Childhood cancer mutagenesis caused by transposase-derived PGBD5.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

KMT2D suppresses Sonic hedgehog-driven medulloblastoma progression and metastasis.

The major cause of treatment failure and mortality among medulloblastoma patients is metastasis intracranially or along the spinal cord. The molecular mechanisms driving tumor metastasis in Sonic hedgehog-driven medulloblastoma (SHH-MB) patients, however, remain largely unknown. In this study we define a tumor suppressive role of KMT2D (MLL2), a gene frequently mutated in the most metastatic ß-subtype. Strikingly, genetic mouse models of SHH-MB demonstrate that heterozygous loss of Kmt2d in conjunction with activation of the SHH pathway causes highly penetrant disease with decreased survival, increased hindbrain invasion and spinal cord metastasis. Loss of Kmt2d attenuates neural differentiation and shifts the transcriptional/chromatin landscape of primary and metastatic tumors toward a decrease in differentiation genes and tumor suppressors and an increase in genes/pathways implicated in advanced stage cancer and metastasis (TGFß, Notch, Atoh1, Sox2, and Myc). Thus, secondary heterozygous KMT2D mutations likely have prognostic value for identifying SHH-MB patients prone to develop metastasis.

CSF1R inhibition depletes tumor-associated macrophages and attenuates tumor progression in a mouse sonic Hedgehog-Medulloblastoma model.

The immune microenvironment of tumors can play a critical role in promoting or inhibiting tumor progression depending on the context. We present evidence that tumor-associated macrophages/microglia (TAMs) can promote tumor progression in the sonic hedgehog subgroup of medulloblastoma (SHH-MB). By combining longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) and immune profiling of a sporadic mouse model of SHH-MB, we found the density of TAMs is higher in the ~50% of tumors that progress to lethal disease. Furthermore, reducing regulatory T cells or eliminating B and T cells in Rag1 mutants does not alter SHH-MB tumor progression. As TAMs are a dominant immune component in tumors and are normally dependent on colony-stimulating factor 1 receptor (CSF1R), we treated mice with a CSF1R inhibitor, PLX5622. Significantly, PLX5622 reduces a subset of TAMs, prolongs mouse survival, and reduces the volume of most tumors within 4 weeks of treatment. Moreover, concomitant with a reduction in TAMs the percentage of infiltrating cytotoxic T cells is increased, indicating a change in the tumor environment. Our studies in an immunocompetent preclinical mouse model demonstrate TAMs can have a functional role in promoting SHH-MB progression. Thus, CSF1R inhibition could have therapeutic potential for a subset of SHH-MB patients.

The in vivo genetic program of murine primordial lung epithelial progenitors.

Multipotent Nkx2-1-positive lung epithelial primordial progenitors of the foregut endoderm are thought to be the developmental precursors to all adult lung epithelial lineages. However, little is known about the global transcriptomic programs or gene networks that regulate these gateway progenitors in vivo. Here we use bulk RNA-sequencing to describe the unique genetic program of in vivo murine lung primordial progenitors and computationally identify signaling pathways, such as Wnt and Tgf-ß superfamily pathways, that are involved in their cell-fate determination from pre-specified embryonic foregut. We integrate this information in computational models to generate in vitro engineered lung primordial progenitors from mouse pluripotent stem cells, improving the fidelity of the resulting cells through unbiased, easy-to-interpret similarity scores and modulation of cell culture conditions, including substratum elastic modulus and extracellular matrix composition. The methodology proposed here can have wide applicability to the in vitro derivation of bona fide tissue progenitors of all germ layers.

Lateral cerebellum is preferentially sensitive to high sonic hedgehog signaling and medulloblastoma formation.

The main cell of origin of the Sonic hedgehog (SHH) subgroup of medulloblastoma (MB) is granule cell precursors (GCPs), a SHH-dependent transient amplifying population in the developing cerebellum. SHH-MBs can be further subdivided based on molecular and clinical parameters, as well as location because SHH-MBs occur preferentially in the lateral cerebellum (hemispheres). Our analysis of adult patient data suggests that tumors with Smoothened (SMO) mutations form more specifically in the hemispheres than those with Patched 1 (PTCH1) mutations. Using sporadic mouse models of SHH-MB with the two mutations commonly seen in adult MB, constitutive activation of Smo (SmoM2) or loss-of-Ptch1, we found that regardless of timing of induction or type of mutation, tumors developed primarily in the hemispheres, with SmoM2-mutants indeed showing a stronger specificity. We further uncovered that GCPs in the hemispheres are more susceptible to high-level SHH signaling compared with GCPs in the medial cerebellum (vermis), as more SmoM2 or Ptch1-mutant hemisphere cells remain undifferentiated and show increased tumorigenicity when transplanted. Finally, we identified location-specific GCP gene-expression profiles, and found that deletion of the genes most highly expressed in the hemispheres (Nr2f2) or vermis (Engrailed1) showed opposing effects on GCP differentiation. Our studies thus provide insights into intrinsic differences within GCPs that impact on SHH-MB progression.

Thyroid Progenitors Are Robustly Derived from Embryonic Stem Cells through Transient, Developmental Stage-Specific Overexpression of Nkx2-1.

The clinical importance of anterior foregut endoderm (AFE) derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs). Here, we identify transient overexpression of the transcription factor (TF) NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex) and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd) at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates.