Loss of histone 3 lysine 9 trimethylation drives recurrence in high-risk pediatric medulloblastoma

Background: Pediatric Group 3 medulloblastoma (G3 MB) recurs in more than 40% of cases. Initial treatment typically includes surgical resection followed by craniospinal irradiation (RT) and chemotherapy with cisplatin and cyclophosphamide. Recurrent G3 MB is usually metastatic, has poor long-term survival, and no survival-altering treatment. Treatment of recurrent G3 MB impacts only a portion of tumor cells, or worse, may facilitate the emergence of treatment resistant cell clones. G3 MB's cells of origin correspond to neural stem or progenitor cells that originate early in normal development. This suggests that pluripotency genes whose expression is normally epigenetically silenced are reactivated in G3 MB oncogenesis and recurrence. Histone 3 post-translational modifications, particularly trimethylation of histone 3 lysine 9 (H3K9me3), silence expression of pluripotency genes when no longer needed during development. We hypothesize that loss of H3K9me3 at key pluripotency genes facilitates G3 MB recurrence. Methods: To model recurrence, we developed treatment-resistant cell lines, tumoroids and orthotopic patient-derived xenograft (PDX) mouse models of G3 MB. Using single cell RNA-Seq and multiomics we analyzed gene accessibility and expression changes between primary-recurrent patient sample pairs. To define cell and tumor micro-environment changes during recurrence, we conducted spatial transcriptomics of primary-recurrent patient sample pairs using a customized gene list. Results: Analysis of primary-recurrent patient sample pairs revealed that most cell types are conserved during recurrence, mesenchymal cells begin to outnumber angiogenic populations, hypoxic populations increase, and neural stemlike cells replace progenitor-like populations. Treatment of G3 MB cells with near-IC 50 levels of cisplatin or RT + cisplatin produces a treatment resistant / recurrent phenotype with enriched DNA repair capability, enriched MAPK and autophagy pathways, and depleted p53 activity. Cisplatin + cyclophosphamide treatment of mice with PDX tumors extends survival but ultimately results in aggressive treatment resistant tumors with divergent stemlike populations as compared to the primary tumors. Expression of SETDB1, a demethylase (DM) that removes H3K9me3, decreased upon G3 MB recurrence, while expression of EHMT2 , a methyltransferase (MT) that places H3K9me2, increased. Similar trends were not observed in non-G3 MB samples. Conclusions and ongoing work: H3K9 MT and DM alterations suggest the loss of H3K9me3 leads to emergence of pluripotent gene expression during G3 MB recurrence. We are analyzing CUT & RUN data to verify whether H3K9me3 occupancy is altered through changes in expression of H3K9 MTs and DMs. Longer term, we plan to evaluate whether inhibiting the loss of H3K9me3 diminishes the frequency or severity of recurrence in G3 MB models.