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Glioblastoma multiform (GBM) is the most common brain tumor characterized by a dismal prognosis. GBM cancer stem cells (gCSC) or tumor-initiating cells are the cell population within the tumor-driving therapy resistance and recurrence. While temozolomide (TMZ), an alkylating agent, constitutes the first-line chemotherapeutic significantly improving survival in GBM patients, resistance against this compound commonly leads to GBM recurrence and treatment failure. Although the roles of protein-coding transcripts, proteins and microRNA in gCSC, and therapy resistance have been comprehensively investigated, very little is known about the role of long noncoding RNAs (lncRNAs) in this context. Using nonoverlapping, independent RNA sequencing and gene expression profiling datasets, we reveal that TP73-AS1 constitutes a clinically relevant lncRNA in GBM. Specifically, we demonstrate significant overexpression of TP73-AS1 in primary GBM samples, which is particularly increased in the gCSC. More importantly, we demonstrate that TP73-AS1 comprises a prognostic biomarker in glioma and in GBM with high expression identifying patients with particularly poor prognosis. Using CRISPRi to downregulate our candidate lncRNA in gCSC, we demonstrate that TP73-AS1 promotes TMZ resistance in gCSC and is linked to regulation of the expression of metabolism- related genes and ALDH1A1, a protein known to be expressed in cancer stem cell markers and protects gCSC from TMZ treatment. Taken together, our results reveal that high TP73-AS1 predicts poor prognosis in primary GBM cohorts and that this lncRNA promotes tumor aggressiveness and TMZ resistance in gCSC.

Rhabdoid brain tumours, also called atypical teratoid rhabdoid tumours, are lethal childhood cancers with characteristic genetic alterations of SMARCB1/hSNF5. Lack of biological understanding of the substantial clinical heterogeneity of these tumours restricts therapeutic advances. We integrated genomic and clinicopathological analyses of a cohort of patients with atypical teratoid rhabdoid tumours to find out the molecular basis for clinical heterogeneity in these tumours. We obtained 259 rhabdoid tumours from 37 international institutions and assessed transcriptional profiles in 43 primary tumours and copy number profiles in 38 primary tumours to discover molecular subgroups of atypical teratoid rhabdoid tumours. We used gene and pathway enrichment analyses to discover group-specific molecular markers and did immunohistochemical analyses on 125 primary tumours to evaluate clinicopathological significance of molecular subgroup and ASCL1-NOTCH signalling. Transcriptional analyses identified two atypical teratoid rhabdoid tumour subgroups with differential enrichment of genetic pathways, and distinct clinicopathological and survival features. Expression of ASCL1, a regulator of NOTCH signalling, correlated with supratentorial location (p=0·004) and superior 5-year overall survival (35%, 95% CI 13–57, and 20%, 6–34, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·033) in 70 patients who received multimodal treatment. ASCL1 expression also correlated with superior 5-year overall survival (34%, 7–61, and 9%, 0–21, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·001) in 39 patients who received only chemotherapy without radiation. Cox hazard ratios for overall survival in patients with differential ASCL1 enrichment treated with chemotherapy with or without radiation were 2·02 (95% CI 1·04–3·85; p=0·038) and 3·98 (1·71–9·26; p=0·001). Integrated analyses of molecular subgroupings with clinical prognostic factors showed three distinct clinical risk groups of tumours with different therapeutic outcomes. An integration of clinical risk factors and tumour molecular groups can be used to identify patients who are likely to have improved long-term radiation-free survival and might help therapeutic stratification of patients with atypical teratoid rhabdoid tumours. C17 Research Network, Genome Canada, b.r.a.i.n.child, Mitchell Duckman, Tal Doron and Suri Boon foundations.

HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and β) in sustaining malignant cells’ growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90β isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.

Diffuse midline glioma H3K27M-altered (DMG) remains a fatal pediatric brain cancer driven by a global loss of histone H3K27 trimethylation. Radiotherapy comprises the most important treatment modality and significantly improves overall survival. Novel therapeutic strategies for DMG patients without or with radiotherapy are urgently needed. Here, we aimed to gain insights into potential radiation response modulators. To identify modulators of radiation response, we performed a high-throughput drug screening (HTS) in seven representative DMG cell lines using conventional chemotherapeutic drugs and phase I-IV drugs ( n  = 687), followed by irradiation with 0 or 2 × 4 Gray (Gy). The ataxia–telangiectasia and Rad3-related (ATR) inhibitor berzosertib emerged as a potent radiosensitizer. Its effects were validated in three DMG cell lines using short-term proliferation assays, long-term limiting dilution assays (LDA), 3D spheroid cultures, and the chorioallantoic membrane (CAM) assay in ovo . Across all three tested DMG cell line models, berzosertib enhanced the antineoplastic effects of clinically relevant radiation doses, significantly reducing proliferation and clonogenic survival, delaying spheroid growth, and suppressing tumor formation in ovo . These findings provide strong preclinical evidence that ATR inhibition increases the sensitivity of DMG cells to radiotherapy. They highlight a novel therapeutic vulnerability and support further exploration of ATR inhibitors in rational combination strategies to improve radiotherapy efficacy for this deadly disease.

Ferroptosis is a recently described type of regulated necrotic cell death whose induction has anti-cancer therapeutic potential, especially in hematological malignancies. However, efforts to uncover novel ferroptosis-inducing therapeutics have been largely unsuccessful. In the current investigation, we classified brightfield microscopy images of tumor cells undergoing defined modes of cell death using deep transfer learning (DTL). The trained DTL network was subsequently combined with high-throughput pharmacological screening approaches using automated live cell imaging to identify novel ferroptosis-inducing functions of the polo-like kinase inhibitor volasertib. Secondary validation showed that subsets of B-cell acute lymphoblastic leukemia (B-ALL) cell lines, namely 697, NALM6, HAL01, REH and primary patient B-ALL samples were sensitive to ferroptosis induction by volasertib. This was accompanied by an upregulation of ferroptosis-related genes post-volasertib treatment in cell lines and patient samples. Importantly, using several leukemia models, we determined that volasertib delayed tumor growth and induced ferroptosis in vivo. Taken together, we have applied DTL to automated live-cell imaging in pharmacological screening to identify novel ferroptosis-inducing functions of a clinically relevant anti-cancer therapeutic.

Background New therapies are urgently needed in melanoma, particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors. To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen and identified 5-Nonyloxytryptamine (5-NL), a serotonin agonist, as increasing the ability of T cells to target tumor cells. Methods The pharmacological screen utilized lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells and melanoma B16.F10 cells expressing the LCMV gp33 CTL epitope. In vivo tumor growth in C57BL/6 J and NSG mice, in vivo antibody depletion, flow cytometry, immunoblot, CRISPR/Cas9 knockout, histological and RNA-Seq analyses were used to decipher 5-NL’s immunomodulatory effects in vitro and in vivo. Results 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8 + T cells. 5-NL’s pro-immune effects were not directly consequential to T cells. Rather, 5-NL upregulated antigen presenting machinery in melanoma and other tumor cells in vitro and in vivo without increasing PD-L1 expression. Mechanistic studies indicated that 5-NL’s induced MHC-I expression was inhibited by pharmacologically preventing cAMP Response Element-Binding Protein (CREB) phosphorylation. Importantly, 5-NL combined with anti-PD1 therapy showed significant improvement when compared to single anti-PD-1 treatment. Conclusions This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors.

Medulloblastomas arise from undifferentiated precursor cells in the cerebellum and account for about 20% of all solid brain tumors during childhood; standard therapies include radiation and chemotherapy, which oftentimes come with severe impairment of the cognitive development of the young patients. Here, we show that the posttranscriptional regulator Y-box binding protein 1 (YBX1), a DNA- and RNA-binding protein, acts as an oncogene in medulloblastomas by regulating cellular survival and apoptosis. We observed different cellular responses upon YBX1 knockdown in several medulloblastoma cell lines, with significantly altered transcription and subsequent apoptosis rates. Mechanistically, PAR-CLIP for YBX1 and integration with RNA-Seq data uncovered direct posttranscriptional control of the heterochromatin-associated gene CBX5; upon YBX1 knockdown and subsequent CBX5 mRNA instability, heterochromatin-regulated genes involved in inflammatory response, apoptosis and death receptor signaling were de-repressed. Thus, YBX1 acts as an oncogene in medulloblastoma through indirect transcriptional regulation of inflammatory genes regulating apoptosis and represents a promising novel therapeutic target in this tumor entity.

Medulloblastoma (MB) is the most common malignant brain tumor in children that is subdivided into distinct MB groups characterized by specific genetic, epigenetic, and transcriptional profiles. In addition to single nucleotide variants, structural variants (SVs), including DNA deletions, duplications, focal gene amplifications, translocations and inversions, may contribute to MB pathogenesis. However, comprehensive profiling of SVs is challenging, as established cytogenetic methods such as karyotyping and fluorescence in situ hybridization (FISH) are limited by low resolution or restricted to detection of predefined SVs. Moreover, commonly used molecular diagnostic techniques like gene panel next-generation sequencing and microarray-based DNA copy number (CN) analysis may not reveal complex SVs. To more comprehensively characterize SVs in MB, we applied optical genome mapping (OGM) for genome-wide SV detection in 29 MB tissues and six MB cell lines. We found that OGM reliably detected genome-wide DNA CN changes in these samples, as validated by comparison to microarray-based DNA CN profiling. In addition, OGM allowed for the characterization of novel genomic alterations, including amplification and overexpression of the GPC5 gene on chr13q31.3 in a Group 3 MB tumor, as well as recurrent SVs affecting the NRXN1 gene on chr2p16.3 by deletion or translocation in subsets of MB Group 3 and 4 tumors. Taken together, our data indicate that OGM represents a promising approach for comprehensive profiling of SVs in pediatric brain tumors.

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC -overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB. MYC amplification is an independent prognostic factor for the most aggressive subgroup (Group 3) of pediatric medulloblastoma (G3 MB). Here, the authors highlight the role of the RNA-binding protein, Musashi-1 (MSI1) in G3 MB and identify MSI1-bound targets sharing MYC associated pathways.