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.

Leptomeningeal metastases are the primary source of morbidity and mortality for pediatric medulloblastoma patients. Due to limited surgical sampling of metastases in patients, little is understood of the mechanisms of metastasis. Here, we identify biologically distinct quiescent small metastases (designated as micrometastases) and mitotically active larger metastases (macrometastases). Macrometastases are more metabolically active than micrometastases and contain higher levels of lipids, particularly cholesterol. Macrometastases secrete CXCL12, which attracts lipid-laden macrophages into the tumor. Lipid-laden macrophages upregulate the cholesterol transporter ABCG1, promoting the efflux of free cholesterol, which is then taken up by tumor cells via the HDL receptor SCARB1. CXCL12-driven macrophage recruitment and exogenous cholesterol are sufficient and necessary to drive progression of medulloblastoma leptomeningeal metastases in vivo. High fat diets drive metastatic progression in vivo. Dietary or pharmacological interventions targeting the CXCL12-SCARB1-cholesterol axis represent therapeutic strategies to either prevent or treat medulloblastoma leptomeningeal metastases.

Characterizing tumor-specific neoantigen peptides, derived from genomic or transcriptomic aberrations and presented to the immune system, is critical for immuno-oncology studies. To this end, the modular iPepGen immunopeptidogenomics pipeline provides these functions: (1) Neoantigen prediction and protein database generation from genomic or transcriptomic sequencing data; (2) Peptide identification (3) Verification from immunopeptidomic mass spectral data; (4) Neoantigen classification and visualization; (5) Candidate prioritization for further study. Easy access via a publicly available, scalable cloud-based gateway coupled with online, interactive training materials streamlines the adoption by cancer researchers who require immunopeptidogenomic analysis tools but lack advanced computational expertise and resources.

Neurofibromatosis type I (NF1) is a common cancer predisposition syndrome caused by heterozygous loss-of-function mutations in the tumor-suppressor gene NF1. Individuals with NF1 develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell (SC) lineage after somatic loss of the wild-type NF1 allele, some of which progress further to malignant peripheral nerve sheath tumors (MPNST). There is only one FDA-approved targeted therapy for symptomatic plexiform neurofibromas and none approved for MPNSTs. The genetic basis of NF1 syndrome makes associated tumors ideal for using synthetic drug sensitivity approaches to uncover therapeutic vulnerabilities. We developed a drug discovery pipeline to identify therapeutics for NF1-related tumors using isogeneic pairs of NF1-proficient and NF1-deficient immortalized human SCs. We utilized these in a large-scale high-throughput screen for drugs that preferentially kill NF1-deficient cells, through which we identified 23 compounds capable of killing NF1-deficient SCs with selectivity. Multiple hits from this screen clustered into classes defined by the method of action. Four clinically interesting drugs from these classes were tested in vivo using both a genetically engineered mouse model of high-grade PNSTs and human MPNST xenografts. All drugs tested showed single-agent efficacy in these models as well as significant synergy when used in combination with the MEK inhibitor selumetinib. This high-throughput screen platform yielded novel therapeutically relevant compounds for the treatment of NF1-associated tumors and can serve as a tool to rapidly evaluate new compounds and combinations in the future.

