Sox2+ cells in Sonic Hedgehog-subtype medulloblastoma resist p53-mediated cell-cycle arrest response and drive therapy-induced recurrence

Abstract Background High-intensity therapy effectively treats most TP53 wild-type (TP53-WT) Sonic Hedgehog-subgroup medulloblastomas (SHH-MBs), but often cause long-term deleterious neurotoxicities in children. Recent clinical trials investigating reduction/de-escalation of therapy for TP53-WT SHH-MBs caused poor overall survival. Here, we investigated whether reduced levels of p53-pathway activation by low-intensity therapy potentially contribute to diminished therapeutic efficacy. Methods Using mouse SHH-MB models with different p53 activities, we investigated therapeutic efficacy by activating p53-mediated cell-cycle arrest versus p53-mediated apoptosis on radiation-induced recurrence. Results Upon radiation treatment, p53WT-mediated apoptosis was sufficient to eliminate all SHH-MB cells, including Sox2+ cells. The same treatment eliminated most Sox2− bulk tumor cells in SHH-MBs harboring p53R172P, an apoptosis-defective allele with cell-cycle arrest activity, via inducing robust neuronal differentiation. Rare quiescent Sox2+ cells survived radiation-enhanced p53R172P activation and entered a proliferative state, regenerating tumors. Transcriptomes of Sox2+ cells resembled quiescent Nestin-expressing progenitors in the developing cerebellum, expressing Olig2 known to suppress p53 and p21 expression. Importantly, high SOX2 expression is associated with poor survival of all four SHH-MB subgroups, independent of TP53 mutational status. Conclusions Quiescent Sox2+ cells are efficiently eliminated by p53-mediated apoptosis, but not cell-cycle arrest and differentiation. Their survival contributes to tumor recurrence due to insufficient p53-pathway activation.


Sox2 + cells in Sonic Hedgehog-subtype medulloblastoma resist p53-mediated cell-cycle arrest response and drive therapy-induced recurrence
Medulloblastoma (MB) is the most common malignant brain tumor in children, 30% of which are Sonic Hedgehog-subgroup MB (SHH-MB). [1][2][3] Prior human and mouse research have demonstrated that SHH-MBs arise from the granule cell precursor (GCP) lineage in the developing cerebellum. 1,[4][5][6][7] GCPs are a neuronal-restricted progenitor population transiently located in the external granular layer (EGL). 8 During development, SHH signaling drives GCP proliferation, which then migrate into the internal granular layer (IGL) and differentiate into granule cells. 8,9 Genetic alterations in the components of the SHH signaling pathway, including PTCH1, SMOOTHENED, SUFU, GLI2, or N-MYC, cause aberrant SHH-pathway activation, driving SHH-MB formation. 1,2,4,5 Current standard treatment, including surgery, high-intensity radiation and chemotherapy, leads to 70%-80% survival for average-risk SHH-MBs, which frequently retain wild-type TP53 alleles (TP53-WT SHH-MBs). 2,10,11 However, standard high-intensity therapies often cause devastating side effects including neurocognitive, neuroendocrine, and motor deficits, diminishing the quality of life for long-term survivors. 10 Clinical de-escalation of therapy trials, including elimination of radiation and reduced chemotherapy, has been proposed for MBs in infants and young children to avoid neurocognitive toxicities. 12,13 However, recent de-escalation trials were suspended due to poor overall survival. 14 These clinical observations suggest that a therapeutic threshold(s), reached by standard high-intensity therapeutic protocols, was not reached by de-escalation protocols. Given that most TP53-mutant SHH-MBs are in the SHHα subgroup and highly resistant to standard highintensity therapy, 2,[9][10][11] the critical therapeutic threshold could be activation of p53-mediated tumor suppressive responses, including apoptosis, cell-cycle arrest, and senescence. 15 Consistently, genetic studies using mouse models have demonstrated that disruption of p53-mediated apoptosis confers radiation resistance to SHH-MBs. 16,17 Moreover, radiation exposure induces p53-mediated apoptosis in the developing cerebellum, leading to neurological damages. 18 Together, we propose a model wherein activation of p53-mediated apoptosis by standard high-intensity therapy overcomes a therapeutic threshold to eliminate SHH-MB cells and prevent recurrence, but p53-mediated apoptosis may concurrently cause therapy-associated neu rotoxicities. 12,13,18 However, a recent study suggested that p53-mediated cell-cycle arrest and senescence, but not apoptosis, are sufficient to suppress SHH-MB formation. 19 This study suggests that de-escalation of therapy may reach a lower therapeutic threshold that activates p53mediated cell-cycle arrest and senescence, while avoiding neurotoxic p53-mediated apoptosis.
Here, we used genetically engineered mouse (GEM) models of SHH-MBs to compare therapeutic efficacy of p53-mediated cell-cycle arrest versus p53-mediated apoptosis during SHH-MB formation and radiation-induced recurrence ( Figure 1A). We show that p53-mediated cellcycle arrest response induces neuronal differentiation of SHH-MB cells, driving bulk tumor cells out of the tumor bed and dramatically reducing tumor volume. In contrast to apoptosis, however, p53-mediated cell-cycle arrest and neuronal differentiation failed to completely eliminate a previously described quiescent Sox2 + stem cell-like population. 20,21 Following radiation treatment, Sox2 + cells entered the cell cycle and regenerated tumors. Furthermore, we provided a molecular mechanism by which Sox2 + SHH-MB cells are resistant to p53/p21-mediated cell-cycle arrest response via Olig2 expression. Importantly, we showed that high SOX2 expression is associated with poor survival in all four SHH-MB subgroups, independent of TP53 mutational status. Thus, our study provides important insights into patient stratification in the design of future de-escalation of therapy trials.

