Imaging and multi-omics datasets converge to define different neural progenitor origins for ATRT-SHH subgroups.

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.

AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Network-Based Integration of Multi-Omics Data Identifies the Determinants of miR-491-5p Effects.

The identification of miRNAs' targets and associated regulatory networks might allow the definition of new strategies using drugs whose association mimics a given miRNA's effects. Based on this assumption we devised a multi-omics approach to precisely characterize miRNAs' effects. We combined miR-491-5p target affinity purification, RNA microarray, and mass spectrometry to perform an integrated analysis in ovarian cancer cell lines. We thus constructed an interaction network that highlighted highly connected hubs being either direct or indirect targets of miR-491-5p effects: the already known EGFR and BCL2L1 but also EP300, CTNNB1 and several small-GTPases. By using different combinations of specific inhibitors of these hubs, we could greatly enhance their respective cytotoxicity and mimic the miR-491-5p-induced phenotype. Our methodology thus constitutes an interesting strategy to comprehensively study the effects of a given miRNA. Moreover, we identified targets for which pharmacological inhibitors are already available for a clinical use or in clinical trials. This study might thus enable innovative therapeutic options for ovarian cancer, which remains the leading cause of death from gynecological malignancies in developed countries.

SMARCA4-deficient rhabdoid tumours show intermediate molecular features between SMARCB1-deficient rhabdoid tumours and small cell carcinomas of the ovary, hypercalcaemic type.

Extracranial rhabdoid tumours (ECRTs) are an aggressive malignancy of infancy and early childhood. The vast majority of cases demonstrate inactivation of SMARCB1 (ECRTSMARCB1 ) on a background of a remarkably stable genome, a low mutational burden, and no other recurrent mutations. Rarely, ECRTs can harbour the alternative inactivation of SMARCA4 (ECRTSMARCA4 ) instead of SMARCB1. However, very few ECRTSMARCA4 cases have been published to date, and a systematic characterization of ECRTSMARCA4 is missing from the literature. In this study, we report the clinical, pathological, and genomic features of additional cases of ECRTSMARCA4 and show that they are comparable to those of ECRTSMARCB1. We also assess whether ECRTSMARCB1 , ECRTSMARCA4 , and small cell carcinomas of the ovary, hypercalcaemic type (SCCOHT) represent distinct or overlapping entities at a molecular level. Using DNA methylation and transcriptomics-based tumour classification approaches, we demonstrate that ECRTSMARCA4 display molecular features intermediate between SCCOHT and ECRTSMARCB1 ; however, ECRTSMARCA4 appear to be more closely related to SCCOHT by DNA methylation. Conversely, both transcriptomics and DNA methylation show a larger gap between SCCOHT and ECRTSMARCB1 , potentially supporting their continuous separate classification. Lastly, we show that ECRTSMARCA4 display concomitant lack of SMARCA4 (BRG1) and SMARCA2 (BRM) expression at the protein level, similar to what is seen in SCCOHT. Overall, these results expand our knowledge on this rare tumour type and explore the similarities and differences among entities from the 'rhabdoid tumour' spectrum. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Atypical teratoid/rhabdoid tumors (ATRTs) with SMARCA4 mutation are molecularly distinct from SMARCB1-deficient cases.

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.

Molecular subgrouping of atypical teratoid/rhabdoid tumors-a reinvestigation and current consensus.

BACKGROUND: Atypical teratoid/rhabdoid tumors (ATRTs) are known to exhibit molecular and clinical heterogeneity even though SMARCB1 inactivation is the sole recurrent genetic event present in nearly all cases. Indeed, recent studies demonstrated 3 molecular subgroups of ATRTs that are genetically, epigenetically, and clinically distinct. As these studies included different numbers of tumors, various subgrouping techniques, and naming, an international working group sought to align previous findings and to reach a consensus on nomenclature and clinicopathological significance of ATRT subgroups. METHODS: We integrated various methods to perform a meta-analysis on published and unpublished DNA methylation and gene expression datasets of ATRTs and associated clinicopathological data. RESULTS: In concordance with previous studies, the analyses identified 3 main molecular subgroups of ATRTs, for which a consensus was reached to name them ATRT-TYR, ATRT-SHH, and ATRT-MYC. The ATRT-SHH subgroup exhibited further heterogeneity, segregating further into 2 subtypes associated with a predominant supratentorial (ATRT-SHH-1) or infratentorial (ATRT-SHH-2) location. For each ATRT subgroup we provide an overview of its main molecular and clinical characteristics, including SMARCB1 alterations and pathway activation. CONCLUSIONS: The introduction of a common classification, characterization, and nomenclature of ATRT subgroups will facilitate future research and serve as a common ground for subgrouping patient samples and ATRT models, which will aid in refining subgroup-based therapies for ATRT patients.