Based on transcriptional expression, most studies divide glioblastoma roughly into proneural (PDGFRA), mesenchymal (NF1) and classical (EGFR) subtypes. We created genetically engineered, spontaneous glioblastoma-like tumors in mice by Sleeping Beauty (SB) transposable element. These genetically induced mouse models of different glioblastoma subtypes showed increased survival in the NRAS oncogene-driven mesenchymal-like subtype (median survival: 70.5 days) compared with the PDGFB-driven proneural-like subtype (median survival: 32 days). To investigate the difference in overall survival, the migration of glioblastoma cells and immune cell content was examined. The tumor cells in mesenchymal subtype of glioblastoma-like tumors were more infiltrative and migrated faster. The primary glioblastoma cells from mesenchymal-like mouse models and human patient-derived xenograft (PDX) lines with mesenchymal characteristics both showed significantly higher random motility coefficient (p<0.001) and cell area (p<0.001) compared with proneural-like mouse models and human patient-derived xenograft (PDX) lines (Shamsan et. al, 2022, BioRxiv). Based on these observations, we hypothesized that cytotoxic T lymphocytes (CTLs) contribute to the differential survival in mesenchymal and proneural subtypes of glioblastoma-like mice tumor models, and the difference can be computationally modeled. Endogenous CTL migration profiles were collected and the survival of adaptive immune system deficient mice with glioblastoma-like tumors of different subtypes is ongoing. It is expected that adaptive immune system deficient mice with mesenchymal glioblastoma-like tumors live shorter than wildtype mice. The Brownian Dynamics Tumor Simulator (BDTS) is a three-dimensional computation model for glioblastoma that incorporates a wide range of parameters involved in tumor growth. This computer-based simulator consists of tumor cells and T cells, in which both cell types migrate, proliferate, and undergo apoptosis. The migration profiles of cells under the microscope can help parameterize the simulator and make it more resemble in vivo tumor development. The BDTS successfully modeled tumor growth and predicted anti-migratory therapy responses in glioblastoma.

SUMMARY Glioblastoma remains a deadly cancer driven by invasion of tumor cells into the brain. Transcriptomic analyses have revealed distinct molecular subtypes, but mechanistic differences that explain clinical differences are not clear. Here, we show that, as predicted by the motor-clutch model for cell migration, mesenchymal glioma cells are more spread, generate larger traction forces, and migrate faster in brain tissue compared to proneural cells. Despite their fast migration and comparable proliferation rate in vitro, mice with mesenchymal tumors live longer than mice with proneural tumors, which was correlated with an immune response in the mesenchymal mice that included T cell-mediated killing of cancer cells, similar to human tumors. Thus, mesenchymal tumors have aggressive migration, but are relatively 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.

Background/Objectives: NRAS mutations are found in approximately 10% of patients with acute myeloid leukemia (AML), with nearly half of those occurring at codon 12, but little is known about how differing G12 mutants affect cancer cell activity. Methods: A novel bioinformatic technique, differential expression and pathway ranking (DEAPR), was used to identify the most prominent changes in terms of both individual genes and associated pathways when comparing AML THP-1 cells containing an NRASG12D mutation with B11 cells, which are THP-1-derived cells with the NRASG12D allele removed and a dox-inducible NRASG12V allele introduced. Results: In total, 1456 differentially expressed (DE) protein-coding genes were uniquely associated to the NRASG12D mutation, while 585 DE protein-coding genes were specific to the NRASG12V mutation. The innate immune system pathway was prominent in both mutant-specific lists, even though the genes involved were not in both lists. Furthermore, the two calprotectin genes (S100A8 and S100A9), also associated with innate immunity, were upregulated in the NRASG12D mutant and downregulated in the NRASG12V mutant. Conclusions: This study, using the DEAPR strategy, clearly demonstrates the dramatic changes associated with two seemingly similar NRAS mutations, suggesting the deployment of different treatment strategies based on the type of NRAS mutation present.

RAD18 is a conserved eukaryotic E3 ubiquitin ligase that promotes genome stability through multiple pathways. One of these is gap-filling DNA synthesis at active replication forks and in post-replicative DNA. RAD18 also regulates homologous recombination (HR) repair of DNA breaks; however, the current literature describing the contribution of RAD18 to HR in mammalian systems has not reached a consensus. To investigate this, we examined three independent RAD18-null human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e., HCT116 mutants were hyper-recombinogenic (hyper-rec). Interestingly, these phenotypes were linked to RAD18’s role in PCNA K164 ubiquitination, as HCT116 PCNAK164R/+ mutants were also hyper-rec, consistent with previous studies in rad18−/− and pcnaK164R avian DT40 cells. Importantly, the knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. We propose that the hierarchy of post-replicative repair and HR, intrinsic to each cell type, dictates whether RAD18 is required for suppression of hyper-recombination and that this function is linked to PCNA K164 ubiquitination.

