Malignant glioma is incurable. Using a mouse genetic mosaic system to generate sporadic Trp53,Nf1-null OPCs, we previously identified oligodendrocyte precursor cell (OPC) as a cell-of-origin of glioma. Here, we report that pre-malignant Trp53,Nf1-null OPCs outcompete wildtype counterparts during their expansion. Blocking competition by mutating/strengthening wildtype OPCs impeded both pre-malignant progression and malignant expansion of glioma. "In-tissue" phosphoproteomic profiling revealed an enrichment of phosphopeptides related to RNA splicing and protein translation at the peak of cell competition, suggesting that competitiveness may stem from unique protein species. Among candidates was mTORC1, whose pharmacological inhibition or genetic disruption resulted in a loss of competitiveness in our mouse model. Finally, analysis of patient biopsies and interrogating the role of individual gliomagenic mutations in OPC competition supported its relevance in human gliomas. Together, these findings identified the driving role of competitive interactions among OPCs in gliomagenesis, and suggest unconventional therapeutic strategies to target this process.

Purpose The purpose of this study was to determine the safety, feasibility, and immunologic responses of treating grade 4 astrocytomas with multiple infusions of anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) armed T cells (EGFR BATs) in combination with radiation and chemotherapy. Methods This phase I study used a 3 + 3 dose escalation design to test the safety and feasibility of intravenously infused EGFR BATs in combination with radiation and temozolomide (TMZ) in patients with newly diagnosed grade 4 astrocytomas (AG4). After finding the feasible dose, an expansion cohort with unmethylated O 6 -methylguanine-DNA methyltransferase (MGMT) tumors received weekly EGFR BATs without TMZ. Results The highest feasible dose was 80 × 10 9 EGFR BATs without dose-limiting toxicities (DLTs) in seven patients. We could not escalate the dose because of the limited T-cell expansion. There were no DLTs in the additional cohort of three patients with unmethylated MGMT tumors who received eight weekly infusions of EGFR BATs without TMZ. EGFR BATs infusions induced increases in glioma specific anti-tumor cytotoxicity by peripheral blood mononuclear cells ( p < 0.03) and NK cell activity ( p < 0.002) ex vivo, and increased serum concentrations of IFN-γ ( p < 0.03), IL-2 ( p < 0.007), and GM-CSF ( p < 0.009). Conclusion Targeting AG4 with EGFR BATs at the maximum feasible dose of 80 × 10 9 , with or without TMZ was safe and induced significant anti-tumor-specific immune responses. These results support further clinical trials to examine the efficacy of this adoptive cell therapy in patients with MGMT-unmethylated GBM. ClinicalTrials.gov Identifier : NCT03344250

Hallmark MSigDB enrichments for differentially expressed transcripts between control and mutant bulk samples at 150 dpi (Sheets 1, 2, 7, and 8), 12 dpi (Sheets 3, 4, 9, and 10), and 90 dpi (Sheets 5, 6, 11, and 12). Increased (up, Sheets 1, 3, 5, 7, 9, and 11) and decreased (down, Sheets 2, 4, 6, 8, 10, 12) transcripts were analyzed separately without any fold-change cutoff (Sheets 1-6) or with a two-fold cutoff (fc2.0, Sheets 7-12).

Supplementary Figures S1-S15, Supplementary Table S1, and captions for Supplementary Files S1-S3.

PDF file - 247K, Transfection efficiency of U87 cells infected with lentiviruses encoding anti-miR-148, mCherry, and hygromycin resistance gene.

Relative transcript abundance changes (log2 fold change between mutant and control) in bulk samples at 150 dpi (Sheet 1), 12 dpi (Sheet 2), and 90 dpi (Sheet 3). Comparisons are separately broken down according to sex at each dpi time point (Sheets 4-9) .

PDF file - 23K, miR-148a inhibits MIG6 and BIM mRNA expression.

PDF file - 25K, MIG6 mediates the effects of miR-148a on cell proliferation.

PDF file - 20K, Transfection efficiency of U87 cells infected with lentiviruses encoding anti-miR-148, mCherry, and hygromycin resistance gene.

PDF file - 30K, miR-148a levels after transfection with pre-miR-148a.

Candidate male- and female-specific regulatory heterogeneities in mutant cells at 12 dpi (Sheet 1) and 90 dpi (Sheet 2). Shared candidates are shown as RHEGs for each day.

Abstract Cancer evolves from premalignant clones that adopt unusual cell states to achieve transformation. We previously pinpointed the oligodendrocyte precursor cell (OPC) as a cell of origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection, determined global gene-expression changes by bulk RNA sequencing, and compared with cell-state fluctuations at the single-cell level by stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1-Trp53 deletion, bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, and stochastic profiling revealed a spectrum of stem-progenitor (Axl, Aldh1a1), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, mutant OPCs that strongly expressed key effectors of nonsense-mediated decay (Upf3b) and homology-dependent DNA repair (Rad51c, Slx1b, Ercc4) were identified along with DNA-damage markers, suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease (Mus81) and Rin1, a Ras-Raf antagonist and possible counterbalance to Nf1 loss, which was microdeleted or downregulated in gliomas at 150 days. These hidden cell-state variations uncover replication stress as a potential bottleneck that must be resolved for glioma initiation. Significance: Profiling premalignant cell states in a mouse model of glioma uncovers regulatory heterogeneity in glioma cells-of-origin and defines a state of replication stress that precedes tumor initiation. See related articles by Singh and colleagues, p. 1840 and Schaff and colleagues, p. 1853

Cancer evolves from premalignant clones that accumulate mutations and adopt unusual cell states to achieve transformation. Previously, we pinpointed the oligodendrocyte precursor cell (OPC) as a cell-of-origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1–Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection and determined gene-expression changes by bulk RNA sequencing and a fluctuation analysis, called stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1–Trp53 deletion, while bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, stochastic profiling revealed a spectrum of stem-progenitor ( Axl, Aldh1a1 ), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected very few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, we identified mutant OPCs that strongly expressed key effectors of nonsense-mediated decay ( Upf3b ) and homology-dependent DNA repair ( Rad51c, Slx1b, Ercc4 ) along with DNA-damage markers suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease ( Mus81 ) and Rin1, a Ras–Raf antagonist and possible counterbalance to Nf1 loss. At 150 days, Rin1 was microdeleted in some gliomas and downregulated in all others. Replication stress may pose a considerable bottleneck that must be resolved for gliomas to initiate. Statement of significance In situ stochastic profiling of heterogeneous cell states in a mouse model of glioma uncovers regulatory confusion in a glioma cell-of-origin and defines a state of replication stress that precedes tumor initiation.

When brain metastasis develops, the prognosis of cancer is dismal. Insights into the biology of the primary cancer and the brain metastasis are necessary to inform more effective and targeted treatments. To study the role of microRNAs in brain metastasis, we performed differential expression profiling of 12 primary tumors and their paired brain metastases using smRNAseq (the 12 primary tumors included three non-small cell carcinomas, three melanomas, three endometrial carcinomas, one breast carcinoma, one thyroid carcinoma and one renal-cell carcinoma). To start, we identified microRNAs that were either highly upregulated or downregulated in the brain metastasis samples as compared to the paired primary tumors. After confirmation with real-time quantitative PCR, we further investigated the top microRNAs from both groups through functional assays performed in cell lines generated from primary melanoma, melanoma lymph node metastasis, and melanoma brain metastasis. From this top-down, patient sample to model cell-line approach we identified two microRNAs that are potentially important regulators in the development of brain metastasis. Characterization of their targets and their interactions may offer a therapeutic opportunity to improve the prognosis of patients with brain metastasis.

Single-cell transcriptomic methods classify new and existing cell types very effectively, but alternative approaches are needed to quantify the individual regulatory states of cells in their native tissue context. We combined the tissue preservation and single-cell resolution of laser capture with an improved preamplification procedure enabling RNA sequencing of 10 microdissected cells. This in situ 10-cell RNA sequencing (10cRNA-seq) can exploit fluorescent reporters of cell type in genetically engineered mice and is compatible with freshly cryoembedded clinical biopsies from patients. Through recombinant RNA spike-ins, we estimate dropout-free technical reliability as low as ~250 copies and a 50% detection sensitivity of ~45 copies per 10-cell reaction. By using small pools of microdissected cells, 10cRNA-seq improves technical per-cell reliability and sensitivity beyond existing approaches for single-cell RNA sequencing (scRNA-seq). Detection of low-abundance transcripts by 10cRNA-seq is comparable to random 10-cell groups of scRNA-seq data, suggesting no loss of gene recovery when cells are isolated in situ. Combined with existing approaches to deconvolve small pools of cells, 10cRNA-seq offers a reliable, unbiased, and sensitive way to measure cell-state heterogeneity in tissues and tumors.

Overcoming the highly immunosuppressive glioblastoma (GBM) microenvironment is critical to successful immunotherapy. In a recently-launched phase I trial we are examining the safety and feasibility of a novel adoptive cell immunotherapy platform, activated T cells (ATC) with anti-CD3 X anti-EGFR bispecific antibody (EGFR BATs), for patients with newly-diagnosed GBM. Autologous T cells obtained before radiation and chemotherapy are expanded and activated, becoming cytotoxic T lymphocytes. We postulate that these activated T cells armed with bispecific antibody will enhance the response to radiation/temozolomide and potentially increase trafficking to the GBM microenvironment. In a 3 + 3 design dose-escalation trial, we will determine the maximum tolerated dose (MTD) of three dose tiers of EGFR BATs (80, 120 and 160 billion cells divided in eight infusions) in combination with radiation/temozolomide in patients with newly-diagnosed GBM. Patients undergo leukapheresis prior to initiating radiation/temozolomide. Patients receive EGFR BATs on days 14 and 21 after finishing concurrent RT/ temozolomide and on day 21 of six cycles of adjuvant temozolomide. We are measuring peripheral blood immune response before and after immunotherapy and correlating the results with patient characteristics and clinical outcomes. The first four patients have been treated at the first dose tier (80 billion cells) without any dose-limiting toxicity. Updated safety and feasibility results on the patients in the first two dose tiers will be presented, as well as immune monitoring studies, imaging studies, and clinical outcomes.