Materials and Methods
Details are available in Supplementary Materials and Methods.

Importance of the Study
TP53-WT SHH-MBs have excellent prognosis, but long-term neurotoxicities are common. De-escalation of therapy was proposed to reduce neurotoxicities while retaining efficacy. However, recent clinical trials found that de-escalation of therapy instead caused poor overall survival. We hypothesize that therapeutic efficacy in TP53-WT SHH-MBs and neurotoxicities in the developing brain following standard high-intensity treatment are caused by a common mechanism-p53-mediated apoptosis. We found that radiation-enhanced p53mediated apoptosis eliminated all SHH-MB cells and prevented tumor recurrence. Radiationenhanced p53-mediated cell-cycle arrest, despite inducing neuronal differentiation in bulk tumor cells, failed to eliminate Sox2 + SHH-MB cells with transcriptomic similarity to quiescent Nestin-expressing progenitors. Importantly, high SOX2 expression is associated with poor survival of all four SHH-MB subgroups, independent of TP53 mutational status. Thus, our study suggests that the failure in eliminating SOX2 + cells may contribute to tumor recurrence, providing insights into patient stratification in future de-escalation of therapy trials.

Histology, IHC, and Western Blotting
Mice were collected as described previously. 22,23 Tumors were classified using the WHO guidebook for MB as diagnostic criteria, based on the most severe region observed, and lesions were assessed based on previously established criteria. 19,26 Immunohistochemistry and immunofluorescence were performed as described previously. 22 For western blotting, samples were prepared as described previously. 23

Genetic Analysis and Next Generation Sequencing
Total RNA and genomic DNA was isolated from MBs and cerebellum using the AllPrep DNA/RNA Mini Kit (Qiagen), and cDNA synthesis was performed using QuantiTect Reverse Transcription Kit (Qiagen). The cDNA product, following quality assessment and measurement of concentration, was used to sequence exons of the p53 allele, as described previously. 22 Amplified PCR products were submitted to the University of Michigan DNA sequencing core or Genewiz L.L.C. for Sanger sequencing. Genomic DNA samples were then analyzed by next generation sequencing to detect point mutants in Ptch1 and p53 as described previously. 23

