CK1α agonists attenuate medulloblastoma stemness and relapse risk

CK1α agonists attenuate medulloblastoma stemness and relapse risk | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CK1α agonists attenuate medulloblastoma stemness and relapse risk Jezabel Rodriguez-Blanco, Kendell Peterson, Maria Turos-Cabal, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7915551/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract While outcomes for most children with medulloblastoma (MB) are relatively favorable, those in the Sonic Hedgehog (SHH) subgroup with Tumor Protein P53 ( TP53 ) mutations–known as the SHHα subtype–face a much poorer prognosis. SHHα patients relapse more frequently and rapidly, underscoring the need for therapies that prevent recurrence. We recently identified a Gli-driven Sox2⁺ cell population that promotes relapse in SHH MB. However, few Gli-targeting strategies have shown clinical promise to date. One translational Gli inhibitor is pyrvinium, an FDA-approved compound known to destabilize Gli through increasing Casein Kinase 1α (CK1α) activity. In this study, we tested whether pyrvinium and a brain-permeable derivative, SSTC3, affect stemness and relapse risk in mouse and human-derived SHHα MB models. We found that pyrvinium suppresses the Gli-driven proliferation of Sox2⁺ cells. Unlike other SHH/Gli-targeting approaches, pyrvinium also impaired MB self-renewal by depleting Cluster of Differentiation 15 (CD15)⁺ cells. Mechanistic studies revealed that CD15⁺ cell self-renewal is WNT-dependent and driven by the loss of p53/ microRNA-34a –mediated repression of WNT signaling. Remarkably, pyrvinium and SSTC3 reduced Sox2⁺, CD15⁺, and dual Sox2/CD15-labeled populations in mouse and patient-derived SHHα models. Consistent with their ability to diminish tumor stemness, pyrvinium also impaired primary and secondary tumor engraftment. Together, these findings highlight the translational potential of the CK1α agonist pyrvinium and its derivatives for patients with very high-risk SHHα MB. Biological sciences/Cancer/CNS cancer Biological sciences/Cancer/Paediatric cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Brain tumors are the leading cause of cancer-related death in children, with medulloblastoma (MB) being the most common malignant form( 1 ). From the four major molecular subgroups, the Sonic Hedgehog (SHH) subgroup accounts for approximately 30% of cases( 2 ). Clinical outcomes in this subgroup are strongly influenced by co-occurring genetic alterations. In particular, Tumor Protein P53 ( TP53 ) mutations, present in ~ 7–30% of cases at diagnosis( 3 – 5 ) and ~ 30% at recurrence( 4 ), are associated with increased relapse risk and shorter time to relapse( 3 ), and thus these tumors are classified as very high-risk( 6 ). Given their dismal outcomes, there is an urgent need to understand the mechanisms driving relapse in TP53 -mutant SHH MB, also known as SHHα( 7 ). Emerging evidence suggests that a rare population of MB progenitor cells (MPCs), often marked by the stemness factor SRY-box transcription factor 2 (Sox2), may contribute to disease recurrence( 8 ). Our previous work in SHH MB showed that the expansion of Sox2⁺ cells relies on non-canonical Gli activity, rendering them resistant to Smoothened (Smo) inhibitors in clinical development( 9 ). By contrast, compounds that block Gli-dependent transcription via bromodomain and extra-terminal (BET) inhibition reduced Sox2⁺ cells and relapse risk in SHH MB animal models( 9 ). Nevertheless, the toxicity associated with BET inhibitors in clinical settings( 10 ) underscores the need for alternative Gli-targeting approaches to prevent MB recurrence. One translational approach to target Gli involves activating Casein kinase 1α (CK1α). CK1α is a serine/threonine kinase that phosphorylates Gli, promoting its degradation( 11 ). Small-molecule CK1α agonists, such as the FDA-approved anti-helminthic drug pyrvinium, selectively bind CK1α and function as allosteric activators( 12 ). Through CK1α activation, pyrvinium and a brain-permeable derivative, SSTC3, attenuate SHH-driven MB growth( 13 , 14 ). However, CK1α also phosphorylates β-catenin, an essential transcriptional activator in the Wingless-related integration site (WNT) pathway, targeting it for proteasomal degradation( 15 ). Thus, CK1α agonists extend their effect beyond Gli, as both pyrvinium and SSTC3 block WNT signaling and suppress the growth of WNT-driven malignancies( 16 , 17 ). Given their dependency on Gli for propagation, we hypothesized that CK1α agonists deplete Sox2⁺ cells and reduce SHHα relapse risk. Accordingly, the CK1α agonist pyrvinium reduced Gli levels and the proliferation of Sox2-enriched MB cultures, specifically targeting Sox2 + cells. Curiously, while other SHH-targeting strategies failed to block the self-renewal of SHHα MB cultures, pyrvinium did so at low nanomolar doses. As we had previously shown that self-renewal in SHHα MB is WNT-driven( 18 ), we investigated whether pyrvinium’s effects on secondary sphere formation assays were WNT-dependent. Consistently, pyrvinium reduced WNT signaling in SHHα MB cultures, and its impact on self-renewal was rescued by constitutive WNT activation. Thus, similar to pyrvinium, a compound blocking β-catenin-driven transcription( 19 ), PKF115-584, inhibited the self-renewal of CD15-sorted cells. In vivo, pyrvinium reduced the numbers of Gli1 + /Sox2⁺ and β-catenin⁺/CD15⁺ cells, as well as those of Sox2 + /CD15 + cells in a subcutaneous SHHα MB model. Similarly, the pyrvinium derivative SSTC3 reduced Sox2⁺, CD15⁺, and Sox2⁺/CD15⁺ cells in orthotopic mouse- and human-derived SHHα MB. Furthermore, pyrvinium reduced primary sphere formation and tumor engraftment. Our data underscores the potential of repurposing the FDA-approved CK1α agonist pyrvinium or translating its derivatives for very high-risk SHHα MB. MATERIAL AND METHODS Mouse studies All procedures were approved by the IACUC at the Medical University of South Carolina (MUSC) and the University of Miami (UM). Ptch1 TM1MPS /J 66 ( Ptch1-LacZ ) and B6.129S2-Trp53 tm1Tyj /J 67 ( Trp53 -mutant) mice (Jackson Laboratory) were crossed to establish a colony. Spontaneous tumors were expanded and maintained as allografts in CD1-Foxn1 nu mice (Charles River). For pyrvinium (Enzo or Sigma-Aldrich) treatment, 1×10 6 viable cells (trypan blue–excluded) were implanted subcutaneously (s.c.) into similar mice. Once tumors reached ~ 200 mm³, mice received pyrvinium (0.8 mg/kg, s.c.) near the tumor every other day (q.o.d.). Tumors were harvested 6 hours (h) after the final dose and either fixed or dissociated with Papain (Worthington) or Accutase (Invitrogen) for FACS, sphere formation, or re-engraftment assays. For flank re-engraftment, indicated viable cells were implanted s.c. into CD1-Foxn1 nu mice. For orthotopic re-engraftment, 1×10 4 viable cells from pyrvinium-treated tumors were resuspended in 3 µL Neurobasal-A media and implanted into the cerebellum( 18 ). For primary engraftment, MPC-2 cultures were treated with 200 nM pyrvinium for 24h before s.c. implantation. For SSTC3 (StemSynergy Therapeutics) studies, mice were orthotopically implanted with 1×10 5 Ptch1-LacZ; Trp53 -mutant cells or 1×10 6 TP53 -mutant patient-derived xenograft (PDOX) cells (SJSHHMB-14-7196, courtesy of Dr. Roussel, St. Jude). Ten- and thirty-day post-implantation, respectively, mice received daily (q.d.) intraperitoneal (i.p.) injections of vehicle or SSTC3 (10 mg/kg) for 3 days. Brains were then harvested for flow cytometry (Accutase) or IHC. Bioinformatic analyses GSE85217 (Cavalli 2017 dataset)( 7 ) and GSE68015 (Gump 2015 dataset)( 20 ) transcriptomic data were downloaded from the GlioVis data portal( 21 ). Statistical Analysis Results represent the mean ± SEM from at least three independent experiments. For BrdU staining, four fields per condition from three experiments were quantified. IHC quantification reflects the mean ± SEM from at least three fields across three or more mice. Flow cytometry data from tumor-derived cell suspensions represent the mean ± SEM from at least three mice per condition. Multiple group comparisons used one-way ANOVA with post-hoc Dunnett analysis. Two-sample comparisons used one-tailed Student’s t-tests. Symptom-free survival was assessed using Log-rank (Mantel-Cox) tests. Patient outcome analysis employed maximally ranked statistics to identify the optimal CSNK1A1 expression cut point (9.1). Tumor engraftment significance was determined using a χ² test. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. Additional Material and Methods are described in Supplemental Methods. RESULTS Pyrvinium blocks Gli1-driven Sox2⁺ cell proliferation We previously showed that the propagation of MB cultures enriched in Sox2 + cells, herein referred to as MPC cultures, is driven by non-canonical Gli activation downstream of Smo( 9 ). Because CK1α destabilizes Gli( 14 , 16 ), we tested the efficacy of pyrvinium, a known CK1α agonist( 12 ), in MPC cultures. Given that most SHH MB relapses are associated with TP53 mutations( 3 , 5 ), we focused on Trp53 -mutant cultures. According to its mechanism of action, pyrvinium not only reduced Gli1 -driven promoter activity in a reporter cell line (Fig. 1 A) but also decreased the SHH-driven cell proliferation of the granule layer of the developing cerebellum in organotypic cultures (Fig. 1 B; Supplemental Fig. 1A ). In MPC cultures, nanomolar concentrations of pyrvinium reduced Gli1 levels (Fig. 1 C, D) and the number of Gli1⁺ cells (Fig. 1 E), thereby attenuating the expression of SHH target genes (Fig. 1 F). Furthermore, pyrvinium reduced the number of viable cells in MPC cultures, with an average EC50 of 17 nM (Fig. 1 G), which is consistent with their dependency on Gli to propagate( 9 ). Given pyrvinium’s ability to reduce the number of viable cells in MPC cultures, we investigated whether this effect was due to decreased proliferation or increased cell death. BrdU incorporation assays suggested that pyrvinium’s effects are primarily attributed to reduced proliferation, as evidenced by a drop in BrdU + cells (Fig. 1 H; Supplemental Fig. 1B ). This was further supported by the reduction in levels of proliferation markers, such as Cyclin-D1 