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Purpose: Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed. Experimental Design: We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2–high and OLIG2–low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models. Results: We found that MYC-associated MB can be stratified into OLIG2–high and OLIG2–low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2–low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2–high tumors were resistant to radiation and consistently developed recurrence. In OLIG2–high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2– tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells. Conclusions: Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.

Fig. S1, S2, S3 and Table S1.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MYC-driven MBs, commonly found in the group 3 MB, are aggressive and metastatic with the worst prognosis. Modeling MYC-driven MB is the foundation of therapeutic development. Here, we applied a synthetic mRNA-driven strategy to generate neuronal precursors from human-induced pluripotent stem cells (iPSCs). These neuronal precursors were transformed by the MYC oncogene combined with p53 loss of function to establish an MYC-driven MB model recapitulating the histologic and transcriptomic hallmarks of group 3 MB. We further show that the marine compound Frondoside A (FA) effectively inhibits this MYC-driven MB model without affecting isogenic neuronal precursors with undetectable MYC expression. Consistent results from a panel of MB models support that MYC levels are positively correlated with FA's antitumor potency. Next, we show that FA suppresses MYC expression and its downstream gene targets in MB cells, suggesting a potential mechanism underlying FA's inhibitory effects on MYC-driven cancers. In orthotopic xenografts of MYC-driven MB, intratumoral FA administration potently induces cytotoxicity in tumor xenografts, significantly extends the survival of tumor-bearing animals, and enhances the recruitment of microglia/macrophages and cytotoxic T lymphocytes to tumors. Moreover, we show that MYC levels also predict FA potency in glioblastoma and non–small cell lung cancer cells. Taken together, this study provides an efficient human iPSC-based strategy for personalizable cancer modeling, widely applicable to mechanistic studies (e.g., genetic predisposition to cancer) and drug discovery. Our preclinical results justify the clinical translation of FA in treating MYC-driven MB and other human cancers.

Purpose:Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed.Experimental Design:We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2–high and OLIG2–low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models.Results:We found that MYC-associated MB can be stratified into OLIG2–high and OLIG2–low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2–low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2–high tumors were resistant to radiation and consistently developed recurrence. In OLIG2–high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2– tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells.Conclusions:Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.

Supplementary Table from OLIG2 Is a Determinant for the Relapse of MYC-Amplified Medulloblastoma

Supplementary Data from OLIG2 Is a Determinant for the Relapse of MYC-Amplified Medulloblastoma

A subset of group 3 medulloblastoma frequently harbors amplification or overexpression of MYC lacking additional focal aberrations, yet it remains unclear whether MYC overexpression alone can induce tumorigenesis and which cells give rise to these tumors. Here, we showed that astrocyte progenitors in the early postnatal cerebellum were susceptible to transformation by MYC. The resulting tumors specifically resembled human group 3 medulloblastoma based on histology and gene-expression profiling. Gene-expression analysis of MYC-driven medulloblastoma cells revealed altered glucose metabolic pathways with marked overexpression of lactate dehydrogenase A (LDHA). LDHA abundance correlated positively with MYC expression and was associated with poor prognosis in human group 3 medulloblastoma. Inhibition of LDHA significantly reduced growth of both mouse and human MYC-driven tumors but had little effect on normal cerebellar cells or SHH-associated medulloblastoma. By generating a new mouse model, we demonstrated for the first time that astrocyte progenitors can be transformed by MYC and serve as the cells of origin for group 3 medulloblastoma. Moreover, we identified LDHA as a novel, specific therapeutic target for this devastating disease.Significance:Insights from a new model identified LDHA as a novel target for group 3 medulloblastoma, paving the way for the development of effective therapies against this disease.

RNA-Seq analysis

Figure S1 shows MYC driven tumors generated from early postnatal cerebellar cells. Figure S2 shows IHC staining of tdt+ or tdt- MYC-driven tumors. Figure S3 shows gene expression of mouse MYC-driven tumors compared to that of human medulloblastomas. Figure S4 shows properties of Sox2+/Aldh1L1high, Sox2+/Aldh1L1low and Sox2+/Aldh1L1neg cells. Figure S5 shows properties of Sox2+/CD133+, Sox2+/CD133- or Sox2-/CD133+ cells. Figure S6 shows gene expression of SOX family members in tumors or normal cerebellar cells. Figure S7 shows that LDHA is a potential target for treating human MYC-amplified medulloblastoma.

Background: The causes of the rising incidence of early-onset colorectal cancer (EOCRC), defined as CRC in patients aged 60). Methods: The international cohort included 13,262 patients diagnosed with stages I-III colon or rectal cancer who had ctDNA testing using a personalized and tumor-informed multiplex PCR assay (Signatera™ 16-plex bespoke mPCR NGS assay), from which whole-exome sequencing (WES) on the surgically resected tumor was performed. Tumor mutational burden (TMB) and microsatellite instability (MSI) status were derived from WES analysis. The prevalence of gene-wide mutations, pathogenic gene variants, and mutations in known oncogenic pathways was compared between EOCRC and LOCRC groups, stratified by TMB and MSI status. Fisher’s exact test was used to test significance between the groups and p-values were adjusted using the FDR method for multiple test correction. Results: A total of 3,093 patients with EOCRC (70.8% colon, 27.4% rectal, 1.9% unknown) and 10,169 patients with LOCRC (79.9% colon, 18.3% rectal, 1.7% unknown) were included, where 9.0%/37.3%/53.7% were AJCC stages I, II, and III, respectively. Early-onset patients compared to late-onset patients had fewer cases of stage II CRC (30.7% vs. 39.3%, p Conclusion: Patients with LOCRC were more likely to have pathogenic gene variants and mutations in established pathways of CRC carcinogenesis. Tumors in EOCRC cases harbored unique genomic alterations that varied between the TMB-low/MSS, TMB-H/MSI-H, and TMB-H/MSS subpopulations. Citation Format: Eric M. Lander, Samuel Rivero-Hinojosa, Vasily N. Aushev, Jesús Izaguirre-Carbonell, Adham Jurdi, Minetta C. Liu, Cathy Eng. Genomic alterations associated with early-onset and late-onset colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6696.

Medulloblastoma is a common childhood brain tumor for which current therapies fail to cure high-risk subgroups, including metastatic and recurrent disease. Toxicity limits further intensification of conventional chemo-radiation therapy, and most survivors endure significant lifelong sequelae from treatment. There is an urgent need to develop new tools to combat this cancer without increasing the late effects burden. Neoantigens, which are exclusively expressed on tumor cells, are one of the main targets of an effective antitumor T-cell response. The ideal target antigen is abundantly expressed by tumor cells but not by normal tissues, in order to limit off-target effects. Tumors translate a host of highly novel transcripts that are the result of aberrations in tumor DNA and the unmasking of alternative or novel exons. We used a novel proteogenomic approach to identify tumor-restricted peptides and used them to select and expand T cells capable of mounting a tumor-specific cytotoxic immune response, with no off-target effects for patients with MB. Using RNA-seq and WGS data, we created personalized custom searchable databases containing predicted novel proteins from somatic mutations, novel isoforms, and fusion proteins from 56 medulloblastoma tumors. By searching this database with raw mass spectrometry data from the paired medulloblastoma tumor, we have identified tens of peptides arising from the translation of tumor-specific transcripts; these peptides are potential neoantigens. Our data indicate that in cases of tumors with low mutation rates, such as pediatric brain tumors, novel isoforms are the main source of neoantigens. In a pilot study, we have tested these peptides for their ability to select and expand polyclonal populations of T cells from the same patient whose tumor was the source of the peptides. The immunogenicity of each individual peptide was then determined. Flow cytometry cellular characterization reveals populations of both CD4+ and CD8+ cells with an activation profile marked by IFN-γ and TNF-α production. Immunosuppressive marker profiles have also been characterized for these cells. Using cytotoxicity assays, we demonstrated that tumor-specific T cells can eliminate neoantigen-bearing tumor cells. These findings demonstrate an initial proof of principle that proteogenomics can be used to identify immunogenic tumor specific peptides and lay the groundwork for a personalized, targeted T-cell therapy for children with high-risk medulloblastoma. Citation Format: Samuel Rivero-Hinojosa, Melanie Grant, Aswini Panigrahi, Huizhen Zhang, Veronika Caisova, Catherine Bollard, Brian Rood. Proteogenomic discovery of novel tumor proteins as neoantigens for personalized T-cell immunotherapy in pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A23.