Statistical Analysis
Data were analyzed and statistics performed using Graphpad Prism 6. Kaplan-Meier survival curves were compared using the Mantel-Cox test. Significance was calculated using either Student's 2-tailed t-test or ANOVA with Bonferroni's multiple comparisons test. Data were presented as mean ± SEM.
The p53 WT and p53 R172P Alleles Are Retained During Ptch1-Loss Driven SHH-MB Formation Given homozygous loss of p53 dramatically increased the penetrance and accelerated Ptch1 loss-driven SHH-MB formation, we investigated whether p53 R172P and p53 WT alleles were, respectively, inactivated in SHH-MBs arising from Ptch1 +/− p53 R172P and Ptch1 +/− p53 WT models. First, we showed that both p53 R172P and p53 WT alleles were retained in SHH-MBs and SHH-MB-derived cell lines, respectively (Supplementary Figure Figure 1F). 24 As a negative control, no induction of p53 transcriptional targets was observed in Ptch1 −/− p53 ∆E5-6 SHH-MBs ( Figure 1F). Given the intact p53 R172P or p53 WT alleles in tumors, we investigated a potential alternative mechanism(s) for p53 pathway inhibition in SHH-MBs by examining the expression of other known regulators of the p53 pathway. No alteration was observed except for elevated Twist1 expression in a small number of Ptch1 −/− p53 WT MBs analyzed, which was independent of radiation treatment ( Figure 1G and H). Importantly, TP53-WT SHH-MBs from infants and children also exhibited no alteration of these p53-pathway regulators compared with TP53-mutant SHH-MBs ( Figure 1I). Of note, many TP53-WT SHH-MBs in adults often exhibited mutually exclusive gene expression alterations of MDM2, MDM4, TWIST1, TWIST2, or TRIM24, suggesting a potentially attenuated p53 pathway ( Figure 1J). Together, these results demonstrate that the p53 pathway is not genetically disrupted during the formation of TP53/p53-WT SHH-MBs in humans and mice, and more importantly, can be activated to exhibit therapeutic effects upon radiation treatment.

Both p53-Mediated Cell-Cycle Arrest and Apoptosis Robustly Eliminate Proliferating Tumor Cells Following Radiation Treatment
We next assessed therapeutic efficacy of activating p53 R172P -mediated cell-cycle arrest versus p53 WT -mediated apoptosis in SHH-MBs. We designed a clinically relevant treatment protocol in the SHH-MB models, administering 10 fractions of 2 Gray each over 12 days, from P22 to P34, and then investigated therapeutic effects at three time points: 1 day (P35) or 12 days (P46) after the completion of radiation treatment, or long-term follow-up until tumors emerged (Figure 2A). Compared with untreated lesions at P22 (Supplementary Figure S3A-C), radiation treatment shrank lesions in all models at P35, exhibiting 65%, 80%, and 95% of reduction in SHH-MBs with p53 ∆E5-6 , p53 R172P , and p53 WT alleles, respectively (Figure 2B and C; Supplementary Figure S3D). These results suggest that radiation induces both p53-independent and p53-dependent tumor inhibitory effects. However, the radiation-treated lesions were distinctly different in these three models. Radiation-treated lesions in the Ptch1 +/− p53 ∆E5-6 model exhibited widespread proliferation, high levels of mutant p53 expression, and maintained rare quiescent Sox2 + cells. Small focal regions within the proliferating Ptch1 +/− p53 ∆E5-6 tumors morphologically resembled differentiated neurons of the IGL with the expression of the markers for differentiating cells, p27, and neurons, NeuN, but no Ki67 or p53 ∆E5-6 expression ( Figure 2B Figure 2D and E). The chain-like NeuN + cells are reminiscent of differentiating GCPs migrating from the EGL to IGL during cerebellar development. 8 Despite effectively driving tumor cells out of the cell cycle and inducing neuronal differentiation, radiation treatment failed to completely eliminate quiescent Sox2 + cells in Ptch1 −/− p53 R172P tumors ( Figure 2F-H). Strikingly, this radiation treatment protocol almost completely eliminated tumor or tumor-like cells in the Ptch1 +/− p53 WT cerebella ( Figure 2F-H), though apoptotic cells were no longer detected (data not shown). The rare lesion-like cells in radiation-treated Ptch1 +/− p53 WT cerebella morphologically resembled differentiated neurons in the IGL with expression of p27 and NeuN, but not Sox2 ( Figure  2F-H; Supplementary Figure S3F and G). These results demonstrate that radiation-induced p53 R172P -mediated cellcycle arrest induces massive differentiation of bulk tumor cells, but fails to completely eliminate quiescent Sox2 + cells. In contrast, radiation-induced p53 WT -mediated apoptosis and cell-cycle arrest efficiently eliminate all tumor cells, including Sox2 + cells.