and PCNA (Fig. 1 I, J), as well as cells labeled for the proliferation marker Ki67 (Fig. 1 K). In contrast, pyrvinium had only minimal effects on cell death, as determined by the number of cleaved Caspase-3-labeled cells (Fig. 1 H; Supplemental Fig. 1B ) and its protein levels (Fig. 1 I, J). Importantly, similar to other Gli inhibitors( 9 ), pyrvinium also reduced the number of Sox2⁺ cells in MPC cultures (Fig. 1 L), including those Sox2 + cells co-labeled with Gli1 and Ki67 (Fig. 1 M). Together, these data suggest that pyrvinium depletes Sox2⁺ cells by blocking their Gli-driven proliferation. Pyrvinium attenuates MB self-renewal MPCs are known to self-renew to maintain tumor stemness( 22 ). Interestingly, while our previous work showed that Gli inhibition by targeting BET proteins reduced MPC culture proliferation( 9 ), the ability of these drugs to block secondary sphere formation, used as a measure of tumor stemness( 23 ), was not tested at that time. To evaluate this, we used self-renewal protocols in which MPC cultures were exposed to drugs of interest for 24h before disaggregating spheres( 18 ). Equal numbers of viable cells were next re-plated and allowed to form new spheres in the absence of drug (Fig. 2 A). These studies revealed that three structurally distinct BET inhibitors (I-BET151, JQ-1, BMS-986158) fail to block the self-renewal of MPC cultures (Fig. 2 B) at concentrations we previously showed to attenuate SHH signaling and reduce MPC viability( 9 ). Similarly, the compound acting on Smo, vismodegib, failed to impair MPC secondary sphere formation (Fig. 2 C), suggesting that MPC self-renewal is not SHH/Gli-driven. Accordingly, siRNA pools targeting Gli1 and Gli2 also failed to inhibit MB self-renewal (Fig. 2 D). In contrast, pyrvinium attenuated secondary sphere formation in MPC cultures, with an average EC50 of 25 nM (Fig. 2 E). Given pyrvinium’s effect on MB self-renewal ex vivo , we studied whether it similarly affects tumor initiation. MPC cultures were exposed to pyrvinium before implanting limiting dilutions of viable cells into mice. Tumor initiation was observed from the vehicle-treated cultures, even when only 150,000 cells were implanted. However, pyrvinium-treated cells did not form tumors, even when nearly tenfold higher cell numbers were used (Fig. 2 F). Together, these findings suggest that pyrvinium blocks MB self-renewal by targeting a pathway different than SHH/Gli. Pyrvinium inhibits MB self-renewal by targeting WNT signaling In addition to regulating Gli stability( 11 ), CK1α is also known to prime β-catenin for degradation( 15 ), thereby suppressing WNT signaling. Moreover, our previous findings indicated that self-renewal in Trp53 -mutant MB is driven by WNT activation( 18 ). Thus, we wondered whether pyrvinium’s effects on self-renewal operate through WNT inhibition. Supporting this idea, pyrvinium reduced WNT activity in a TCF/LEF1 reporter assay (Fig. 3 A) and decreased both β-catenin levels (Fig. 3 B, C) and expression of WNT target genes (Fig. 3 D) in MPC cultures. Furthermore, overexpression of a constitutively active β-catenin form ( S33Y mutant) increased baseline expression of WNT biomarkers (Fig. 3 E) and reduced pyrvinium’s efficacy on MPC self-renewal (Fig. 3 F), supporting a WNT-dependent mechanism of action. Although our prior work linked WNT activation to p53 loss in MPC cultures( 18 ), the mechanism driving elevated WNT signaling remained unclear. One possible explanation involves microRNAs ( miRNAs ), small non-coding RNAs that bind to 3’ UTRs of target mRNAs and suppress their translation( 24 ). p53 loss has previously been associated with downregulation of miR-34a , leading to de-repression of WNT components such as LEF1( 25 ), a key member of the β-catenin transcriptional complex( 26 ). Accordingly, Trp53 -mutant MPC cultures exhibited reduced levels of miR-34a compared to wild-type counterparts (Fig. 3 G). Introduction of a mimic-miR-34a into Trp53 -mutant MPC cultures decreased LEF1 levels, whereas inhibition of miR-34a using an anti-miR increased them (Fig. 3 H). Accordingly, an anti-miR-34a increased WNT target gene expression in Trp53-wild-type MPCs (Fig. 3 I). Further implicating miR-34a in MB self-renewal, a mimic-miR-34a reduced secondary sphere formation in Trp53 mutant MPC cultures, while an anti-miR-34a had minimal effect (Fig. 3 J). These findings suggest that loss of p53 reduces miR-34a levels in MPC cultures, thereby enabling WNT activation (Fig. 3 K). Pyrvinium depletes a self-renewing CD15⁺ cell population The ability of BET inhibitors to deplete Sox2 cells( 9 ) without affecting self-renewal suggests the involvement of an alternative cell pool. To define the self-renewing compartment targeted by pyrvinium, we focused on CD15⁺ cells, which were previously shown to represent a tumor-initiating population in SHH MB( 27 – 29 ). CD15⁺ cells were sorted from Ptch1-LacZ, Trp53 mutant tumors, using Ter-119 to deplete erythroid lineage cells (Fig. 4 A). Compared to CD15⁻ cells, CD15⁺ cells formed more spheres, and pyrvinium reduced these numbers (Fig. 4 B). Furthermore, linking CD15 + self-renewal to WNT signaling, similar to pyrvinium, a compound that blocks β-catenin/LEF1 interaction, PKF115-584( 19 ), impaired sphere formation in similarly sorted cells, while SHH inhibition by vismodegib failed to do so (Fig. 4 B). These findings suggest that pyrvinium targets a WNT-driven CD15⁺ population. Gene expression analysis further supported this idea, as CD15-sorted cells showed elevated expression of multiple WNT target genes compared to CD15⁻ cells, while SHH target gene levels were similar (Fig. 4 C). Furthermore, treatment of MPC cultures with pyrvinium reduced the CD15⁺ cell population (Fig. 4 D). Like pyrvinium, PKF115-584 reduced CD15 + cell numbers, whereas vismodegib had no effect (Fig. 4 E). Together, these results suggest that pyrvinium targets a WNT-driven CD15⁺ cell population critical for self-renewal. A Sox2⁺/CD15⁺ population was previously mentioned in the literature( 30 ), but its regulation remains unclear. Given its ability to deplete both Sox2⁺ and CD15⁺ cells, we asked whether the CD15⁺ cells targeted by pyrvinium also express Sox2. Flow cytometry analyses showed that most CD15⁺ cells in MPC cultures are Sox2⁺, and pyrvinium reduced the number of these double-positive cells (Fig. 4 F). WNT inhibition with PKF115-584 similarly reduced Sox2⁺ cells (Fig. 4 G) and Sox2⁺/CD15⁺ cells (Fig. 4 H), whereas vismodegib did not (Fig. 4 G, H). In contrast, Gli inhibition by targeting BET reduced Sox2⁺ cells, but did not affect CD15⁺ or Sox2⁺/CD15⁺ cells (Fig. 4 I). Together, these findings suggest that pyrvinium depletes Smo inhibitor–resistant, Gli1-driven Sox2⁺ cells, including a WNT-driven CD15⁺ subset (Fig. 4 J). CK1α agonists reduce MB stemness Given pyrvinium’s ability to deplete Sox2⁺ and CD15⁺ cells in MPC cultures, we tested whether it does the same in vivo. Mice were implanted subcutaneously with Ptch1-LacZ, Trp53 -mutant MB cells, and allowed to form tumors. Because of its poor systemic bioavailability( 31 ), pyrvinium was administered subcutaneously near the tumor site( 16 ). This local dosing suppressed tumor growth (Fig. 5 A; Supplemental Fig. 1C ), reduced proliferative index, and decreased the number of Gli1⁺ and Sox2⁺ cells, including double-positive ones (Fig. 5 B; Supplemental Fig. 2 ). Pyrvinium also depleted CD15⁺ and β-catenin⁺ cells, including a WNT-labeled CD15⁺ subset, and diminished the Sox2⁺/CD15⁺ population within these tumors (Fig. 5 B; Supplemental Fig. 2 ). Pyrvinium’s poor bioavailability limits its use to subcutaneous tumor models( 31 ). To test whether CK1α agonists reduce MB stemness in orthotopic MB, we used a brain-permeable derivative of pyrvinium, SSTC3( 14 , 17 ). Like pyrvinium, SSTC3 suppressed SHH and WNT target gene expression (Fig. 5 C), and reduced cell viability (Fig. 5 D) and self-renewal (Fig. 5 E) in MPC cultures. In mice bearing orthotopic Ptch1-LacZ, Trp53-mutant tumors, SSTC3 reduced the proliferation index and the numbers of cells labeled for SHH as well as for WNT makers (Fig. 5 F; Supplemental Fig. 3 ). SSTC3 also reduced overall Sox2⁺ cells, including Gli1⁺ ones, and decreased CD15⁺, CD15⁺/β-catenin⁺, as well as Sox2⁺/CD15⁺ populations (Fig. 5 F; Supplemental Fig. 3 ). To determine whether the efficacy of CK1α agonists goes beyond murine models, we tested SSTC3 in a TP53 -mutant SHH PDOX model. Despite a higher baseline of Sox2⁺ cells compared to mouse-derived tumors, SSTC3 maintained its ability to reduce Sox2⁺, CD15⁺ cells, and Sox2⁺/CD15⁺ cells (Fig. 5 G; Supplemental Fig. 1D ), supporting efficacy across systems. Pyrvinium reduces MB relapse risk Based on their ability to reduce MB stemness and ex vivo self-renewal, we next examined whether CK1α agonists would also impair tumor-propagating potential using relapse-risk protocols in which primary sphere formation is assessed along with secondary tumor engraftment and time to engraftment (Fig. 6 A). Due to its FDA approval and ongoing cancer clinical trials (NCT05055323, NCT06782048, NCT06590454), we focused these studies on pyrvinium. In primary sphere formation assays, in which equal numbers of viable cells were plated and allowed to form spheres, results showed that pyrvinium-treated tumors fail to propagate in culture (Fig. 6 B). Similarly, limiting dilution assays in which equal numbers of viable tumor cells were subcutaneously re-implanted showed differences in engraftment ability between vehicle and pyrvinium-treated tumors (Fig. 6 C). Moreover, orthotopic re-engraftment showed that tumors derived from pyrvinium-treated residual disease took longer to establish (Fig. 6 D). These results show pyrvinium’s potential to reduce SHHα MB relapse risk. Furthermore, analysis of patient datasets showed that high expression of the gene coding for CK1α ( CSNK1A1 ) correlates with better MB outcomes (Fig. 6 E) and, suggestive of a likely therapeutic window, CSNK1A1 is more expressed in MB versus normal brain (Fig. 6 F). SHH tumors expressed higher CSNK1A1 than other MB subgroups (Fig. 6 G), with SHHα cases showing the highest expression (Fig. 6 H). Since high basal CK1α may favor agonist activity, these findings suggest SHH MB, especially