The use of CRISPR to knockdown or knockout genes is a powerful tool for understanding the specific role of a gene in disease development. However, it can cause many unanticipated changes to the transcriptome that are not detected by DNA amplification and Sanger sequencing of the target site. Various RNA-sequencing techniques can be used to identify these changes and effectively gauge the full impact of the CRISPR knockout, thereby providing a means of selecting appropriate clones for further experimentation. Background/Objectives: RNA-seq data from 4 CRISPR knockout experiments were analyzed and techniques developed to both confirm the success of the CRISPR modifications and identify potential issues. Methods: A broad-based analysis of RNA-sequencing data identified many CRISPR-based changes not identified by PCR amplification of DNA around the CRISPR target site. These changes included an inter-chromosomal fusion event, exon skipping, chromosomal truncation, and the unintentional transcriptional modification and amplification of a neighboring gene. Conclusions: The inadvertent modifications identified by the evaluation of 4 CRISPR experiments highlight the value of using RNA-seq to identify transcriptional changes to cells altered by CRISPR, many of which cannot be recognized by evaluating DNA alone. Specific guidelines are presented for designing and analyzing CRISPR experiments using RNA-seq data.

Neurofibromatosis type 1 (NF1) is a common genetic disorder that predisposes individuals to a range of nervous system tumors, including central nervous system and malignant peripheral nerve sheath tumors (MPNST). MPNSTs are the leading cause of NF1-related mortality, with an 8-13% lifetime risk and 5-year survival rate below 50%. Complete surgical resection is the only curative option but is often infeasible due to tumor location. Effective treatment of MPNST presents a critical unmet clinical need, with no targeted therapies or cellular immunotherapies approved for this cancer. Using mass spectrometry-based surface proteomics, we identified common human MPNST-enriched antigens suitable for chimeric antigen receptor (CAR) development, including B7-H3 and PTK7, both of which have been clinically explored in other settings. We found gamma delta (γδ) T cells carrying a B7-H3 CAR were able to target and kill human MPNST cells in vitro. We have also developed a syngeneic immune-proficient mouse model of MPNST, allowing us to evaluate NF1 heterozygous T cells as a platform for autologous CAR-T development, and have assessed potential synergy between CAR-T therapy and MEK inhibition. Interestingly, several antigens, including PTK7, are also expressed in our novel NF1-driven mouse model, which mimics global NF1 heterozygosity and enables preclinical testing in an immunocompetent background. This platform supports evaluation of MPNST-specific CAR-T activity and directly addresses the feasibility of using NF1+/– T cells for autologous immunotherapy. We also explore whether MEK inhibition, currently used in NF1 patients with plexiform neurofibromas, enhances CAR-T efficacy. NF1+/– T cell function may be boosted through PI3K/AKT pathway feedback activation following MEK blockade. Additionally, we show MEK inhibitors upregulate MHC class I expression in MPNSTs, potentially improving immunogenicity via epitope spreading. Together, these studies aim to expand the applicability of cellular therapies to solid tumors, with translational relevance for pediatric and adult patients with NF1-associated cancers.

Osteosarcoma (OSA) is the most common primary bone tumor in children and adolescents, yet outcomes have remained largely unchanged for over 40 years. While chimeric antigen receptor (CAR) T cell therapy has shown success in blood cancers, it faces major limitations in solid tumors due to immune evasion, antigen loss, and immunosuppressive tumor microenvironments. Natural killer (NK) cells offer several advantages over T cells, including multiple killing mechanisms and lower risks of graft-versus-host disease, neurotoxicity, and cytokine release syndrome, making them promising candidates for off-the-shelf cell therapies. However, unmodified NK cells have shown limited efficacy in clinical settings due to poor engraftment, persistence, and tumor-mediated suppression. To overcome these barriers, we developed a cost-effective method to engineer CAR NK cells targeting CD70, a tumor antigen overexpressed in relapsed and metastatic OSA. We further enhanced these cells by incorporating soluble interleukin-15 (IL-15) and a dominant-negative TGF-β receptor, creating “armored” CAR NK cells. These engineered cells resist transforming growth factor β (TGF-β) suppression, secrete IL-15, and demonstrate improved cytotoxicity, persistence, and tumor homing in both in vitro and in vivo models. Our findings support CD70 CAR NK cells as a promising immunotherapeutic strategy for relapsed and metastatic OSA.

