Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain [1][2][3][4] . Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage [5][6][7][8] . By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL 9,10 . However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage 3,4 . Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental

Co-expression and cross-regulation of the four TCF/LEFs render their redundant and unique functions ambiguous. Here, we describe quadruple-knockout (QKO) mouse ESCs lacking all full-length TCF/LEFs and cell lines rescued with TCF7 or TCF7L1. QKO cells self-renew, despite gene expression patterns that differ significantly from WT, and display delayed, neurectoderm-biased, embryoid body (EB) differentiation. QKO EBs have no contracting cardiomyocytes and differentiate poorly into mesendoderm but readily generate neuronal cells. QKO cells and TCF7L1-rescued cells cannot efficiently activate TCF reporters, whereas TCF7-rescued cells exhibit significant reporter responsiveness. Surprisingly, despite dramatically different transactivation capacities, re-expression of TCF7L1 or TCF7 in QKO cells restores their tri-lineage differentiation ability, with similar lineage marker expression patterns and beating cardiomyocyte frequencies observed in EBs. Both factors also similarly affect the transcriptome of QKO cells. Our data reveal that a single TCF, regardless of its activation capacity, is sufficient for effective trilineage differentiation of ESCs.

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Medulloblastoma (MB) is the most common primary malignant pediatric brain cancer. We recently identified novel roles for the MEK/MAPK pathway in regulating human Sonic Hedgehog (SHH) MB tumorigenesis. The MEK inhibitor, selumetinib, decreased SHH MB growth while extending survival in mouse models. However, the treated mice ultimately succumbed to disease progression. Here, we perform RNA sequencing on selumetinib-treated orthotopic xenografts to identify molecular pathways that compensate for MEK inhibition specifically in vivo. Notably, the JAK/STAT3 pathway exhibits increased activation in selumetinib-treated tumors. The combination of selumetinib and the JAK/STAT3 pathway inhibitor, pacritinib, further reduces growth in two xenograft models and also enhances survival. Multiplex spatial profiling of proteins in drug-treated xenografts reveals shifted molecular dependencies and compensatory changes following combination drug treatment. Our study warrants further investigation into MEK and JAK/STAT3 inhibition as a novel combinatory therapeutic strategy for SHH MB.

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How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell–driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2−/− fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.

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