TP53 -mutant, could be particularly responsive to CK1α agonists, providing a rationale for advancing intrathecal pyrvinium dosing or systemic administration of improved derivatives into clinical trials for this very high-risk MB subtype. DISCUSSION Here, we demonstrate that compounds activating CK1α attenuate MB stemness and, consequently, reduce relapse risk in animal models. Our findings suggest that these drugs function as dual inhibitors, simultaneously blocking Gli and WNT signaling, key drivers of Sox2⁺ cell proliferation and CD15⁺ self-renewal, respectively. The ability of CK1α agonists to attenuate MB stemness is particularly critical in SHHα MB, which accounts for ~ 75% of SHH MB recurrences( 3 ), and shows 0–41% five-year-survival after relapse( 3 , 5 ). While we cannot exclude the possibility that pyrvinium and SSTC3 also attenuate stemness in TP53 -wild-type SHH MB, this study was focused on the mutant subtype due to the clinical need they represent. We previously showed that Sox2⁺ MB cells depend on the activation of Gli downstream of Smo, and that these cells are susceptible to depletion by BET inhibitors, which block Gli transcriptional activity( 32 , 33 ). However, efforts to translate these findings have been limited by the clinical toxicity of BET inhibitors( 34 ) and the recent termination of a pediatric brain tumor trial testing them (NCT03936465). Dose-limiting toxicities of BET inhibitors underscores the need for alternative approaches to suppress Gli transcriptional activity. Because Gli proteins are transcription factors and therefore difficult to target directly( 35 ), we focused instead on regulatory mechanisms controlling Gli stability. Since CK1α phosphorylates GLI, priming it for degradation( 36 ), we tested pyrvinium—a previously characterized CK1α agonist( 12 )—in MB cultures enriched for Sox2⁺ cells( 9 ). Pyrvinium suppressed SHH signaling and reduced the proliferation of Sox2⁺ cells. Thus, this drug shows efficacy like that of BET inhibitors, but with the potential advantage of lower toxicity. Besides breast cancer( 37 ), CK1α agonists have been reported to attenuate glioblastoma stemness by targeting CD133⁺ cells ( 38 ). Although early work described CD133⁺ MB cells as the self-renewing pool( 39 ), subsequent studies pointed to CD15⁺ cells instead ( 28 , 29 ). Thus, based on our data showing that pyrvinium blocks MB self-renewal, we studied whether pyrvinium targets CD15⁺ cells. Here, we showed that pyrvinium depletes a self-renewing CD15 + cell pool, and it does so by targeting WNT signaling. This aligns with previous evidence implicating WNT signaling in regulating cancer stemness( 40 ). However, while previous works implicated CD15 cells with WNT signaling in retina progenitors( 41 ), to our knowledge, this is the first study to link CD15⁺ cells with WNT signaling in cancer. Mechanistically, we found that, similar to previous observations( 25 ), WNT activation in SHHα MPC cultures is associated with the loss of p53 /miR-34a -mediated repression of WNT components such as LEF1. While additional studies are needed to clarify why WNT signaling is specifically required in CD15⁺ cells or which other WNT signaling components are repressed by miR-34a in MB, our findings identified a connection between p53 loss, WNT signaling, and CD15-driven MB self-renewal. Our data support that pyrvinium exerts its effects on stemness by inhibiting Gli and WNT signaling. However, pyrvinium has been reported to inhibit AKT signaling( 42 ), a pathway known to promote survival and maintenance of tumor stem-like cells( 43 ), including those in MB( 44 ). Similarly, pyrvinium can attenuate STAT3 activity( 45 , 46 ), which is a critical regulator of stemness and self-renewal in multiple cancers( 47 ), including MB( 48 ). Thus, the anti-stemness effects of pyrvinium may involve the suppression of a broader group of oncogenic signaling programs that converge on regulating stemness. While our work primarily focused on pyrvinium due to its FDA approval and its use in recently launched trials for pancreatic and gastric cancer (NCT05055323, NCT06782048, NCT06590454), we also evaluated its brain-permeable derivative, SSTC3. In orthotopic SHHα MB models, SSTC3 reduced stemness markers. Moreover, we previously showed that this compound also debulks tumor tissues and prolongs survival in SHH MB orthotopic models, including in a TP53 -mutant PDOX( 14 ). These previous findings suggest that the efficacy of CK1α agonists extends beyond the herein described stemness regulation and supports their further development for very high-risk SHHα MB patients. Declarations CONFLICTS OF INTEREST The authors declare no conflicts of interest. ACKNOWLEDGMENTS We are deeply grateful to the Childhood Brain Tumor Foundation (CBTF) and the families supporting this initiative, which funded the naive idea of a postdoc for testing the effect of pyrvinium on MB stemness. We also thank StemSynergy Therapeutics Inc. for providing SSTC3 and the laboratories of Drs. Robbins, Capobianco, Lee, and Guttridge for their helpful insights and discussions regarding this manuscript. We would also like to thank the Histology and Immunohistochemistry Laboratory (Dept. of Pathology and Laboratory Medicine), and the Biorepository & Tissue Analysis, the Translational Science Lab, and the Flow Cytometry & Cell Sorting shared resources at the HCC (MUSC) supported by a CA138313 P30 grant, and the UM Flow Cytometry Shared Resource and the Department of Surgery Tissue and Pathology core for their work on tissue processing and flow analyses. Schematics were created using BioRender.com. Grammarly and ChatGPT were used to assist in the editing phase of this manuscript. This work was supported by CBTF (to J. R.-B.), Monka Foundation (to J.R.-B.), NINDS K01NS119351 and 1R01NS138021 grants (to J.R.-B.), ALSF “A” Award 23-28298 (to J.R.-B.), FICYT POST10-27 (to J.R.-B.), NCI R00 CA241367 (to T.B.), SREB SC15321 (to K.P.), NCATS TL1 TR001451 & UL1 TR001450TL1 (to K.P.), HCC LOWVELO (to V.K. and M.T.-C.), MUSC College of Graduate Studies Odyssey (to P.S.), and HCC BLOCKS (to L.F.). DATA AVAILABILITY All data presented in this manuscript will be made available upon request. References Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016;131(6):803–20. Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S, et al. Medulloblastoma Comprises Four Distinct Molecular Variants. J Clin Oncol. 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Additional Declarations There is no duality of interest Supplementary Files SupplementalMethodsPeterson.docx Supplemental Methods PetersonBlots.pdf Uncropped Blots SupplementalCK1a.pdf Supplemental Figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7915551","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":535900410,"identity":"527691c3-111a-44b3-9ec2-29f272701819","order_by":0,"name":"Jezabel 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17:15:32","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18021,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMethodsPeterson.docx","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/3c7585b9779cb0f5e087bb4f.docx"},{"id":95669833,"identity":"14816137-fb7c-426e-b7f2-f5007adfcbd6","added_by":"auto","created_at":"2025-11-11 17:15:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":752407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePyrvinium attenuates SHH signaling in MPC cultures\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e. SHH signaling was induced in \u003cem\u003eGli1\u003c/em\u003e reporter cells with 100 nM SAG for 24h, followed by treatment with the indicated concentrations of pyrvinium for an additional 24h. \u003cstrong\u003eB\u003c/strong\u003e. Organotypic cerebellar slices from 7-day-old mice were treated with 200 nM pyrvinium for 24h, followed by a BrdU pulse. Quantification and representative BrdU staining images (scale bar 100 µm) \u0026nbsp;are shown. Data was analyzed using an unpaired t-test. Calbindin was used to label the Purkinje layer. \u003cstrong\u003eC\u003c/strong\u003e. Gli1 levels were assessed by immunoblotting in MPC-2 cultures treated with increasing concentrations of pyrvinium for 24h. \u003cstrong\u003eD\u003c/strong\u003e. Gli1 levels were similarly evaluated in the indicated MPC cultures treated with 200 nM pyrvinium for 24h. \u003cstrong\u003eE\u003c/strong\u003e. MPC-1 cultures were treated with the indicated concentrations of pyrvinium for 24h, and Gli1-labeled cells were quantified by flow cytometry. \u003cstrong\u003eF\u003c/strong\u003e. Expression of SHH target genes was measured by RT-qPCR in MPC-2 cultures treated with increasing concentrations of pyrvinium for 24h. EC\u003csub\u003e50\u003c/sub\u003es were calculated using non-linear regression.\u003cstrong\u003e G\u003c/strong\u003e. MPC cultures were treated with pyrvinium for 72h, and cell viability was assessed using an MTT assay. EC50s were calculated using non-linear regression analyses.\u003cstrong\u003e H\u003c/strong\u003e. MPC-2 cultures were exposed to 200 nM pyrvinium for 24h before assessing BrdU incorporation and cleaved Caspase-3⁺ (C-Casp3). Quantification of positive cells and representative images (scale bar 200 µm) are shown. Data was analyzed using an unpaired t-test. \u003cstrong\u003eI\u003c/strong\u003e. Levels of the indicated proteins were assessed in extracts from MPC-2 cultures treated with increasing concentrations of pyrvinium for 24h. \u003cstrong\u003eJ\u003c/strong\u003e. Levels of the indicated proteins were assessed in extracts from the indicated MPC cultures treated with 200 nM pyrvinium for 24h.\u003cstrong\u003e K\u003c/strong\u003e. MPC-1 cultures were treated with the indicated concentrations of pyrvinium for 24h, and Ki67-labeled cells were quantified by flow cytometry. \u003cstrong\u003eL\u003c/strong\u003e. MPC-1 cultures were exposed to the indicated concentrations of pyrvinium, and numbers of Sox2⁺ cells were determined by flow cytometry.