Abstract BACKGROUND Our previous analysis of established cancer predisposition genes in medulloblastoma (MB) identified pathogenic germline variants in ~5% of all patients. Here, we extended our analysis to include all protein-coding genes. METHODS Case-control analysis performed on 795 MB patients against >118,000 cancer-free children and adults was performed to identify an association between rare germline variants and MB. RESULTS Germline loss-of-function variants of Elongator Complex Protein 1 (ELP1; 9q31.3) were strongly associated with SHH subgroup (MBSHH). ELP1-associated-MBs accounted for ~15% (29/202) of pediatric MBSHH cases and were restricted to the SHHα subtype. ELP1-associated-MBs demonstrated biallelic inactivation of ELP1 due to somatic chromosome 9q loss and most tumors exhibited co-occurring somatic PTCH1 (9q22.32) alterations. Inheritance was verified by parent-offspring sequencing (n=3) and pedigree analysis identified two families with a history of pediatric MB. ELP1-associated-MBSHH were characterized by desmoplastic/nodular histology (76%; 13/17) and demonstrated a favorable clinical outcome when compared to TP53-associated-MBSHH (5-yr OS 92% vs 20%; p-value=1.3e-6) despite both belonging to the SHHα subtype. ELP1 is a subunit of the Elongator complex, that promotes efficient translational elongation through tRNA modifications at the wobble (U34) position. Biochemical, transcriptional, and proteomic analyses revealed ELP1-associated-MBs exhibit destabilization of the core Elongator complex, loss of tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response. CONCLUSIONS We identified ELP1 as the most common MB predisposition gene, increasing the total genetic predisposition for pediatric MBSHH to 40%. These results mark MBSHH as an overwhelmingly genetically-predisposed disease and implicate disruption of protein homeostasis in MBSHH development.
Abstract Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events in childhood medulloblastoma (MB). ELP1 germline carriers develop SHH-MBs that exhibit coincident somatic PTCH1 mutations and universal loss-of-heterozygosity of the remaining ELP1 allele through chromosome 9q deletion. The molecular, biochemical, and cellular mechanisms by which germline ELP1/Elongator deficiency contribute to SHH-MB tumorigenesis remain largely unknown. Herein, we report that mice engineered to mimic germline Elp1 LOF (i.e., Elp1HET) seen in SHH-MB patients exhibit hallmark features of premalignancy events in cycling cerebellar granule neuron progenitors (GNPs), the lineage-of-origin for SHH-MB. Compared to wild-type counterparts, Elp1HET GNPs exhibit increased replication stress-associated DNA damage, homologous recombination-associated genomic instability, accelerated cell cycle kinetics, reduced p53-dependent apoptosis in response to genotoxic stress, and slowed differentiation. Orthotopic transplantation of Elp1HET GNPs harboring somatic Ptch1 inactivation into the cerebella of immunocompromised mice promotes onset of SHH-MB tumors with incomplete penetrance that exhibit reduced p53 transcriptional activity through a currently unknown mechanism(s). Concomitant Elp1 and Ptch1 gene targeting in p53-null GNPs reproduces highly penetrant cerebellar tumors recapitulating the molecular and phenotypic features of ELP1-associated SHH-MB. Finally, reactivation of the p53 pathway through preclinical treatment with an MDM2 inhibitor promotes cell death and prolongs the survival of patient-derived xenograft tumor (PDX) models harboring deleterious ELP1 mutations. Together, our findings reveal that germline Elp1 LOF heightens genomic instability and malignant transformation in cycling GNPs, providing a mechanistic model for the subgroup-restricted pattern of predisposition associated with pathogenic ELP1 germline carriers. These results provide essential mechanistic insight into the molecular and cellular basis of SHH-MB predisposition driven by ELP1 LOF and nominate therapies that overcome p53 pathway inhibition as a rational treatment option for affected children.