Clonally Expanded T Cells Reveal Immunogenicity of Rhabdoid Tumors.

Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.

An autocrine ActivinB mechanism drives TGFß/Activin signaling in Group 3 medulloblastoma.

Medulloblastoma (MB) is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFß/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. We showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation. Beside genetic alterations, we demonstrated that an ActivinB autocrine stimulation is responsible for pathway activation in a subset of Group 3 MB characterized by high PMEPA1 levels. Importantly, Galunisertib, a kinase inhibitor of the cognate receptors currently tested in clinical trials for Glioblastoma patients, showed efficacy on orthotopically grafted MB-PDX. Our data demonstrate that the TGFß/Activin pathway is active in a subset of Group 3 MB and can be therapeutically targeted.

Characterization of the grapevine Shaker K+ channel VvK3.1 supports its function in massive potassium fluxes necessary for berry potassium loading and pulvinus-actuated leaf movements.

In grapevine, climate changes lead to increased berry potassium (K+ ) contents that result in must with low acidity. Consequently, wines are becoming 'flat' to the taste, with poor organoleptic properties and low potential aging, resulting in significant economic loss. Precise investigation into the molecular determinants controlling berry K+ accumulation during its development are only now emerging. Here, we report functional characterization by electrophysiology of a new grapevine Shaker-type K+ channel, VvK3.1. The analysis of VvK3.1 expression patterns was performed by qPCR and in situ hybridization. We found that VvK3.1 belongs to the AKT2 channel phylogenetic branch and is a weakly rectifying channel, mediating both inward and outward K+ currents. We showed that VvK3.1 is highly expressed in the phloem and in a unique structure located at the two ends of the petiole, identified as a pulvinus. From the onset of fruit ripening, all data support the role of the VvK3.1 channel in the massive K+ fluxes from the phloem cell cytosol to the berry apoplast during berry K+ loading. Moreover, the high amount of VvK3.1 transcripts detected in the pulvinus strongly suggests a role for this Shaker in the swelling and shrinking of motor cells involved in paraheliotropic leaf movements.

High-Throughput Drug Screening Identifies Pazopanib and Clofilium Tosylate as Promising Treatments for Malignant Rhabdoid Tumors.

Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/ß and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs.

Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells.

The grape berry provides a model for investigating the physiology of non-climacteric fruits. Increased K(+) accumulation in the berry has a strong negative impact on fruit acidity (and quality). In maturing berries, we identified a K(+) channel from the Shaker family, VvK1.2, and two CBL-interacting protein kinase (CIPK)/calcineurin B-like calcium sensor (CBL) pairs, VvCIPK04-VvCBL01 and VvCIPK03-VvCBL02, that may control the activity of this channel. VvCBL01 and VvCIPK04 are homologues of Arabidopsis AtCBL1 and AtCIPK23, respectively, which form a complex that controls the activity of the Shaker K(+) channel AKT1 in Arabidopsis roots. VvK1.2 remained electrically silent when expressed alone in Xenopus oocytes, but gave rise to K(+) currents when co-expressed with the pairs VvCIPK03-VvCBL02 or VvCIPK04-VvCBL01, the second pair inducing much larger currents than the first one. Other tested CIPK-CBL pairs expressed in maturing berries were found to be unable to activate VvK1.2. When activated by its CIPK-CBL partners, VvK1.2 acts as a voltage-gated inwardly rectifying K(+) channel that is activated at voltages more negative than -100 mV and is stimulated upon external acidification. This channel is specifically expressed in the berry, where it displays a very strong induction at veraison (the inception of ripening) in flesh cells, phloem tissues and perivascular cells surrounding vascular bundles. Its expression in these tissues is further greatly increased upon mild drought stress. VvK1.2 is thus likely to mediate rapid K(+) transport in the berry and to contribute to the extensive re-organization of the translocation pathways and transport mechanisms that occurs at veraison.