\u003cstrong\u003e M\u003c/strong\u003e. MPC-1 cultures were exposed to the indicated concentrations of pyrvinium, and numbers of Sox2⁺/Ki67⁺ and Sox2⁺/Gli1⁺ cells were determined by flow cytometry. In all cases, representative flow cytometry plots are shown. Unless otherwise indicated, mean ± SEM of data normalized to DMSO were analyzed using a one-way ANOVA followed by Dunnett’s post-hoc. * p\u0026lt; 0.05, ** p\u0026lt; 0.01, *** p\u0026lt; 0.001, **** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/b57158a9ed95ff7c8e0ac467.png"},{"id":95669834,"identity":"9a17aba7-46db-4123-b210-548bd6165ee7","added_by":"auto","created_at":"2025-11-11 17:15:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePyrvinium attenuates MB self-renewal.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Schematic of the self-renewal protocol. \u003cstrong\u003eB.\u003c/strong\u003e MPC-2 cultures were exposed to increasing concentrations of indicated BET inhibitors for 24h before allowing equal numbers of viable cells to form new spheres for one week. \u003cstrong\u003eC.\u003c/strong\u003e Similar cultures were exposed to increasing concentrations of vismodegib before completing self-renewal protocols. \u003cstrong\u003eD.\u003c/strong\u003e MPC-2 cultures were transfected with siRNA pools targeting \u003cem\u003eGli1\u003c/em\u003e and \u003cem\u003eGli2\u003c/em\u003e, as well as scramble \u003cem\u003esiRNA\u003c/em\u003e (\u003cem\u003esiSC\u003c/em\u003e) and GFP-labeled controls. Seventy-two hours after transfection, spheres were dissociated, and equal numbers of viable cells were replated. A one-way ANOVA was used to analyze data normalized to \u003cem\u003esiSC\u003c/em\u003e. \u003cstrong\u003eE.\u003c/strong\u003e MPC cultures were allowed to form secondary spheres following a 24h incubation with the indicated concentrations of pyrvinium. EC\u003csub\u003e50\u003c/sub\u003es were calculated using non-linear regression. \u003cstrong\u003eF.\u003c/strong\u003e Indicated numbers of viable cells from MPC-2 cultures treated with 200 nM pyrvinium for 24h were subcutaneously implanted. The frequency of tumor engraftment was analyzed using a one-sided χ\u003csup\u003e2 \u003c/sup\u003etest. In all cases, representative images of self-renewal assays are shown. Unless otherwise indicated, mean ± SEM of data normalized to DMSO were analyzed using non-linear regression tests. * p\u0026lt; 0.05, ** p\u0026lt; 0.01, *** p\u0026lt; 0.001, **** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/2336c8e80865d7c0f1ddebb5.png"},{"id":95669835,"identity":"e04a3006-b112-4b1f-9b3d-e9a7a5e953b4","added_by":"auto","created_at":"2025-11-11 17:15:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePyrvinium acts on WNT signaling to block MB self-renewal. A\u003c/strong\u003e. WNT signaling was induced in TCF/LEF1 reporter cells by 100 ng/ml WNT3a and 10 mM LiCl for 24h before treatment with pyrvinium for an additional 24h. Data was analyzed using a one-way ANOVA followed by Dunnett’s post-hoc. \u003cstrong\u003eB\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eThe levels of b-catenin following 24h incubation with increasing concentrations of pyrvinium were determined in MPC-2 cultures by immunoblotting. \u003cstrong\u003eC\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eThe levels of b-catenin following 24h incubation with 200 nM pyrvinium were similarly determined in the indicated MPC cultures. \u003cstrong\u003eD\u003c/strong\u003e. MPC-2 cultures were exposed to indicated concentrations of pyrvinium for 24h before determining the expression of indicated WNT target genes by RT-qPCR. EC\u003csub\u003e50\u003c/sub\u003es were calculated using non-linear regression.\u003cstrong\u003e E. \u003c/strong\u003eMPC-2 cultures were transfected with the indicated vectors and expression of WNT target genes was determined 48h later. Data was normalized to \u003cem\u003epcDNA\u003c/em\u003e control. \u003cstrong\u003eF.\u003c/strong\u003e MPC-2 cultures were transfected with the indicated vectors. 3 days post-transfection cells were treated with 200 nM pyrvinium for an additional 24h before sphere dissociation and re-plating of equal numbers of viable cells. Numbers of secondary spheres were quantified 7 days later. Data was normalized to \u003cem\u003epcDNA\u003c/em\u003e control. \u003cstrong\u003eG\u003c/strong\u003e. The expression of \u003cem\u003emiR-34a-5p\u003c/em\u003e was determined in MPC-2 (\u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e) and MPC-47 (\u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e) \u003cem\u003ePtch1-LacZ\u003c/em\u003e cultures by RT-qPCR and normalized to MPC-2 data. \u003cstrong\u003eH\u003c/strong\u003e. MPC-2 cultures were transfected with specific \u003cem\u003emimic-\u003c/em\u003e or \u003cem\u003eanti-miR\u003c/em\u003e sequences along with respective scramble (\u003cem\u003emiR-SC\u003c/em\u003e) controls, and LEF1 levels were determined by immunoblotting 48h later. \u003cstrong\u003eI\u003c/strong\u003e. MPC-47 cells were transfected with \u003cem\u003eanti-miR-34a\u003c/em\u003e or a scramble control, and the expression of WNT target genes was determined 48h later by RT-qPCR. Results were normalized to the corresponding scramble sequence. \u003cstrong\u003eJ\u003c/strong\u003e. MPC-2 cultures were transfected with indicated \u003cem\u003emimic\u003c/em\u003e- and \u003cem\u003eanti-miR\u003c/em\u003e sequences, and secondary sphere formation was determined 72h later. Results were normalized to the corresponding scramble sequence. \u003cstrong\u003eK\u003c/strong\u003e. Schematic of the mechanism of activation of WNT signaling in \u003cem\u003eTrp53\u003c/em\u003e mutant MPC cultures. In all cases, representative images of wells from sphere formation assays are shown.\u003cstrong\u003e \u003c/strong\u003eUnless otherwise indicated, mean ± SEM data were analyzed using an unpaired t-test. * p\u0026lt; 0.05, ** p\u0026lt; 0.01, *** p\u0026lt; 0.001, **** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/d12bb97a51323f4b0ce80cc7.png"},{"id":95669840,"identity":"2eee48cc-d9ed-4580-92e2-d31bc7d1aa40","added_by":"auto","created_at":"2025-11-11 17:15:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePyrvinium depletes a self-renewing CD15⁺ cell population\u003c/strong\u003e. \u003cstrong\u003eA.\u003c/strong\u003e CD15⁺/Ter-119⁻ cells were flow-sorted from \u003cem\u003ePtch1-LacZ,\u003c/em\u003e \u003cem\u003eTrp53\u003c/em\u003e-mutant MB tumors. \u003cstrong\u003eB.\u003c/strong\u003e Similarly sorted cells were plated\u003cstrong\u003e \u003c/strong\u003ein the presence of vismodegib (200 nM), PKF115-584 (1000 nM), or pyrvinium (200 nM), and their ability to form spheres was determined. \u003cstrong\u003eC.\u003c/strong\u003e RNA was extracted from CD15⁺/Ter-119⁻ and CD15⁻/Ter-119⁻ sorted pools, and expression of WNT and SHH target genes was determined by RT-qPCR. Data were normalized to CD15⁻ cells and analyzed using an unpaired t-test.\u003cstrong\u003e D.\u003c/strong\u003e MPC-1 cultures were exposed for 24h to increasing concentrations of pyrvinium and numbers of CD15⁺ cells were determined by flow analysis. \u003cstrong\u003eE.\u003c/strong\u003e Similar cultures were exposed to vismodegib (200 nM) or PKF115-584 (1000 nM) for 24h, and CD15⁺ cell numbers were determined by flow analysis.\u003cstrong\u003e F\u003c/strong\u003e. The number of Sox2\u003csup\u003e+\u003c/sup\u003e/CD15\u003csup\u003e+\u003c/sup\u003e cells was determined by flow cytometry in MPC-1 cultures treated with increasing concentrations of pyrvinium for 24h.\u003cstrong\u003e G\u003c/strong\u003e. Similar cultures were exposed to vismodegib (200 nM) or PKF115-584 (1000 nM) for 24h, and Sox2⁺ cell numbers were determined by flow analysis.\u003cstrong\u003e H\u003c/strong\u003e. The number of Sox2\u003csup\u003e+\u003c/sup\u003e/CD15\u003csup\u003e+\u003c/sup\u003e cells was determined by flow cytometry in similarly treated cultures. \u003cstrong\u003eI\u003c/strong\u003e. MPC-1 cultures were exposed to I-BET151 (500 nM) for 24h, and the numbers of indicated positive cells were determined by flow analysis. Data was analyzed using an unpaired t-test. \u003cstrong\u003eJ. \u003c/strong\u003eA schematic of the mechanism of action of pyrvinium in MPC cultures. Unless otherwise indicated, mean ± SEM of data normalized to DMSO were analyzed using a one-way ANOVA followed by Dunnett’s post-hoc. * p\u0026lt; 0.05, ** p\u0026lt; 0.01, *** p\u0026lt; 0.001, **** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/afd7651c849cd13aac3eed84.png"},{"id":95797374,"identity":"759cb647-0cb4-4e12-bf0a-da4da91fce01","added_by":"auto","created_at":"2025-11-13 08:04:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":797122,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCK1α agonists reduce MB stemness.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e \u003cem\u003ePtch1-LacZ, Trp53\u003c/em\u003e-mutant MB cells were subcutaneously implanted. Once tumors reached ~200 mm³, mice were treated with pyrvinium (0.8 mg/kg, s.c., q.o.d.) or vehicle, and tumor volumes were measured over time. Representative H\u0026amp;E staining (scale bar 500 µm) at the endpoint is shown. \u003cstrong\u003eB.\u003c/strong\u003e Mice harboring similar tumors received three doses of pyrvinium, and cells positive for the indicated markers were quantified by flow analysis. \u003cstrong\u003eC\u003c/strong\u003e. MPC-2 cultures treated with 1000 nM SSTC3 for 24h were analyzed by RT-qPCR for SHH and WNT target gene expression. \u003cstrong\u003eD\u003c/strong\u003e. Similar cultures were exposed to increasing SSTC3 concentrations, and cell viability was measured by MTT assay 72h later. EC\u003csub\u003e50\u003c/sub\u003es were calculated by non-linear regression. \u003cstrong\u003eE\u003c/strong\u003e. Similar cultures were allowed to form secondary spheres after 24h SSTC3 treatment, and EC50 values were calculated using non-linear regression. \u003cstrong\u003eF\u003c/strong\u003e. Mice were orthotopically implanted with \u003cem\u003ePtch1-LacZ; Trp53\u003c/em\u003e-mutant MB cells, then treated with SSTC3 (10 mg/kg, i.p., q.d.) for 3 days starting 10 days post-surgery. Brains were harvested, and numbers of cells positive for the indicated markers were quantified by flow cytometry. \u003cstrong\u003eG\u003c/strong\u003e. Mice with orthotopic SJSHHMB-14-7196 tumors (grown for 30 days) were treated with SSTC3 (10 mg/kg, i.p., q.d.) for 3 days before IHC staining for Sox2 and CD15. Unless otherwise indicated, representative images (scale bar 20 µm) are shown. Unless noted, mean ± SEM of data normalized to DMSO were analyzed using an unpaired t-test. *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001, ****p\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/efe6ae96e1870b4050e09519.png"},{"id":95797346,"identity":"9edf60aa-16f4-47e7-bf53-bf15ae595519","added_by":"auto","created_at":"2025-11-13 08:03:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":141561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePyrvinium reduces MB relapse risk.