiling, and single nuclear RNA-sequencing (snRNA-seq) of human patient somatic NF1 mutant, IDH-wildtype glioblastomas with single cell RNA-sequencing (scRNA-seq), genome-wide clustered regularly interspaced short palindromic repeats interference (CRISPRi) screens, and pharmacologic studies in cell lines and mouse intracranial glioblastoma models to define molecular subgroups and functional mediators of MEK inhibitor response. Targeted DNA sequencing of NF1 mutant, IDH-wildtype glioblastomas (n=186 tumors) revealed CDKN2A/B deletion was associated with poor outcomes in NF1 mutant, but not NF1 wildtype, glioblastomas. DNA methylation profiling (n=129 tumors) demonstrated three epigenetic subgroups with distinct clinical features, co- mutation patterns across cell cycle genes, and reference methylation classifier identities. Genome-wide CRISPRi screens in mouse SB28 and human GBM43 glioblastoma cells identified a conserved cell cycle gene network mediating cell growth, consistent with the clinical importance of additional hits affecting the cell cycle in human somatic NF1 mutant glioblastomas. Moreover, genome-wide mediators of selumetinib response converged upon two Ras pathway effectors mediating selumetinib sensitivity: BRAF and SHOC2. SHOC2 repression in glioblastoma cells significantly improved selumetinib response both in vitro and in intracranial allografts in vivo . Single cell RNA-sequencing (scRNA-seq) of mouse intracranial glioblastomas treated with the MEK inhibitor selumetinib revealed MES-like tumor cells correlated with CDKN2A retention and the CRISPRi screen selumetinib sensitivity signature, with selumetinib resistant cells displaying Ras pathway induction. In contrast, non-MES like tumor cells were CDKN2A deficient and lacked expression of the CRISPRi screen selumetinib sensitivity signature, with selumetinib resistant cells inducing a glial de-differentiation program. Finally, snRNA-seq of NF1 mutant, CDKN2A/B deleted, IDH-wildtype glioblastomas (n=9) showed non MES-like tumor cells exhibit increased cell cycle progression and were not associated with the CRISPRi screen selumetinib sensitivity signature. MES-like tumor cells within newly diagnosed, but not recurrent, tumors retained expression of the CRISPRi screen selumetinib sensitivity signature, suggesting resistance can arise both between and within specific transcriptomic glioblastoma cell tumor cell subpopulations. Taken together, our data identifies clinically important subgroups of NF1 mutant, IDH-wildtype glioblastomas and supports a model in which heterogeneity between tumors and within tumor cell subpopulations underlies MEK inhibitor response, supporting the need for additional synergistic therapeutic approaches beyond maximal Ras pathway blockade for NF1 mutant glioblastomas. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-5 [5]: #ref-6 [6]: #ref-7 [7]: #ref-8 [8]: #ref-9 [9]: #ref-10 [10]: #ref-11 [11]: #ref-12 [12]: #ref-13 [13]: #ref-14 [14]: #ref-15 [15]: #ref-16 [16]: #ref-17 [17]: #ref-20

Abstract The tumor suppressor NF1 is frequently mutated in IDH-wildtype glioblastoma. However, the molecular subgroups and clinically relevant biomarkers within somatic NF1 mutant, IDH-wildtype glioblastomas remain incompletely understood. Here, we combine methylation arrays and targeted DNA sequencing to identify epigenetic subgroups and clinical biomarkers within non-syndromic NF1 mutant IDH-wildtype glioblastoma. We identified 186 patients treated at the University of California San Francisco between 2016-2024 with newly diagnosed IDH-wildtype glioblastoma containing a somatic pathogenic NF1 mutation on a CLIA certified targeted DNA sequencing assay. Illumina EPIC DNA methylation arrays (n=129 patients) were processed using the minfi package and Heidelberg random forest classifier (v12.8). Downstream analyses including hierarchical clustering and survival analyses were performed in R. DNA sequencing identified 12 recurrently altered genes in greater than 10% of cases across cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic modifiers (ATRX, SETD2), and growth factor signaling (EGFR, PDGFRA, PTPN11). DNA methylation analysis revealed three epigenetic subgroups. Group 1 showed lower median patient age and significantly increased TP53 co-mutation rate. Group 2 contained a significantly higher proportion of male patients and was significantly enriched for RB1 and TERT promoter alterations, with a majority of tumors classified as GBM-MES using the Heidelberg classifier. Group 3 showed a significantly higher proportion of female patients and was significantly enriched for homozygous CDKN2A/B deletion. Homozygous CDKN2A/B loss was associated with significantly worse overall survival across the entire non-syndromic NF1 mutant glioblastoma cohort but not in a propensity score matched NF1 wildtype glioblastoma cohort. DNA methylation profiling revealed three epigenetic subgroups characterized by distinct clinical features, co-mutation patterns, and reference DKFZ methylation classifier identities. CDKN2A/B loss may be a negative prognostic biomarker specifically in NF1 mutant glioblastomas; future multi-institutional studies are required to validate these findings and their therapeutic implications in a larger, representative cohort.

We integrate targeted epigenome editing with unbiased genome-wide approaches to build a novel discovery and therapeutic platform in glioblastoma, a framework that is well suited for targeting diseases with known or suspected epigenetic vulnerabilities.

Our data indicate additional genetic hits beyond NF1 loss may be required for RT-associated malignant transformation of pNFs and radioresistance in MPNSTs. Analysis of transcriptomic responses to RT suggests that upregulated growth factor signaling and TGFβ-associated immunosuppression are distinct features of MPNST. Future work will focus on CRISPRi screens to unbiasedly nominate functional modifiers of RT response in NF1/CDKN2AB deficient tumors, which may be broadly useful in cancer.

Abstract The tumor suppressor NF1 is recurrently mutated in sporadic IDH-wildtype glioblastoma and activates Ras/Raf/MEK signaling, motivating the use of targeted MEK inhibition. Here, we combine multiplatform molecular analysis of NF1 mutant glioblastoma human resection specimens with functional genomic CRISPRi screens to the MEK inhibitor selumetinib. METHODS: We identified 123 consecutive patients treated at the University of California San Francisco between 2016-2022 with NF1 mutant, IDH-wildtype glioblastoma. DNA methylation data was processed using the minfi package, and hierarchical clustering, principal component analysis (PCA), and survival analysis was performed in R. Human NF1 mutant GBM43 and mouse NRAS mutant SB28 GBM cells expressing CRISPRi machinery were used for genome wide selumetinib-resistance screens. Immunoblotting was performed to validate CRISPRi screen hits and selumetinib responses in cell lines. RESULTS: DNA sequencing of human glioblastomas identified 12 recurrent alterations in greater than 10% of cases including cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic regulators (ATRX, SETD2), and Ras signaling (EGFR, PDGFRA, PTPN11). Methylation data (n=55 tumors) identified three groups marked by differences in MGMT methylation (p=0.04, chi-square test) and CDKN2A/B loss (p=0.01, chi-square test). CRISPRi screens in GBM43 and SB28 cells identified driver phenotypes in cell cycle regulators (TP53, CDKN2A, CDK4), PI3K signaling (PTEN, MTOR), and Ras signaling (RASA2, PTPN11) consistent with co-occurring mutations in the human NF1 mutant glioblastoma cohort. In contrast, mediators of selumetinib response converged on regulators of Ras/Raf/MEK signaling including the Ras effector SHOC2. Biochemical analysis validated SHOC2 repression is sufficient for selumetinib sensitization. CONCLUSIONS: Using a combination of human tumor genomic analysis, functional genetic screens, and biochemical validation, we identify three epigenetic groups of NF1 mutant glioblastoma with distinct co-mutation patterns and validate compensatory Ras/Raf/MEK activation as a conserved mechanism of selumetinib resistance, suggesting combination molecular therapy as a rational therapeutic combination.

Glioblastomas are comprised of dynamic malignant cell states and microenvironment cell types. Mechanistic and functional studies of glioblastoma resistance to therapy have been limited by a lack of in vivo approaches for multiplexed interrogation of single cells. Here we use genome-wide and targeted CRISPR interference (CRISPRi) to identify therapeutic vulnerabilities in glioblastomas in vivo by coupling convection enhanced delivery (CED) with functional genomics and single-cell transcriptomics (perturb-seq). Human (GBM43, LN18) or mouse (GL261, SB28) glioblastomas expressing CRISPRi machinery were established intracranially in mice, and unmodified mouse glioblastomas were established intracranially in immunocompetent mice expressing CRISPRi machinery in the tumor microenvironment. Perturb-seq gene prioritization was performed using genome-wide CRISPRi screens ± radiotherapy in cell cultures. Dual-sgRNA Perturb-seq lentivirus libraries were transduced in vivo using CED, and single-cell RNA sequencing with sgRNA capture was performed ± radiotherapy. RESULTS: were validated using RNA sequencing of 254,288 single nuclei from 43 pairs of primary and recurrent patient-matched human glioblastomas. Perturb-seq ± radiotherapy of 98 genes underlying glioblastoma radiotherapy responses was performed across 414,092 single cells in vivo. Genetic perturbations reprogrammed tumor and microenvironment cell states in response to radiotherapy, and high dimensional manifolds revealed genetic dependencies in metabolic and DNA damage response pathways. The DNA-dependent protein kinase (DNA-PK) component PRKDC was identified as a driver of glioblastoma radiotherapy resistance through regulation of malignant cell-intrinsic growth and stress pathways, and regulation of malignant cell-extrinsic interferon pathways that controlled cell-cell interactions with the tumor microenvironment. PRKDC inhibition with nedisertib sensitized glioblastomas to radiotherapy and extended survival in vivo, and single-cell RNA sequencing revealed hyperactivation of cell stress and cytokine signatures with combination treatment. In summary, we report in vivo perturb-seq as a platform for simultaneous discovery and functional interrogation of therapeutic vulnerabilities in glioblastoma, and show DNA-PK inhibition sensitizes glioblastomas to radiotherapy in vivo.

