Largaespada, David A.

Background Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. Methods We developed transgenic mice carrying D oxycycline (Dox)- i nducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8 + T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 + T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. Results Under chronic stimulation, SB -mutagenized CD8 + T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Conclusions Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

Abstract Glioblastoma (GBM) remains a deadly cancer driven in part by invasion of tumor cells into the brain. Transcriptomic analyses have identified distinct molecular subtypes, but mechanistic differences that account for clinical differences are not clear. In this study, we show that, as predicted by the motor-clutch model of cell migration, mesenchymal glioma cells are more spread, generate larger traction forces, and migrate faster in brain tissue compared with proneural cells. Despite their rapid migration and comparable proliferation rates in vitro, mice with mesenchymal tumors survive longer than those with proneural tumors. This improved survival correlated with an immune response in mesenchymal tumors, including T cell–mediated. Consistently, inducing mesenchymal tumors in immunodeficient mice resulted in shorter survival, supporting a protective immune role in mesenchymal tumors. Thus, mesenchymal tumors have aggressive migration but are immunologically “hot,” which suppresses net proliferation. These two features counteract each other and may explain the lack of a strong survival difference between subtypes clinically, while also opening up new opportunities for subtype-specific therapies. Significance: This study highlights new mechanical and immunologic insights into GBM molecular subtypes using an integrated modeling–genome engineering strategy, which can potentially facilitate GBM subtype-specific therapeutic strategies.

Adoptive cell therapy (ACT) has shown remarkable success in the treatment of some malignancies, particularly leukemia. However, there are multiple factors that limit the durability of ACT in solid tumors, including dose-limiting toxicities, the immunosuppressive tumor microenvironment, and T-cell exhaustion. As the manufacture and preparation of adoptive T-cell therapies allows time and adequate conditions for ex vivo T-cell engineering, forward genetic screens can identify novel genetic targets that could improve their effectiveness. CRISPR is a commonly used functional genomics tool that has been successfully used to both enhance our understanding of mechanisms of resistance and to discover potential genetic edits to improve ACT. A complementary approach, Sleeping Beauty transposon mutagenesis provides additional opportunities to identify novel genetic edits without being constrained by the annotated human genome. Here, we summarize forward genetic screens and their tools to uncover strategies to enhance ACT. Complementary approaches can be combined and improved on to identify translatable genetic editing strategies through studies that accurately recapitulate disease-specific challenges.

Abstract Background Malignant peripheral nerve sheath tumors (MPNSTs) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic add-back experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1-mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells (SCs) has not been defined. We hypothesized that PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs). Methods We engineered NF1-deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis, we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways. Results We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog (SHH) signaling in PRC2-deficient iHSCs. Functional studies confirm that Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines. Conclusions We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches.

Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer predisposition syndrome defined by germline deletion of one NF1 allele. Somatic loss of heterozygosity in the remaining NF1 allele in Schwann lineage cells gives rise to benign tumors known as plexiform neurofibromas. These tumors have a high risk of transforming into malignant peripheral nerve sheath tumors (MPNST) through the sequential deletion of CDKN2A/B followed by recurrent mutations in SUZ12 or EED, constituting the inactivation of polycomb repressor complex 2 (PRC2). PRC2 is a transcriptional repressor complex involved in the silencing of several development and differentiation genes. Loss of this complex leads to a depletion in trimethylation marks on Histone H3 Lysine 27 (H3K27me3) and the subsequent gain of acetylation marks on the same lysine residue (H3K27Ac). These molecular hallmarks are shared with diffuse intrinsic pontine gliomas (DIPG) harboring the H3K27M mutation which prevents effective deposition of H3K27me3, thus having similar molecular phenotypes to MPNST. However, the full range of epigenetic consequences of these events are currently not well characterized. Additionally, given that MPNSTs originate from cells of neural crest origin and DIPGs from cells of neural origin, it is not know to what degree epigenetic changes stemming from H3K27me3-loss are general across tumors or are context-dependent based on cell-of-origin. In order to contribute to this effort, the Largaespada lab has engineered Schwann cell lines to harbor deletions in NF1 and either SUZ12 or EED and assayed the H3K27ac landscape of these cells in addition to patient-derived MPNST cell lines. Utilizing the ranked ordering of super enhancers (ROSE) algorithm, we have identified common super enhancers within each MPNST cell line. These super enhancer elements are located nearby a variety of putative oncogenes responsible for the maintenance of cell identity. These elements were then compared to publicly available data from DIPG primary and patient-derived samples to understand common regulatory events driven by loss of H3K27me3 as well as molecular signatures present only in MPNST or in DIPG that may be a result of PRC2 or H3K27M alone. We further characterized these super enhancers by assessing the expression levels of their associated genes to understand their role in transcriptional activation. These efforts provide a roadmap of the super enhancer landscape of both MPNSTs and DIPGs to investigate novel mechanisms of H3K27me3-dependent tumorigenesis while also contributing to knowledge of potential effects of H3K27me3-loss that dependent on the cell-of-origin for each respective tumor. Citation Format: Christopher M. Stehn, Minu Bhunia, Kyle Williams, Alex T. Larsson, Ariel Caride, Jeong-Heon Lee, Tamas Ordog, David Largaespada. Characterizing the super enhancer landscape of H3K27me3 loss in MPNST and DIPG [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B009.