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Schematic of relapse-risk protocols: mice bearing subcutaneous \u003cem\u003ePtch1-LacZ,\u003c/em\u003e \u003cem\u003eTrp53\u003c/em\u003e-mutant MB received 3 doses of pyrvinium before preparing single-cell suspensions for primary sphere assays or subcutaneous and orthotopic engraftment. \u003cstrong\u003eB.\u003c/strong\u003e Quantification and representative images of primary spheres from vehicle- and pyrvinium-treated tumors are shown. The data were normalized to one vehicle-treated animal. \u003cstrong\u003eC.\u003c/strong\u003e Tumor engraftment frequency using limiting cell numbers. Data was analyzed with a one-sided χ\u003csup\u003e2 \u003c/sup\u003etest. \u003cstrong\u003eD.\u003c/strong\u003e Time to relapse was assessed by symptom-free survival and analyzed by Log-rank (Mantel-Cox) tests. \u003cstrong\u003eE\u003c/strong\u003e. Correlation between \u003cem\u003eCSNK1A1\u003c/em\u003e expression and MB patient outcome was assessed using the Cavalli 2017 dataset. Log-rank (Mantel–Cox) tests with optimal cutoff settings were used for data analyses. \u003cstrong\u003eF\u003c/strong\u003e. \u003cem\u003eCSNK1A1\u003c/em\u003e expression was compared between MB and normal brain in the Gump 2015 dataset. \u003cstrong\u003eG\u003c/strong\u003e. \u003cem\u003eCSNK1A1\u003c/em\u003e levels were compared across MB subgroups in the Cavalli 2017 dataset by one-way ANOVA with Dunnett’s post hoc test. \u003cstrong\u003eH\u003c/strong\u003e. \u003cem\u003eCSNK1A1\u003c/em\u003e expression in SHHα MB was compared with all other MB subtypes together. Unless noted, mean ± SEM of data were analyzed using an unpaired t-test. *p\u0026lt; 0.05, **p\u0026lt; 0.01, ***p\u0026lt; 0.001, ****p\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/72bacfaae069c5204bfaac60.png"},{"id":95804591,"identity":"5741af6b-d701-4099-b009-33f151888709","added_by":"auto","created_at":"2025-11-13 08:38:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3437902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/5d8741a3-ce0f-4cf0-b4b1-1b286109216f.pdf"},{"id":95669832,"identity":"7e019f0a-c55f-4b86-ba42-9693849efba5","added_by":"auto","created_at":"2025-11-11 17:15:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18021,"visible":true,"origin":"","legend":"Supplemental Methods","description":"","filename":"SupplementalMethodsPeterson.docx","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/34927666cc5ae269f55c541a.docx"},{"id":95669841,"identity":"82f4ccaa-44d0-4d49-aa5d-51149feeda50","added_by":"auto","created_at":"2025-11-11 17:15:32","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":198797,"visible":true,"origin":"","legend":"Uncropped Blots","description":"","filename":"PetersonBlots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/042cd2208dc827aa9ce9be6d.pdf"},{"id":95669843,"identity":"55ca7506-adf3-4fb5-b5ce-3b048196c763","added_by":"auto","created_at":"2025-11-11 17:15:32","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2681568,"visible":true,"origin":"","legend":"Supplemental Figures","description":"","filename":"SupplementalCK1a.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7915551/v1/eb6fcd2550c64979e00cc965.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"CK1α agonists attenuate medulloblastoma stemness and relapse risk","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBrain tumors are the leading cause of cancer-related death in children, with medulloblastoma (MB) being the most common malignant form(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). From the four major molecular subgroups, the Sonic Hedgehog (SHH) subgroup accounts for approximately 30% of cases(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Clinical outcomes in this subgroup are strongly influenced by co-occurring genetic alterations. In particular, \u003cem\u003eTumor Protein P53\u003c/em\u003e (\u003cem\u003eTP53\u003c/em\u003e) mutations, present in ~\u0026thinsp;7\u0026ndash;30% of cases at diagnosis(\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) and ~\u0026thinsp;30% at recurrence(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), are associated with increased relapse risk and shorter time to relapse(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and thus these tumors are classified as very high-risk(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Given their dismal outcomes, there is an urgent need to understand the mechanisms driving relapse in \u003cem\u003eTP53\u003c/em\u003e-mutant SHH MB, also known as SHHα(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEmerging evidence suggests that a rare population of MB progenitor cells (MPCs), often marked by the stemness factor SRY-box transcription factor 2 (Sox2), may contribute to disease recurrence(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Our previous work in SHH MB showed that the expansion of Sox2⁺ cells relies on non-canonical Gli activity, rendering them resistant to Smoothened (Smo) inhibitors in clinical development(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). By contrast, compounds that block Gli-dependent transcription via bromodomain and extra-terminal (BET) inhibition reduced Sox2⁺ cells and relapse risk in SHH MB animal models(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Nevertheless, the toxicity associated with BET inhibitors in clinical settings(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) underscores the need for alternative Gli-targeting approaches to prevent MB recurrence.\u003c/p\u003e\u003cp\u003eOne translational approach to target Gli involves activating Casein kinase 1α (CK1α). CK1α is a serine/threonine kinase that phosphorylates Gli, promoting its degradation(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Small-molecule CK1α agonists, such as the FDA-approved anti-helminthic drug pyrvinium, selectively bind CK1α and function as allosteric activators(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Through CK1α activation, pyrvinium and a brain-permeable derivative, SSTC3, attenuate SHH-driven MB growth(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, CK1α also phosphorylates β-catenin, an essential transcriptional activator in the Wingless-related integration site (WNT) pathway, targeting it for proteasomal degradation(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Thus, CK1α agonists extend their effect beyond Gli, as both pyrvinium and SSTC3 block WNT signaling and suppress the growth of WNT-driven malignancies(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven their dependency on Gli for propagation, we hypothesized that CK1α agonists deplete Sox2⁺ cells and reduce SHHα relapse risk. Accordingly, the CK1α agonist pyrvinium reduced Gli levels and the proliferation of Sox2-enriched MB cultures, specifically targeting Sox2\u003csup\u003e+\u003c/sup\u003e cells. Curiously, while other SHH-targeting strategies failed to block the self-renewal of SHHα MB cultures, pyrvinium did so at low nanomolar doses. As we had previously shown that self-renewal in SHHα MB is WNT-driven(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), we investigated whether pyrvinium\u0026rsquo;s effects on secondary sphere formation assays were WNT-dependent. Consistently, pyrvinium reduced WNT signaling in SHHα MB cultures, and its impact on self-renewal was rescued by constitutive WNT activation. Thus, similar to pyrvinium, a compound blocking β-catenin-driven transcription(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), PKF115-584, inhibited the self-renewal of CD15-sorted cells. In vivo, pyrvinium reduced the numbers of Gli1\u003csup\u003e+\u003c/sup\u003e/Sox2⁺ and β-catenin⁺/CD15⁺ cells, as well as those of Sox2\u003csup\u003e+\u003c/sup\u003e/CD15\u003csup\u003e+\u003c/sup\u003e cells in a subcutaneous SHHα MB model. Similarly, the pyrvinium derivative SSTC3 reduced Sox2⁺, CD15⁺, and Sox2⁺/CD15⁺ cells in orthotopic mouse- and human-derived SHHα MB. Furthermore, pyrvinium reduced primary sphere formation and tumor engraftment. Our data underscores the potential of repurposing the FDA-approved CK1α agonist pyrvinium or translating its derivatives for very high-risk SHHα MB.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMouse studies\u003c/h2\u003e\u003cp\u003e All procedures were approved by the IACUC at the Medical University of South Carolina (MUSC) and the University of Miami (UM). \u003cem\u003ePtch1\u003c/em\u003e\u003csup\u003e\u003cem\u003eTM1MPS\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/J\u003c/em\u003e\u003csup\u003e\u003cem\u003e66\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003ePtch1-LacZ\u003c/em\u003e) and \u003cem\u003eB6.129S2-Trp53\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Tyj\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/J\u003c/em\u003e\u003csup\u003e\u003cem\u003e67\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eTrp53\u003c/em\u003e-mutant) mice (Jackson Laboratory) were crossed to establish a colony. Spontaneous tumors were expanded and maintained as allografts in \u003cem\u003eCD1-Foxn1\u003c/em\u003e\u003csup\u003e\u003cem\u003enu\u003c/em\u003e\u003c/sup\u003e mice (Charles River). For pyrvinium (Enzo or Sigma-Aldrich) treatment, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e viable cells (trypan blue\u0026ndash;excluded) were implanted subcutaneously (s.c.) into similar mice. Once tumors reached\u0026thinsp;~\u0026thinsp;200 mm\u0026sup3;, mice received pyrvinium (0.8 mg/kg, s.c.) near the tumor every other day (q.o.d.). Tumors were harvested 6 hours (h) after the final dose and either fixed or dissociated with Papain (Worthington) or Accutase (Invitrogen) for FACS, sphere formation, or re-engraftment assays.