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quiescent Sox2 + cells, which become highly proliferative and rapidly regenerate tumors.
Consistently, radiation-treated Ptch1 +/− p53 ∆E5-6/∆E5-6 or Ptch1 +/− p53 ∆E5-6/R172P mice developed SHH-MBs with complete penetrance, median survival, p53 expression, and proliferation frequency comparable to untreated SHH-MBs ( Figure 3D and E; Supplementary Figure S4E and F). Of note, although we observed that Sox2 + cells in Ptch1 −/− p53 R172P lesions survived radiation and entered the cell cycle, the Sox2 + cells became a rare quiescent cell population in all recurrent SHH-MBs at end stages, comparable to untreated SHH-MBs ( Figure 3F and G). 20,21 In contrast, radiation treatment dramatically reduced tumor penetrance compared with untreated Ptch1 +/− p53 WT mice (38% to 9%), and tumor latency further increased by 29% in the only two treated mice (that still developed tumors) ( Figure 3D). Next-generation sequencing failed to detect any p53 mutations in these two radiation-treated / Sox2 Ki67  SHH-MBs, suggesting some p53 WT SHH-MB cells survived radiation treatment, supported by the lack of stabilized p53 in the resultant SHH-MBs ( Figure 3E). These results support the model wherein activation of p53 WT -mediated apoptosis is the therapeutic threshold to overcome in order to eliminate Sox2 + SHH-MBs. In contrast, radiation-enhanced p53 R172P activation has no or little benefit on preventing the recurrence of SHH-MBs as a result of failing to eliminate quiescent Sox2 + tumor cells.

The Transcriptome of Sox2 + SHH-MB Cells Resembles Developing Quiescent Nestin-Expressing Progenitors With Olig2 Expression
Our results suggest that Sox2 + SHH-MB cells resist radiation-enhanced p53 R172P -dependent cell-cycle arrest and neuronal differentiation, which is mediated by p21 expression. 24 To investigate the underlying mechanism, we analyzed published microarray data sets of Sox2 + and Sox2 − cells isolated from SHH-MBs in a similar p53-WT Ptch1 +/− model. 21 Between these two populations, 107 genes were significantly upregulated in Sox2 + SHH-MB cells (Supplementary Figure S5A), whereas Sox2 − SHH-MB cells had 7 upregulated genes (P < .01) (Supplementary Figure S5B). Gene Ontology (GO) analysis found Sox2 + SHH-MB cells upregulated several genes involved in negative regulation of neuronal differentiation, confirming that Sox2 + SHH-MB cells are less differentiated with stemcell-like characteristics (Supplementary Figure S5C). Given the quiescence and undifferentiated nature of Sox2 + SHH-MB cells, we next investigated whether they are similar to a recently discovered quiescent Nestin-expressing precursor (NEP) population in the GCP lineage. 29 We therefore analyzed published data sets showing expression patterns of Nestin + Atoh1 − NEPs and Nestin − Atoh1 + GCPs. 29 In P4 cerebella, 716 genes were upregulated in NEPs, whereas 252 were upregulated in GCPs (P < .001). Importantly, the majority of the 716 genes upregulated in NEPs were also upregulated in the Sox2 + SHH-MB cells, whereas the genes upregulated in the Atoh1 + GCPs were upregulated in the Sox2 − SHH-MB cells ( Figure 4A and B; Supplementary Figure S5D and E). Likewise, most of the 107 significantly upregulated genes in the Sox2 + SHH-MB cells were also upregulated in NEPs, but not in GCPs or SHH-MB cells (Supplementary Figure S5A). In contrast, the genes downregulated in Sox2 + SHH-MB cells were also downregulated in NEPs (Supplementary Figure S5B). Comparable genome-wide expression profiles between Sox2 + SHH-MB cells and NEPs as well as between Sox2 − SHH-MB cells and GCPs suggest that a similar process occurs between tumor regeneration from Sox2 + to Sox2 − cells in radiation-treated SHH-MBs and the regeneration of GCPs from NEPs in radiation-treated developing cerebellum. 21,29,30 One of the genes upregulated in both Sox2 + SHH-MB cells and Sox2 + NEPs, encodes transcription repressor Olig2 ( Figure 4C and D). OLIG2 is a pan-glioma marker also expressed in a rare cell population within human MBs. 4 Previous studies demonstrated that Olig2 represses p53-mediated transcriptional activities, by inhibiting acetylation of p53, and directly represses transcription of p21 31,32 . Similar to human MBs, 4 we showed that a minor cell population in tumor lesions-including many Sox2 + cells-expressed Olig2 protein ( Figure 4E; Supplementary Figure S5F). Specific expression of Olig2 in Sox2 + SHH-MB cells may provide a mechanism for the resistance to p53dependent p21-mediated cell-cycle arrest during SHH-MB formation and radiation treatment.