Malignant glioma is one of the deadliest types of cancer. Understanding how the cell of origin progressively evolves toward malignancy in greater detail could provide mechanistic insights and lead to novel concepts for tumor prevention and therapy. Previously we have identified oligodendrocyte precursor cell (OPC) as the cell of origin for glioma following the concurrent deletion of p53 and NF1 using a mouse genetic mosaic system that can reveal mutant cells prior to malignancy. In the current study, we set out to deconstruct the gliomagenic process in two aspects. First, we determined how the individual loss of p53 or NF1 contributes to aberrant behaviors of OPCs. Second, we determined how signaling aberrations in OPCs progressively change from pre-malignant to transformed stages. We found that while the deletion of NF1 leads to mutant OPC expansion through increased proliferation and decreased differentiation, the deletion of p53 impairs OPC senescence. Signaling analysis showed that, while PI3K and MEK pathways go through stepwise over-activation, mTOR signaling remains at the basal level in pre-transforming mutant OPCs but is abruptly up-regulated in tumor OPCs. Finally, inhibiting mTOR via pharmacological or genetic methods, led to a significant blockade of gliomagenesis but had little impact on pre-transforming mutant OPCs, suggesting that mTOR is necessary for final transformation but not early progression. In summary, our findings show that deconstructing the tumorigenic process reveals specific aberrations caused by individual gene mutations and altered signaling events at precise timing during tumor progression, which may shed light on tumor-prevention strategies.

Glioma is a devastating disease because low-grade tumors inevitably progress into incurable high-grade GBMs. Among all probable explanations, such progressive nature could be attributed to the innate properties of its cell-of-origin. An ideal approach to identify the cell-of-origin for glioma is to analyze growth advantages of mutant cells in each brain cell types prior to malignancy. Our lab models glioma using a mouse genetic system called Mosaic Analysis with Double Markers (MADM), which generates GFP-labeled TSG-null cells and RFP-labeled sibling wildtype cells. By design, increased ratio of green-to-red cell numbers (G/R ratio) serves as the indication of tumor initiation long before malignancy. Using MADM, we found that after introducing p53/Nf1 mutations into neural stem cells (NSCs), significant aberrant expansion only occurred in oligodendrocyte precursor cells (OPCs, G/R ratio > 100), but not in any other NSC-derived lineages or NSCs themselves. Furthermore, introducing p53/Nf1 mutations directly into OPCs consistently led to gliomagenesis, further suggesting OPCs as the cell-of-origin in this model [Liu 2011 Cell]. Surprisingly, a close examination revealed a lack of overall OPC density increase in mutant brains in comparison to WT brains despite of the large G/R ratio, suggesting that green mutant OPCs actively out-compete WT OPCs to overtake the brain. Intrinsic competitive nature of OPCs could explain the inevitable progression of low-grade glioma since cells with “advantageous” mutations would kill other OPCs to make space for their own expansion. Under the same notion, traditional cytotoxic treatment could backfire by providing selective pressure that enriches resistant tumor cells. To look for alternative methods, we demonstrated that blocking the competitiveness of mutant OPCs with genetic methods completely prevented gliomagenesis in the mouse model. Currently, we are using cell culture-based assays and in vivo drug testing to identify potential anti-competition drugs that could be translated into clinical settings.

In this study, we uncovered microRNA-148a (miR-148a) as a novel prognostic and oncogenic microRNA in glioblastoma (GBM). MiR-148a expression was significantly higher in human GBM tissues, cell lines and stem cells (GSCs) as compared to normal human brain and astrocytes. High miR-148a levels were a risk indicator for GBM patient survival. MiR-148a increased GBM cell and GSC growth, survival, migration, and invasion as well as GSC neurosphere formation. We identified the EGFR regulator, MIG6, and the apoptosis regulator, BIM as two direct targets of miR-148a and showed with rescue experiments that they mediate the oncogenic effects of miR-148a. Importantly, by inhibiting MIG6 expression, miR-148a reduced EGFR trafficking to the Rab7-expressing late endosome/lysosomal compartments. This coincided with a reduction of EGFR degradation and induction of EGFR expression and activation. Lastly, inhibition of miR-148a expression led to a strong inhibition of GSC and GBM xenograft growth in vivo. These findings represent the first comprehensive analysis of the role of miR-148a in GBM. They show that miR-148a expression levels are predictive of patient survival and that this microRNA enhances malignancy by inhibiting apoptosis and indirectly activating EGFR. These results also suggest that miR-148a is a potential target for GBM therapy. Citation Format: Jungeun Kim, Ying Zhang, Michael Skalski, Josie Hayes, Benjamin Kefas, David Schiff, Benjamin Purow, Sarah Parsons, Sean Lawler, Roger Abounader. microRNA-148a is a prognostic oncomiR that targets MIG6 and BIM to regulate EGFR and apoptosis in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5222. doi:10.1158/1538-7445.AM2014-5222