Abstract
Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.
Highlights
Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma
We use medulloblastomas that form in transgenic, Smo-mutant mice to study the early effects of vismodegib on cellular diversity in SHH-driven tumors and to determine if this diversity may contribute treatment failure
We found that medulloblastomas contain diverse types of tumor cells, including cerebellar granule neuron progenitors (CGNPs)-like cells in a spectrum of differentiation states, and tumor-derived cells with patterns of gene expression typical of astrocytic precursors and oligodendrocytic precursors, fates outside the expected Atoh[1] lineage of the CGNPs
Summary
Vismodegib transiently slows medulloblastoma growth in mice. We generated medulloblastoma-bearing mice by breeding the transgenic SmoM2 mouse line, which harbors a mutant, constitutively active allele of Smo, preceded by a LoxP-STOPLoxP sequence[27] with Math1-Cre mice, that express Cre recombinase in CGNPs, driven by the Atoh[1] (aka Math1) promoter[28,29,30]. The resulting Math1-Cre/SmoM2 (M-Smo) mice developed medulloblastoma with 100% frequency by postnatal day 12 (P12). We administered either vismodegib or vehicle to medulloblastoma-bearing P12 M-Smo mice, daily from P12 to P15, and every other day until symptomatic progression. Vismodegib induced transient tumor regression, with reduced expression of phosphorylated RB (pRB; Fig. 1a, b). For longitudinal measurement of pharmacodynamic response, we administered vismodegib to another medulloblastoma-prone genotype, hGFAP-Cre/SmoM2/Gli-luc, that carries a synthetic, SHH-sensitive luciferase reporter construct (G-Smo/Gli-luc; Fig. 1d). Luciferase imaging showed that the first dose of vismodegib decreased SHH activation, but that a pRB DAPI
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