Sox2 + SHH-MB Cells Are Most Resistant to p53-Pathway Activation
Inhibition of p53 acetylation by Olig2 raises the possibility that Sox2 + SHH-MB cells are more resistant to p53-pathway activation upon radiation treatment. To test this idea, we sought to determine the mechanism for selective accumulation of mutant p53 ΔE5-6 protein in tumor and stressed cells. First, we showed that p53 ΔE5-6 detection by a p53 antibody was specifically eliminated by p53-specific siRNAs, but not mismatched p53 siRNAs with substitutions of three nucleotides (Supplementary Figure S6A and B). These results demonstrate that, despite deletion of the DBD, the other domains of the p53 ΔE5-6 protein are expressed and recognized by this p53 antibody. Second, we showed that MDM2/Mdm2 could bind and degrade mutant p53 protein encoded by TP53 ΔE5-6 /p53 ΔE5-6 in both human and mouse cells, demonstrating a mechanism for preventing p53 ΔE5-6 accumulation in normal cells (Supplementary Figure  S6C-F). 22 Due to a lack of transcriptional activity, however, the p53 ΔE5-6 protein disrupts the p53-Mdm2 negative feedback loop, increasing the half-life of the p53 ΔE5-6 protein in stressed and tumor cells. 33 Thus, these results establish selective accumulation of mutant p53 ΔE5-6 protein as an in vivo marker for p53-pathway activation in tumor and stressed cells. We next explored differential activation of p53-mediated apoptosis in GCPs at P0.5 and P8 following radiation treatment. 34,35 As previously described, no accumulation of p53 WT or p53 ΔE5-6 protein was detected in the GCPs of the cerebella of P0.5 p53 WT/WT or p53 ∆E5-6/∆E5-6 mice 3 h after low (0.25 Gy) or high (3 Gy) dosage radiation treatment ( Figure  5A and B). After treating P8 p53 WT/WT mice with high-dosage radiation, a robust apoptotic response was accompanied by accumulation of p53 WT protein in some cells within the highly proliferative outer layer of the EGL (oEGL), exclusively comprised of proliferating GCPs ( Figure 5C and D). 8,34,35 Importantly, selective accumulation of p53 ΔE5-6 protein was uniformly observed in proliferating GCPs of in the oEGL of radiation-treated p53 ∆E5-6/∆E5-6 mice, despite no evidence of apoptosis ( Figure 5C and D). Furthermore, accumulation of p53 ΔE5-6 , not p53 WT protein, was detected in proliferating GCPs even after low-dosage radiation, which was insufficient to detect apoptosis in proliferating GCPs at P8 (Figure 5C and D). Thus, selective accumulation of p53 ΔE5-6 protein provides a sensitive marker to identify proliferating GCPs that trigger radiation-induced p53-mediated apoptosis, which otherwise would be eliminated and undetectable in the presence of p53 WT function in vivo. 34,35 Using the p53 ΔE5-6 as a marker, we showed that whereas bulk tumor cells exhibited robust expression of mutant p53 ∆E5-6 protein, Sox2 + cells rarely expressed a detectable level of p53 ∆E5-6 protein in SHH-MBs even following high-dose Neuro-Oncology Advances radiation ( Figure 5E). Furthermore, Sox2 + cells were relatively more resistant to p53-mediated apoptosis, despite a widespread apoptotic response in the entire MB areas of Ptch1 +/− p53 WT mice under high doses of radiation treatment ( Figure 5F). These observations demonstrate that, similar to P0.5 GCPs, a lack of mutant p53 ∆E5-6 accumulation in Sox2 + cells within p53-mutant SHH-MBs accurately predicts the resistance to p53-pathway activation observed in Sox2 + MB-stem cells within p53-WT SHH-MBs.