\u003c/p\u003e\u003cp\u003eFor flank re-engraftment, indicated viable cells were implanted s.c. into \u003cem\u003eCD1-Foxn1\u003c/em\u003e\u003csup\u003e\u003cem\u003enu\u003c/em\u003e\u003c/sup\u003e mice. For orthotopic re-engraftment, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e viable cells from pyrvinium-treated tumors were resuspended in 3 \u0026micro;L Neurobasal-A media and implanted into the cerebellum(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). For primary engraftment, MPC-2 cultures were treated with 200 nM pyrvinium for 24h before s.c. implantation.\u003c/p\u003e\u003cp\u003eFor SSTC3 (StemSynergy Therapeutics) studies, mice were orthotopically implanted with 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003ePtch1-LacZ; Trp53\u003c/em\u003e-mutant cells or 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eTP53\u003c/em\u003e-mutant patient-derived xenograft (PDOX) cells (SJSHHMB-14-7196, courtesy of Dr. Roussel, St. Jude). Ten- and thirty-day post-implantation, respectively, mice received daily (q.d.) intraperitoneal (i.p.) injections of vehicle or SSTC3 (10 mg/kg) for 3 days. Brains were then harvested for flow cytometry (Accutase) or IHC.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBioinformatic analyses\u003c/h3\u003e\n\u003cp\u003eGSE85217 (Cavalli 2017 dataset)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and GSE68015 (Gump 2015 dataset)(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) transcriptomic data were downloaded from the GlioVis data portal(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eResults represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three independent experiments. For BrdU staining, four fields per condition from three experiments were quantified. IHC quantification reflects the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three fields across three or more mice. Flow cytometry data from tumor-derived cell suspensions represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three mice per condition. Multiple group comparisons used one-way ANOVA with post-hoc Dunnett analysis. Two-sample comparisons used one-tailed Student\u0026rsquo;s t-tests. Symptom-free survival was assessed using Log-rank (Mantel-Cox) tests. Patient outcome analysis employed maximally ranked statistics to identify the optimal \u003cem\u003eCSNK1A1\u003c/em\u003e expression cut point (9.1). Tumor engraftment significance was determined using a χ\u0026sup2; test. Statistical significance: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003cp\u003eAdditional Material and Methods are described in Supplemental Methods.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003ePyrvinium blocks Gli1-driven Sox2⁺ cell proliferation\u003c/h2\u003e\u003cp\u003eWe previously showed that the propagation of MB cultures enriched in Sox2\u003csup\u003e+\u003c/sup\u003e cells, herein referred to as MPC cultures, is driven by non-canonical Gli activation downstream of Smo(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Because CK1α destabilizes Gli(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), we tested the efficacy of pyrvinium, a known CK1α agonist(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), in MPC cultures. Given that most SHH MB relapses are associated with \u003cem\u003eTP53\u003c/em\u003e mutations(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), we focused on \u003cem\u003eTrp53\u003c/em\u003e-mutant cultures. According to its mechanism of action, pyrvinium not only reduced \u003cem\u003eGli1\u003c/em\u003e-driven promoter activity in a reporter cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) but also decreased the SHH-driven cell proliferation of the granule layer of the developing cerebellum in organotypic cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; \u003cb\u003eSupplemental Fig.\u0026nbsp;1A\u003c/b\u003e). In MPC cultures, nanomolar concentrations of pyrvinium reduced Gli1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D) and the number of Gli1⁺ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), thereby attenuating the expression of SHH target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Furthermore, pyrvinium reduced the number of viable cells in MPC cultures, with an average EC50 of 17 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), which is consistent with their dependency on Gli to propagate(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven pyrvinium\u0026rsquo;s ability to reduce the number of viable cells in MPC cultures, we investigated whether this effect was due to decreased proliferation or increased cell death. BrdU incorporation assays suggested that pyrvinium\u0026rsquo;s effects are primarily attributed to reduced proliferation, as evidenced by a drop in BrdU\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH; \u003cb\u003eSupplemental Fig.\u0026nbsp;1B\u003c/b\u003e). This was further supported by the reduction in levels of proliferation markers, such as Cyclin-D1 and PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J), as well as cells labeled for the proliferation marker Ki67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). In contrast, pyrvinium had only minimal effects on cell death, as determined by the number of cleaved Caspase-3-labeled cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH; \u003cb\u003eSupplemental Fig.\u0026nbsp;1B\u003c/b\u003e) and its protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J). Importantly, similar to other Gli inhibitors(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), pyrvinium also reduced the number of Sox2⁺ cells in MPC cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL), including those Sox2\u003csup\u003e+\u003c/sup\u003e cells co-labeled with Gli1 and Ki67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Together, these data suggest that pyrvinium depletes Sox2⁺ cells by blocking their Gli-driven proliferation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePyrvinium attenuates MB self-renewal\u003c/h2\u003e\u003cp\u003eMPCs are known to self-renew to maintain tumor stemness(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Interestingly, while our previous work showed that Gli inhibition by targeting BET proteins reduced MPC culture proliferation(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), the ability of these drugs to block secondary sphere formation, used as a measure of tumor stemness(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), was not tested at that time. To evaluate this, we used self-renewal protocols in which MPC cultures were exposed to drugs of interest for 24h before disaggregating spheres(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Equal numbers of viable cells were next re-plated and allowed to form new spheres in the absence of drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These studies revealed that three structurally distinct BET inhibitors (I-BET151, JQ-1, BMS-986158) fail to block the self-renewal of MPC cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) at concentrations we previously showed to attenuate SHH signaling and reduce MPC viability(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Similarly, the compound acting on Smo, vismodegib, failed to impair MPC secondary sphere formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that MPC self-renewal is not SHH/Gli-driven. Accordingly, siRNA pools targeting \u003cem\u003eGli1\u003c/em\u003e and \u003cem\u003eGli2\u003c/em\u003e also failed to inhibit MB self-renewal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In contrast, pyrvinium attenuated secondary sphere formation in MPC cultures, with an average EC50 of 25 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Given pyrvinium\u0026rsquo;s effect on MB self-renewal \u003cem\u003eex vivo\u003c/em\u003e, we studied whether it similarly affects tumor initiation. MPC cultures were exposed to pyrvinium before implanting limiting dilutions of viable cells into mice. Tumor initiation was observed from the vehicle-treated cultures, even when only 150,000 cells were implanted. However, pyrvinium-treated cells did not form tumors, even when nearly tenfold higher cell numbers were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Together, these findings suggest that pyrvinium blocks MB self-renewal by targeting a pathway different than SHH/Gli.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePyrvinium inhibits MB self-renewal by targeting WNT signaling\u003c/h3\u003e\n\u003cp\u003eIn addition to regulating Gli stability(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), CK1α is also known to prime β-catenin for degradation(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), thereby suppressing WNT signaling. Moreover, our previous findings indicated that self-renewal in \u003cem\u003eTrp53\u003c/em\u003e-mutant MB is driven by WNT activation(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Thus, we wondered whether pyrvinium\u0026rsquo;s effects on self-renewal operate through WNT inhibition. Supporting this idea, pyrvinium reduced WNT activity in a TCF/LEF1 reporter assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and decreased both β-catenin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C) and expression of WNT target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) in MPC cultures. Furthermore, overexpression of a constitutively active β-catenin form (\u003cem\u003eS33Y\u003c/em\u003e mutant) increased baseline expression of WNT biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and reduced pyrvinium\u0026rsquo;s efficacy on MPC self-renewal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), supporting a WNT-dependent mechanism of action.