Abstract Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events observed in medulloblastoma (MB), accounting for 30% of the Sonic Hedgehog 3 subtype (SHH-3). Molecularly, ELP1-associated SHH-MBs acquire somatic PTCH1 mutations in >80% of cases, and universal loss-of-heterozygosity of the wild-type ELP1 and PTCH1 alleles through loss of chromosome-arm 9q, resulting in their biallelic inactivation. Notably, germline ELP1 LOF occurs mutually exclusive with somatic/germline TP53 mutations in the SHH-3 subtype, suggesting that genetic perturbation of either ELP1 or TP53 may promote similar downstream oncogenic consequences in cerebellar granule neuron progenitors (GNPs), the accepted cellular origin of SHH-MB. Despite these findings, the molecular, biochemical, and cellular mechanism(s) by which ELP1-deficiency provokes malignant pathogenesis remain unknown. In this study, we sought to close this knowledge gap and functionally determine why children harboring pathogenic ELP1 germline variants are at an increased risk of developing SHH-MB. Mice were genetically engineered to mimic heterozygous Elp1 LOF (Elp1HET). We studied the effect of loss of ELP1 in GNPs using a combination of molecular profiling, immunophenotyping, and cellular assays. GNPs from Elp1HET exhibited a spectrum of molecular and biochemical hallmarks of malignant transformation including increased DNA replication stress, DNA damage, accelerated cell cycle progression, and stalled differentiation. Orthotopic transplantation of Elp1HET GNPs engineered to harbor somatic Ptch1 inactivation yielded SHH-MB-like tumors with compromised p53 signaling, providing an explanation for the specificity of ELP1-associated tumors in SHH-3, and their exclusivity with TP53-mutant tumors. Treatment of ELP1-mutant patient-derived xenografts with an FDA-approved MDM2 inhibitor reactivated p53-dependent apoptosis and extended survival. Collectively, our findings functionally substantiate the role of ELP1 deficiency in predisposition to SHH-3 MB and nominate therapeutic strategies to overcome p53 inhibition as a rational treatment option.
<div>Abstract<p>Medulloblastoma is the most common malignant pediatric brain tumor and there is an urgent need for molecularly targeted and subgroup-specific therapies. The stem cell factor SOX9, has been proposed as a potential therapeutic target for the treatment of Sonic Hedgehog medulloblastoma (SHH-MB) subgroup tumors, given its role as a downstream target of Hedgehog signaling and in functionally promoting SHH-MB metastasis and treatment resistance. However, the functional requirement for SOX9 in the genesis of medulloblastoma remains to be determined. Here we report a previously undocumented level of SOX9 expression exclusively in proliferating granule cell precursors (GCP) of the postnatal mouse cerebellum, which function as the medulloblastoma-initiating cells of SHH-MBs. Wild-type GCPs express comparatively lower levels of SOX9 than neural stem cells and mature astroglia and SOX9<sup>low</sup> GCP-like tumor cells constitute the bulk of both infant (Math1Cre:<i>Ptch1<sup>lox/lox</sup></i>) and adult (<i>Ptch1<sup>LacZ/+</sup></i>) SHH-MB mouse models. Human medulloblastoma single-cell RNA data analyses reveal three distinct <i>SOX9</i> populations present in SHH-MB and noticeably absent in other medulloblastoma subgroups: <i>SOX9</i><sup>+</sup><i>MATH1</i><sup>+</sup> (GCP), <i>SOX9</i><sup>+</sup><i>GFAP</i><sup>+</sup> (astrocytes) and <i>SOX9</i><sup>+</sup><i>MATH1</i><sup>+</sup><i>GFAP</i><sup>+</sup> (potential tumor-derived astrocytes). To functionally address whether SOX9 is required as a downstream effector of Hedgehog signaling in medulloblastoma tumor cells, we ablated <i>Sox9</i> using a Math1Cre model system. Surprisingly, targeted ablation of <i>Sox9</i> in GCPs (Math1Cre:<i>Sox9<sup>lox/lox</sup></i>) revealed no overt phenotype and loss of <i>Sox9</i> in SHH-MB (Math1Cre:<i>Ptch1<sup>lox/lox</sup>;Sox9<sup>lox/lox</sup></i>) does not affect tumor formation.</p>Implications:<p>Despite preclinical data indicating SOX9 plays a key role in SHH-MB biology, our data argue against SOX9 as a viable therapeutic target.</p></div>
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