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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

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.

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.

Cancer testis antigens are ideal for tumor immunotherapy due to their testis-restricted expression. We previously showed that an immunotherapeutic vaccine targeting the germ cell-specific transcription factor BORIS (CTCFL) was highly effective in treating aggressive breast cancer in the 4T1 mouse model. Here, we further tested the therapeutic efficacy of BORIS in a rat 13762 breast cancer model. We generated a recombinant VEE-VRP (Venezuelan Equine Encephalitis-derived replicon particle) vector-expressing modified rat BORIS lacking a DNA-binding domain (VRP-mBORIS). Rats were inoculated with the 13762 cells, immunized with VRP-mBORIS 48 h later, and then, subsequently, boosted at 10-day intervals. The Kaplan–Meier method was used for survival analysis. Cured rats were re-challenged with the same 13762 cells. We demonstrated that BORIS was expressed in a small population of the 13762 cells, called cancer stem cells. Treatment of rats with VRP-BORIS suppressed tumor growth leading to its complete disappearance in up to 50% of the rats and significantly improved their survival. This improvement was associated with the induction of BORIS-specific cellular immune responses measured by T-helper cell proliferation and INFγ secretion. The re-challenging of cured rats with the same 13762 cells indicated that the immune response prevented tumor growth. Thus, a therapeutic vaccine against rat BORIS showed high efficacy in treating the rat 13762 carcinoma. These data suggest that targeting BORIS can lead to the elimination of mammary tumors and cure animals even though BORIS expression is detected only in cancer stem cells.

427 Background: Gastric and esophageal cancers (GECs) together account for a significant global burden in terms of new diagnoses and deaths. Over the last several years, the utility of ctDNA has ranged from estimating tumor burden and characterizing the genomic landscape of tumor biology and response to therapy in the metastatic setting to detection of minimal residual disease (MRD) and cancer surveillance in the locally advanced setting. We have previously studied the role of a commercial ctDNA assay for MRD detection and surveillance in locally advanced GECs. Herein, we present our experience with the same ctDNA assay in advanced stage settings for GECs. Methods: In this retrospective analysis of real-world data obtained from commercial circulating tumor DNA (ctDNA) testing, 53 patients with recurrent/ metastatic esophageal cancer were analyzed. A total of 216 plasma samples were collected between 10/29/2008 and 08/23/2022. The patients were divided into 3 cohorts: Cohort A (N=30) included patients with stage II/III disease who had confirmed clinical recurrence (R). Cohort B (N=25) included recurrent and Stage IV patients who achieved a state of no evidence of disease (NED) on imaging. Cohort C (N=5) included recurrent and Stage IV patients who transiently achieved NED on imaging. A personalized, tumor-informed multiplex PCR-based next-generation sequencing assay (Signatera) was used to quantify ctDNA either postoperatively, on adjuvant or palliative therapy, or during active surveillance. Results: Of 53 patients, 30 patients with recurrent stage II/III esophageal cancer had ctDNA status available postoperatively within 150 days of confirmed clinical recurrence. Cohort A: Among these cases, 25 patients were ctDNA-positive ahead of clinical recurrence (sensitivity 83.3%) with a median of 31 days (range: 1-147 days). Cohort B: Next, we explored the correlation between ctDNA status and imaging, wherein, 96% (24/25) of patients showed a significant correlation between ctDNA status (positive or negative) and disease status by imaging (Fisher exact test p=0.0001). Of the 23 patients, 17 patients were ctDNA-negative and showed NED on imaging (negative predictive value of 100%; 17/17). Cohort C: 100% of patients (N=6) who demonstrated recurrent disease on imaging after NED were ctDNA-positive prior to imaging (positive predictive value of 100%). Conclusions: Our data demonstrate the utility of ctDNA in accurately predicting disease progression and support the potential use of ctDNA to inform treatment decisions or prompt early radiographic imaging. ctDNA may ultimately supersede traditional radiographic surveillance with the advantage of being minimally invasive and cost-effective in monitoring patients during/post-treatment.