Abstract Alkylating chemotherapies exhibit survival benefit for patients with glioblastoma (GBM). CRISPRoff is a programmable epigenetic memory writer that stably and heritably silences genes through DNA methylation. Epigenetic silencing of MGMT via promoter methylation predicts response to chemotherapy and is prognostic for overall survival. Here, we performed epigenome editing using CRISPRoff to stably silence MGMT through induced promoter methylation as a therapeutically tractable approach for potentiating GBM to chemotherapy. We then used genome-wide CRISPR interference (CRISPRi) screens to broadly define novel chemosensitizing targets of GBM cells. Epigenome editing was performed through electroporation of CRISPRoff mRNA and sgRNAs into MGMT hypomethylated GBM cell lines (LN18 and T98G) and primary GBM cells. Whole genome bisulfite sequencing, RT-qPCR, and western blot assessed gene silencing. Cell viability assays measured drug sensitivity. GBM xenografts with CRISPRoff-induced silencing of MGMT were intracranially transplanted in mice and treated with temozolomide. Genome-wide functional genomics screens were performed using CRISPR interference. CRISPRoff against MGMT reduced MGMT mRNA and protein levels by 99.97%, generating up to 150-fold sensitization to temozolomide and 9-fold sensitization to lomustine in GBM cells. Silencing remained stable and heritable in clonally isolated GBM cells at 145 days after transient delivery of CRISPRoff, and gene repression and drug sensitization were equivalent to Cas9-mediated homozygous deletion of MGMT in vitro and in vivo. CRISPRoff of MGMT in intracranial xenografts exhibited significant reduction in tumor growth with temozolomide treatment compared to temozolomide treatment with control CRISPRoff (p = 0.0047). CRISPRi screens validated MGMT and defined 266 and 238 additional modifiers of temozolomide and lomustine response, respectively, including the ATR (BRCA2), DNA repair (REV1), cell cycle (PSMD13), and Fanconi anemia pathways (FANCI, FANCD2). In summary, we integrate targeted epigenome editing with unbiased genome-wide approaches to establish a novel discovery and therapeutic platform against glioblastoma.

Approximately 20 TP53 retrogenes exist in the African and Asian elephant genomes ( Loxodonta Africana, Elephas Maximus ) in addition to a conserved TP53 gene that encodes a full-length protein. Elephant TP53-RETROGENE 9 ( TP53-R9 ) encodes a p53 protein (p53-R9) that is truncated in the middle of the canonical DNA binding domain. This C-terminally truncated p53 retrogene protein lacks the nuclear localization signals and oligomerization domain of its full-length counterpart. When expressed in human osteosarcoma cells (U2OS), p53-R9 binds to Tid1, the chaperone protein responsible for mitochondrial translocation of human p53 in response to cellular stress. Tid1 expression is required for p53-R9-induced apoptosis. At the mitochondria, p53-R9 binds to the pro-apoptotic BCL-2 family member Bax, which leads to caspase activation, cytochrome c release, and cell death. Our data show, for the first time, that expression of this truncated elephant p53 retrogene protein induces apoptosis in human cancer cells. Understanding the molecular mechanism by which the additional elephant TP53 retrogenes function may provide evolutionary insight that can be utilized for the development of therapeutics to treat human cancers.

We establish in vivo Perturb-seq in orthotopic GBM models as a platform for simultaneous functional genomic discovery and characterization of therapeutic targets, revealing an underappreciated role for Prkdc in GBM tumors in vivo that is targetable using small molecules. These tools are adaptable for a wide range of disease models and treatment modalities.