High SOX2 Expression Is Associated With Poor Survival of TP53-WT SHH-MBs
We sought to determine whether SOX2 expression affected patient survival in four recently identified SHH-MB subgroups. 2 High levels of SOX2 expression were only observed in SHH-MBs, but not WNT, Group 3 or 4 subtypes ( Figure 6A). However, significant variations in SOX2 expression were observed between the 4 SHH-MB subgroups: SHH-MBα and SHH-MBδ subgroups expressed SOX2 at higher levels than SHH-MBβ and SHH-MBγ subgroups ( Figure 6A). Accordingly, we investigated the association between SOX2 expression and patient survival within each SHH-MB subgroup. In the SHH-MBα subgroup, TP53 mutations are associated with poor survival; thus, we investigated the association between differential SOX2 expression levels and survival in TP53-WT SHH-MBα, as well as overall ( Figure 6B; Supplementary Figure S7A). Importantly, more TP53-WT SHH-MBα patients with high SOX2 expression (3 of 8 [37.5%]) died than those with low levels of SOX2 expression (1 of 17 [6%]) ( Figure 6B). As TP53 mutation is not a prognostic factor for the three non-SHH-MBα subgroups, we investigated the relation between SOX2 expression and prognosis in each of these subgroups without excluding any tumors ( Figure 6C and D). Patient death was rare in the SHH-MBδ subgroup (0/18, 0%) and the SHH-MBγ subgroup (1/27, 4%) with low levels of SOX2 expression, whereas significantly more deaths occurred in tumors with high levels of SOX2 expression in SHH-MBδ (10/39, 26%) and in SHH-MBγ (3 of 9, 33%) subgroup ( Figure 6C and D). As the SHH-MBβ subgroup has been shown to see more frequent metastases which directly correspond to worse prognosis, we investigated SOX2 expression levels and survival in non-metastatic (M0) SHH-MBβ, as well as overall ( Figure   Atoh1 Atoh1  Figure  6E). These patient data demonstrate that the high SOX2 expression corresponds to poor survival, supporting our observation that Sox2 + SHH-MB cells may contribute to tumor recurrence.