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough our prior work linked WNT activation to p53 loss in MPC cultures(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), the mechanism driving elevated WNT signaling remained unclear. One possible explanation involves \u003cem\u003emicroRNAs\u003c/em\u003e (\u003cem\u003emiRNAs\u003c/em\u003e), small non-coding RNAs that bind to 3\u0026rsquo; UTRs of target \u003cem\u003emRNAs\u003c/em\u003e and suppress their translation(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). p53 loss has previously been associated with downregulation of \u003cem\u003emiR-34a\u003c/em\u003e, leading to de-repression of WNT components such as LEF1(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), a key member of the β-catenin transcriptional complex(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Accordingly, \u003cem\u003eTrp53\u003c/em\u003e-mutant MPC cultures exhibited reduced levels of \u003cem\u003emiR-34a\u003c/em\u003e compared to wild-type counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Introduction of a \u003cem\u003emimic-miR-34a\u003c/em\u003e into \u003cem\u003eTrp53\u003c/em\u003e-mutant MPC cultures decreased LEF1 levels, whereas inhibition of \u003cem\u003emiR-34a\u003c/em\u003e using an \u003cem\u003eanti-miR\u003c/em\u003e increased them (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Accordingly, an \u003cem\u003eanti-miR-34a\u003c/em\u003e increased WNT target gene expression in \u003cem\u003eTrp53-wild-type\u003c/em\u003e MPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Further implicating \u003cem\u003emiR-34a\u003c/em\u003e in MB self-renewal, a \u003cem\u003emimic-miR-34a\u003c/em\u003e reduced secondary sphere formation in \u003cem\u003eTrp53\u003c/em\u003e mutant MPC cultures, while an \u003cem\u003eanti-miR-34a\u003c/em\u003e had minimal effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). These findings suggest that loss of p53 reduces \u003cem\u003emiR-34a\u003c/em\u003e levels in MPC cultures, thereby enabling WNT activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK).\u003c/p\u003e\n\u003ch3\u003ePyrvinium depletes a self-renewing CD15⁺ cell population\u003c/h3\u003e\n\u003cp\u003eThe ability of BET inhibitors to deplete Sox2 cells(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) without affecting self-renewal suggests the involvement of an alternative cell pool. To define the self-renewing compartment targeted by pyrvinium, we focused on CD15⁺ cells, which were previously shown to represent a tumor-initiating population in SHH MB(\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). CD15⁺ cells were sorted from \u003cem\u003ePtch1-LacZ, Trp53\u003c/em\u003e mutant tumors, using Ter-119 to deplete erythroid lineage cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared to CD15⁻ cells, CD15⁺ cells formed more spheres, and pyrvinium reduced these numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Furthermore, linking CD15\u003csup\u003e+\u003c/sup\u003e self-renewal to WNT signaling, similar to pyrvinium, a compound that blocks β-catenin/LEF1 interaction, PKF115-584(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), impaired sphere formation in similarly sorted cells, while SHH inhibition by vismodegib failed to do so (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings suggest that pyrvinium targets a WNT-driven CD15⁺ population. Gene expression analysis further supported this idea, as CD15-sorted cells showed elevated expression of multiple WNT target genes compared to CD15⁻ cells, while SHH target gene levels were similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, treatment of MPC cultures with pyrvinium reduced the CD15⁺ cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Like pyrvinium, PKF115-584 reduced CD15\u003csup\u003e+\u003c/sup\u003e cell numbers, whereas vismodegib had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Together, these results suggest that pyrvinium targets a WNT-driven CD15⁺ cell population critical for self-renewal.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA Sox2⁺/CD15⁺ population was previously mentioned in the literature(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), but its regulation remains unclear. Given its ability to deplete both Sox2⁺ and CD15⁺ cells, we asked whether the CD15⁺ cells targeted by pyrvinium also express Sox2. Flow cytometry analyses showed that most CD15⁺ cells in MPC cultures are Sox2⁺, and pyrvinium reduced the number of these double-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). WNT inhibition with PKF115-584 similarly reduced Sox2⁺ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) and Sox2⁺/CD15⁺ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), whereas vismodegib did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H). In contrast, Gli inhibition by targeting BET reduced Sox2⁺ cells, but did not affect CD15⁺ or Sox2⁺/CD15⁺ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Together, these findings suggest that pyrvinium depletes Smo inhibitor\u0026ndash;resistant, Gli1-driven Sox2⁺ cells, including a WNT-driven CD15⁺ subset (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCK1α agonists reduce MB stemness\u003c/h2\u003e\u003cp\u003eGiven pyrvinium\u0026rsquo;s ability to deplete Sox2⁺ and CD15⁺ cells in MPC cultures, we tested whether it does the same in vivo. Mice were implanted subcutaneously with \u003cem\u003ePtch1-LacZ, Trp53\u003c/em\u003e-mutant MB cells, and allowed to form tumors. Because of its poor systemic bioavailability(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), pyrvinium was administered subcutaneously near the tumor site(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). This local dosing suppressed tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; \u003cb\u003eSupplemental Fig.\u0026nbsp;1C\u003c/b\u003e), reduced proliferative index, and decreased the number of Gli1⁺ and Sox2⁺ cells, including double-positive ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; \u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). Pyrvinium also depleted CD15⁺ and β-catenin⁺ cells, including a WNT-labeled CD15⁺ subset, and diminished the Sox2⁺/CD15⁺ population within these tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; \u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePyrvinium\u0026rsquo;s poor bioavailability limits its use to subcutaneous tumor models(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). To test whether CK1α agonists reduce MB stemness in orthotopic MB, we used a brain-permeable derivative of pyrvinium, SSTC3(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Like pyrvinium, SSTC3 suppressed SHH and WNT target gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and reduced cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and self-renewal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) in MPC cultures. In mice bearing orthotopic \u003cem\u003ePtch1-LacZ, Trp53-mutant\u003c/em\u003e tumors, SSTC3 reduced the proliferation index and the numbers of cells labeled for SHH as well as for WNT makers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF; \u003cb\u003eSupplemental Fig.\u0026nbsp;3\u003c/b\u003e). SSTC3 also reduced overall Sox2⁺ cells, including Gli1⁺ ones, and decreased CD15⁺, CD15⁺/β-catenin⁺, as well as Sox2⁺/CD15⁺ populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF; \u003cb\u003eSupplemental Fig.\u0026nbsp;3\u003c/b\u003e). To determine whether the efficacy of CK1α agonists goes beyond murine models, we tested SSTC3 in a \u003cem\u003eTP53\u003c/em\u003e-mutant SHH PDOX model. Despite a higher baseline of Sox2⁺ cells compared to mouse-derived tumors, SSTC3 maintained its ability to reduce Sox2⁺, CD15⁺ cells, and Sox2⁺/CD15⁺ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG; \u003cb\u003eSupplemental Fig.\u0026nbsp;1D\u003c/b\u003e), supporting efficacy across systems.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePyrvinium reduces MB relapse risk\u003c/h2\u003e\u003cp\u003eBased on their ability to reduce MB stemness and ex vivo self-renewal, we next examined whether CK1α agonists would also impair tumor-propagating potential using relapse-risk protocols in which primary sphere formation is assessed along with secondary tumor engraftment and time to engraftment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Due to its FDA approval and ongoing cancer clinical trials (NCT05055323, NCT06782048, NCT06590454), we focused these studies on pyrvinium. In primary sphere formation assays, in which equal numbers of viable cells were plated and allowed to form spheres, results showed that pyrvinium-treated tumors fail to propagate in culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Similarly, limiting dilution assays in which equal numbers of viable tumor cells were subcutaneously re-implanted showed differences in engraftment ability between vehicle and pyrvinium-treated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Moreover, orthotopic re-engraftment showed that tumors derived from pyrvinium-treated residual disease took longer to establish (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results show pyrvinium\u0026rsquo;s potential to reduce SHHα MB relapse risk.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, analysis of patient datasets showed that high expression of the gene coding for CK1α (\u003cem\u003eCSNK1A1\u003c/em\u003e) correlates with better MB outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and, suggestive of a likely therapeutic window, \u003cem\u003eCSNK1A1\u003c/em\u003e is more expressed in MB versus normal brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). SHH tumors expressed higher \u003cem\u003eCSNK1A1\u003c/em\u003e than other MB subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), with SHHα cases showing the highest expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Since high basal CK1α may favor agonist activity, these findings suggest SHH MB, especially \u003cem\u003eTP53\u003c/em\u003e-mutant, could be particularly responsive to CK1α agonists, providing a rationale for advancing intrathecal pyrvinium dosing or systemic administration of improved derivatives into clinical trials for this very high-risk MB subtype.