Patients with Neurofibromatosis Type 1 (NF1) are predisposed to central and peripheral nervous system tumors, notably malignant peripheral nerve sheath tumors (MPNSTs) and gliomas. In NF1, MPNSTs arise through a stepwise progression: NF1 loss in Schwann cells (SCs) initiates plexiform neurofibromas (PN), which can evolve into atypical neurofibromas (ANF), usually marked by CDKN2A/B deletions. Loss of PRC2 function, usually by loss of SUZ12, is a key driver of ~80% of MPNSTs, profoundly altering gene regulation. To model this progression, we used CRISPR editing in immortalized human SCs, iPSC-derived SCs, and primary mouse SCs from Nf1-/- sciatic nerve. These systems enabled preclinical drug testing and clinical translation. We developed an immunocompetent mouse ANF model via Nf1 and Cdkn2a/b deletion in primary Schwann cells. This model revealed Cdkn2a/b loss enhances immune evasion, alters the tumor metabolome, and confers sensitivity to Mdm2 and folate biosynthesis inhibitors in vitro and in vivo, which synergize with MEK inhibition. Synthetic drug sensitivity screens in NF1- and SUZ12-deficient human SCs revealed new candidates enhancing the effects of MEK inhibition in NF1-deficient SCs in vitro and in vivo and showed PRC2 loss sensitizes SCs to HDAC, AURK, and some DNMT inhibitors. Combining MEK and HDAC inhibitors (e.g., mirdametinib + vorinostat) led to durable tumor regression in vivo and supported our initiation of an early phase window of opportunity trial now open (NCT06693284). Two of our patient cases illustrate clinical potential: one with refractory, H3K27me3-negative MPNST had pain relief and reduced PET activity on selumetinib + vorinostat and remained alive more than two years after failure of all other therapies; another, an 11-year-old with NF1 and H3K27M-mutant spinal glioma, remained recurrence-free for over two years on the same combination. These integrated efforts advance several translational platforms to develop and implement targeted therapies across the NF1 tumor spectrum.

Myeloid-derived suppressor cells (MDSCs) contribute to an immunosuppressive tumor microenvironment that facilitates cancer progression, particularly in patients with high-grade gliomas. We previously reported that increased MDSCs during chemoradiotherapy (chemoRT) is associated with poor outcomes. This study investigates the effect of radiotherapy on immune checkpoint expression on MDSC subsets in patients with recurrent high-grade glioma receiving a second course of RT. Peripheral blood samples were collected on the first and last day of RT. Peripheral blood mononuclear cells (PBMCs) were isolated and subsequently stained with immunofluorescent antibodies to identify and analyze MDSC subsets (HLA-DR, CD33, CD14, CD15) and expression of checkpoint molecules (TIM-3, TIGIT, PD-1, PD-L1, CTLA-4) by flow cytometry. Because MDSCs secrete TGF-β1 and it is a potent immune cell regulator, plasma TGF-β1 concentration was quantified using ELISA in both recurrent and naïve glioma patients. A Kolmogorov-Smirnov assessed normality while paired T-test was used to compare groups, with significance set at p<0.05. Five of 20 and 19 of 25 planned patients have enrolled in our IRB-approved PBMC and plasma protocols at the University of Minnesota Medical Center, respectively. Increased frequency of total MDSCs (12.8%;p< 0.05) and the monocytic(m)-MDSC subset (10.8%;p< 0.05) was measured. Within the total MDSC and mMDSC populations, increased PD-L1 (14.5%, 18.8%, respectively;p< 0.05)) TIM-3 (23.6%, 18.0%, respectively) expression was measured after reirradiation. Increased plasma TGF-β1 was measured in patients with recurrent glioma (+0.143 fold change) compared to patients with naïve glioma (-0.300 fold change) after RT (p<0.05). Preliminary data supports that radiation may negatively influence systemic MDSC-associated immunosuppressive signaling, particularly with recurrent disease. Further understanding of immunodynamic changes secondary to RT will inform personalized immunotherapy strategies for patients with recurrent high-grade glioma.