Abstract Glioblastoma remains a deadly cancer driven in part by invasion of tumor cells into the brain. Transcriptomic analyses have identified distinct molecular subtypes, but mechanistic differences that account for clinical differences are not clear. Here, we show that, as predicted by the motor-clutch model of cell migration, mesenchymal glioma cells are more spread, generate larger traction forces, and migrate faster in brain tissue compared to proneural cells. Despite their rapid migration and comparable proliferation rates in vitro, mice with mesenchymal tumors survive longer than those with proneural tumors. This improved survival correlated with an immune response in the mesenchymal tumors, including T cell-mediated. Consistently, inducing mesenchymal tumors in immunodeficient mice resulted in shorter survival supporting a protective immune role in mesenchymal tumors. Thus, mesenchymal tumors have aggressive migration, but are immunologically ‘hot’ which suppresses net proliferation. These two features counteract each other and may explain the lack of a strong survival difference between subtypes clinically, while also opening up new opportunities for subtype-specific therapies. Significant Statement This study highlights new mechanical and immunological insights into glioblastoma molecular subtypes using an integrated modeling-genome engineering strategy, which can potentially facilitate glioblastoma subtype-specific therapeutic strategies.

The SB transposon system has been used as an insertional mutagen to study tumor development. This novel system involves the mobilization of a transposon (e.g. T2/onc) by a tranposase source (e.g. Rosa26‐SB11) in transgenic mice. We serially sectioned FFPE brains from 154 mice and identified 15 infiltrating gliomas that morphologically resembled small cell astrocytomas, as well as one PNET. The gliomas involved cerebral hemispheres (n=8) or posterior fossa (n=7). The tumors demonstrated remarkable infiltration of underlying brain parenchyma with secondary structuring (i.e. subpial, perivascular and perineuronal aggregation); round to oval cells with irregular nuclear contours, scant cell processes and variable mitotic activity (median 4/10 hpf, range 2–9); and pseudopalisading necrosis (n=5). Endothelial proliferation and calcification were absent. Immunohistochemical stains for GFAP and S100 strongly labeled reactive astrocytes, but minimal immunoreactivity was also seen in occasional small tumor cells. In 14 of 15 mice the genotype was T2/onc/SB11 positive; three mice were p19 Arf +/− , one mouse was p19 Arf −/− and one was Blm −/− . The PNET filling the third ventricle was identified in a control mouse homozygous mutant for p19 Arf , but transgenic only for the T2/Onc concatomer. This tumor, in contrast to the gliomas, demonstrated uniform synaptophysin staining and did not label with GFAP or S100.These results demonstrate that the SB transposon system is a valid model to study gliomagenesis in mice.

Abstract In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p < 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.

Abstract Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. Significance: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

Abstract Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis–associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Cancer Res; 77(23); 6576–88. ©2017 AACR.