\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we demonstrate that compounds activating CK1α attenuate MB stemness and, consequently, reduce relapse risk in animal models. Our findings suggest that these drugs function as dual inhibitors, simultaneously blocking Gli and WNT signaling, key drivers of Sox2⁺ cell proliferation and CD15⁺ self-renewal, respectively. The ability of CK1α agonists to attenuate MB stemness is particularly critical in SHHα MB, which accounts for ~\u0026thinsp;75% of SHH MB recurrences(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and shows 0\u0026ndash;41% five-year-survival after relapse(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). While we cannot exclude the possibility that pyrvinium and SSTC3 also attenuate stemness in \u003cem\u003eTP53\u003c/em\u003e-wild-type SHH MB, this study was focused on the mutant subtype due to the clinical need they represent.\u003c/p\u003e\u003cp\u003eWe previously showed that Sox2⁺ MB cells depend on the activation of Gli downstream of Smo, and that these cells are susceptible to depletion by BET inhibitors, which block Gli transcriptional activity(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). However, efforts to translate these findings have been limited by the clinical toxicity of BET inhibitors(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) and the recent termination of a pediatric brain tumor trial testing them (NCT03936465). Dose-limiting toxicities of BET inhibitors underscores the need for alternative approaches to suppress Gli transcriptional activity. Because Gli proteins are transcription factors and therefore difficult to target directly(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), we focused instead on regulatory mechanisms controlling Gli stability. Since CK1α phosphorylates GLI, priming it for degradation(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), we tested pyrvinium\u0026mdash;a previously characterized CK1α agonist(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u0026mdash;in MB cultures enriched for Sox2⁺ cells(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePyrvinium suppressed SHH signaling and reduced the proliferation of Sox2⁺ cells. Thus, this drug shows efficacy like that of BET inhibitors, but with the potential advantage of lower toxicity.\u003c/p\u003e\u003cp\u003eBesides breast cancer(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), CK1α agonists have been reported to attenuate glioblastoma stemness by targeting CD133⁺ cells (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Although early work described CD133⁺ MB cells as the self-renewing pool(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), subsequent studies pointed to CD15⁺ cells instead (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Thus, based on our data showing that pyrvinium blocks MB self-renewal, we studied whether pyrvinium targets CD15⁺ cells. Here, we showed that pyrvinium depletes a self-renewing CD15\u003csup\u003e+\u003c/sup\u003e cell pool, and it does so by targeting WNT signaling. This aligns with previous evidence implicating WNT signaling in regulating cancer stemness(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). However, while previous works implicated CD15 cells with WNT signaling in retina progenitors(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), to our knowledge, this is the first study to link CD15⁺ cells with WNT signaling in cancer. Mechanistically, we found that, similar to previous observations(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), WNT activation in SHHα MPC cultures is associated with the loss of p53\u003cem\u003e/miR-34a\u003c/em\u003e-mediated repression of WNT components such as LEF1. While additional studies are needed to clarify why WNT signaling is specifically required in CD15⁺ cells or which other WNT signaling components are repressed by \u003cem\u003emiR-34a\u003c/em\u003e in MB, our findings identified a connection between p53 loss, WNT signaling, and CD15-driven MB self-renewal.\u003c/p\u003e\u003cp\u003eOur data support that pyrvinium exerts its effects on stemness by inhibiting Gli and WNT signaling. However, pyrvinium has been reported to inhibit AKT signaling(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), a pathway known to promote survival and maintenance of tumor stem-like cells(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), including those in MB(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Similarly, pyrvinium can attenuate STAT3 activity(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), which is a critical regulator of stemness and self-renewal in multiple cancers(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), including MB(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Thus, the anti-stemness effects of pyrvinium may involve the suppression of a broader group of oncogenic signaling programs that converge on regulating stemness.\u003c/p\u003e\u003cp\u003e While our work primarily focused on pyrvinium due to its FDA approval and its use in recently launched trials for pancreatic and gastric cancer (NCT05055323, NCT06782048, NCT06590454), we also evaluated its brain-permeable derivative, SSTC3. In orthotopic SHHα MB models, SSTC3 reduced stemness markers. Moreover, we previously showed that this compound also debulks tumor tissues and prolongs survival in SHH MB orthotopic models, including in a \u003cem\u003eTP53\u003c/em\u003e-mutant PDOX(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). These previous findings suggest that the efficacy of CK1α agonists extends beyond the herein described stemness regulation and supports their further development for very high-risk SHHα MB patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCONFLICTS OF INTEREST\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e\u003cp\u003eWe are deeply grateful to the Childhood Brain Tumor Foundation (CBTF) and the families supporting this initiative, which funded the naive idea of a postdoc for testing the effect of pyrvinium on MB stemness. We also thank StemSynergy Therapeutics Inc. for providing SSTC3 and the laboratories of Drs. Robbins, Capobianco, Lee, and Guttridge for their helpful insights and discussions regarding this manuscript. We would also like to thank the Histology and Immunohistochemistry Laboratory (Dept. of Pathology and Laboratory Medicine), and the Biorepository \u0026amp; Tissue Analysis, the Translational Science Lab, and the Flow Cytometry \u0026amp; Cell Sorting shared resources at the HCC (MUSC) supported by a CA138313 P30 grant, and the UM Flow Cytometry Shared Resource and the Department of Surgery Tissue and Pathology core for their work on tissue processing and flow analyses. Schematics were created using BioRender.com. Grammarly and ChatGPT were used to assist in the editing phase of this manuscript.\u003c/p\u003e\u003cp\u003eThis work was supported by CBTF (to J. R.-B.), Monka Foundation (to J.R.-B.), NINDS K01NS119351 and 1R01NS138021 grants (to J.R.-B.), ALSF \u0026ldquo;A\u0026rdquo; Award 23-28298 (to J.R.-B.), FICYT POST10-27 (to J.R.-B.), NCI R00 CA241367 (to T.B.), SREB SC15321 (to K.P.), NCATS TL1 TR001451 \u0026amp; UL1 TR001450TL1 (to K.P.), HCC LOWVELO (to V.K. and M.T.-C.), MUSC College of Graduate Studies Odyssey (to P.S.), and HCC BLOCKS (to L.F.).\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e\u003cp\u003eAll data presented in this manuscript will be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLouis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. 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Unraveling the complexity of STAT3 in cancer: molecular understanding and drug discovery. J Exp Clin Cancer Res. 2024;43(1):23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarg N, Bakhshinyan D, Venugopal C, Mahendram S, Rosa DA, Vijayakumar T, et al. CD133(+) brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence. Oncogene. 2017;36(5):606\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7915551/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7915551/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile outcomes for most children with medulloblastoma (MB) are relatively favorable, those in the Sonic Hedgehog (SHH) subgroup with \u003cem\u003eTumor Protein P53\u003c/em\u003e (\u003cem\u003eTP53\u003c/em\u003e) mutations\u0026ndash;known as the SHHα subtype\u0026ndash;face a much poorer prognosis. SHHα patients relapse more frequently and rapidly, underscoring the need for therapies that prevent recurrence. We recently identified a Gli-driven Sox2⁺ cell population that promotes relapse in SHH MB. However, few Gli-targeting strategies have shown clinical promise to date. One translational Gli inhibitor is pyrvinium, an FDA-approved compound known to destabilize Gli through increasing Casein Kinase 1α (CK1α) activity. In this study, we tested whether pyrvinium and a brain-permeable derivative, SSTC3, affect stemness and relapse risk in mouse and human-derived SHHα MB models. We found that pyrvinium suppresses the Gli-driven proliferation of Sox2⁺ cells. Unlike other SHH/Gli-targeting approaches, pyrvinium also impaired MB self-renewal by depleting Cluster of Differentiation 15 (CD15)⁺ cells. Mechanistic studies revealed that CD15⁺ cell self-renewal is WNT-dependent and driven by the loss of p53/\u003cem\u003emicroRNA-34a\u003c/em\u003e\u0026ndash;mediated repression of WNT signaling. Remarkably, pyrvinium and SSTC3 reduced Sox2⁺, CD15⁺, and dual Sox2/CD15-labeled populations in mouse and patient-derived SHHα models. Consistent with their ability to diminish tumor stemness, pyrvinium also impaired primary and secondary tumor engraftment. Together, these findings highlight the translational potential of the CK1α agonist pyrvinium and its derivatives for patients with very high-risk SHHα MB.\u003c/p\u003e","manuscriptTitle":"CK1α agonists attenuate medulloblastoma stemness and relapse risk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 17:15:27","doi":"10.21203/rs.3.rs-7915551/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9306768c-48d8-4959-bb12-35c18a326513","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56978749,"name":"Biological sciences/Cancer/CNS cancer"},{"id":56978750,"name":"Biological sciences/Cancer/Paediatric cancer"}],"tags":[],"updatedAt":"2026-03-06T15:15:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 17:15:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7915551","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7915551","identity":"rs-7915551","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}