Discussion
The effective treatment response frequently observed in TP53-WT SHH-MBs led to the widely held belief that a lower dosage therapeutic approach, which could carry less neurotoxic burden, might be clinically efficacious. 12,13 However, recent clinical trials of de-escalation of therapy have not met with success. 14 Our study revealed that a rare population of SOX2 + /Sox2 + cells may be responsible for treatment resistance in TP53 WT /p53 WT SHH-MBs. Although previous research has shown that Sox2 + cells are more resistant to chemotherapy compared with Sox2 − bulk tumor cells in SHH-MBs with wild-type p53 21 , the mechanism by which Sox2 + cells evade therapy remains unclear. We therefore investigated the mechanism by which these cells remain even when surrounding cells were eliminated. Using SHH-MB models carrying two different p53 mutant alleles, [22][23][24] we show a molecular mechanism for the resistance of Sox2 + cells to chemo/radiation therapy. First, we investigated relative sensitivity of Sox2 + versus Sox2 − cells to p53-pathway activation in vivo by exploring a conditional in-frame p53 deletion mutant allele. Mutant p53 ∆E5-6 protein lacks transcriptional activity, but retains the ability to be bound and degraded by Mdm2. Thus, expression of mutant p53 ∆E5-6 protein was undetectable in most normal cells (likely due to basal Mdm2 activity). However, upon p53-pathway activation, mutant p53 ∆E5-6 protein rapidly accumulated and sustained high levels of expression in tumor cells as-only possible because of a lack of transcriptional activation of Mdm2 and disruption of the negative feedback loop of the p53-Mdm2 regulatory axis. Consequently, we show that Sox2 + cells are more resistant to p53-pathway activation following radiation treatment. In addition, we propose a potential mechanism for this resistance via Olig2 expression in Sox2 + SHH-MB cells, which has shown to acetylate and suppress p53 as well as inhibit p21 expression. 31,32 Second, using the apoptosisdefective p53 R172P mutant allele, we show that Sox2 + cells were resistant to p53-dependent p21-mediated cell-cycle arrest response, whereas radiation-enhanced p53 R172P activation induced massive neuronal differentiation of Sox2 − bulk tumor cells and drove them out of the tumor bed. The similarity in transcriptomes between Sox2 + SHH-MB cells and the recently identified quiescent NEPs, including Olig2 expression, 29 provides a mechanism for the resistance of Sox2 + cells to stress-and therapy-induced p53-pathway activation as well as p21-mediated cell-cycle arrest response. Despite the resistance to p53 R172P -mediated cell-cycle arrest and neuronal differentiation, radiation-enhanced p53 WT

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recurrence. It has been shown that p53 binding targets include both high-affinity binding sites, including cell-cycle arrest targets, and low-affinity sites, including apoptotic targets, are activated at low and high thresholds of p53pathway activation, respectively. 15,18 Since therapeutic activation of p53 leads to the successful treatment of SHH-MBs, we propose that the recent de-escalation trials failed to generate sufficiently high levels of p53-pathway activation to induce apoptosis, and consequently, fail to eliminate Sox2 + cells ( Figure 6F). Following this treatment response, the Sox2 + cells were capable of rapidly reentering the cell cycle and generating a rapidly proliferating recurrent tumor, a behavior comparable to NEPs in the developing cerebellum, which can reenter the cell cycle following injury. 30 These findings emphasize the need to use therapeutic approaches that can effectively eliminate quiescent Sox2 + cells to prevent recurrence. To corroborate this model and the importance of SOX2 + cells would require a clinically challenging   Multiple analyses were conducted in the SOX2 expression datasets to find the expression threshold with the greatest significance (the lowest p-value) between SOX2-high and -low expression values, and the most significant is shown (P-value, Log-rank [Mantel-Cox] test and Bonferroni-corrected Log-rank [Mantel-Cox] test). Frequency of tumor mortality at these thresholds was analyzed (chi-square test). (F) A graphic depicting the overall role of SOX2 + /Sox2 + cells in SHH-MBs.
approach involving analysis of tumor samples at multiple time points following de-escalating therapy. We sought to obtain supporting evidence by investigating the association between high SOX2 expression and overall survival in recently published data sets from human SHH-MBs. 2 We found a significant variation of SOX2 expression among the four SHH-MB subgroups. Strikingly, we found that high SOX2 expression corresponds to poor survival from all four SHH-MB subgroups. Although a larger series of patient data are required to validate these results, these observations provide the evidence supporting the model wherein SOX2 + / Sox2 + SHH-MB cells are more resistant to therapy-induced activation of p53-mediated tumor suppressive responses (e.g., cell-cycle arrest and neuronal differentiation) and responsible for tumor recurrence following de-escalating therapies. Together, our study provides important insights into the stratification of children with TP53-WT SHH-MBs based on the levels of SOX2 expression that may be considered for the design of future de-escalation of therapy trials.

Funding
This work was supported by grants from the National Institutes of Health (2P01 CA085878-10A1 and 1R01 NS053900) and Cancer Biology Training is an NIH -NCI T32 grant (5T32CA009676-22) and the Cellular and Molecular Biology is an NIH -NIGMS T32 grant (5T32GM007315-43), University of Michigan.