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. 2019 Feb 15;25(4):1379-1388.
doi: 10.1158/1078-0432.CCR-18-1319. Epub 2018 Nov 28.

A CK1α Activator Penetrates the Brain and Shows Efficacy Against Drug-resistant Metastatic Medulloblastoma

Jezabel Rodriguez-Blanco  1 , Bin Li  1 , Jun Long  1 , Chen Shen  1 , Fan Yang  1 , Darren Orton  2 , Sara Collins  3 , Noriyuki Kasahara  3   4 , Nagi G Ayad  4   5 , Heather J McCrea  6 , Martine F Roussel  7 , William A Weiss  8 , Anthony J Capobianco  1   4 , David J Robbins  9   4
Affiliations

A CK1α Activator Penetrates the Brain and Shows Efficacy Against Drug-resistant Metastatic Medulloblastoma

Jezabel Rodriguez-Blanco et al. Clin Cancer Res. .

Abstract

Purpose: Although most children with medulloblastoma are cured of their disease, Sonic Hedgehog (SHH) subgroup medulloblastoma driven by TRP53 mutations is essentially lethal. Casein kinase 1α (CK1α) phosphorylates and destabilizes GLI transcription factors, thereby inhibiting the key effectors of SHH signaling. We therefore tested a second-generation CK1α activator against TRP53-mutant, MYCN-amplified medulloblastoma.

Experimental design: The ability of this CK1α activator to block SHH signaling was determined in vitro using GLI reporter cells, granular precursor primary cultures, and PATCHED1 (PTCH1)-mutant sphere cultures. While in vivo efficacy was tested using 2 different medulloblastoma mouse models: PTCH1 and ND2:SMOA1. Finally, the clinical relevance of CK1α activators was demonstrated using a TRP53-mutant, MYCN-amplified patient-derived xenograft.

Results: SSTC3 inhibited SHH activity in vitro, acting downstream of the vismodegib target SMOOTHENED (SMO), and reduced the viability of sphere cultures derived from SHH medulloblastoma. SSTC3 accumulated in the brain, inhibited growth of SHH medulloblastoma tumors, and blocked metastases in a genetically engineered vismodegib-resistant mouse model of SHH medulloblastoma. Importantly, SSTC3 attenuated growth and metastasis of orthotopic patient-derived TRP53-mutant, MYCN-amplified, SHH subgroup medulloblastoma xenografts, increasing overall survival.

Conclusions: Using a newly described small-molecule, SSTC3, we show that CK1a activators could address a significant unmet clinical need for patients with SMO inhibitor-resistant medulloblastoma, including those harboring mutations in TRP53.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

D.J. Robbins is an employee of and has ownership interests (including patents) at StemSynergy Therapeutics, Inc. D. Orton has ownership interests (including patents) in StemSynergy Therapeutics, Inc. and reports receiving commercial research grants from NIH. M.F. Roussel is an employee of University of Tennessee. W.A. Weiss has ownership interests (including patents) in StemSynergy Therapeutics, Inc. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1.
Figure 1.
SSTC3 acts downstream of SMO to attenuate SHH activity. A, LIGHT2 cells were treated with 1 μg/mL recombinant SHH for 24 hours, followed by addition of the indicated concentrations of SSTC3 or SSTC111 for an additional 48 hours. Firefly luciferase activity was then determined and normalized to Renilla luciferase activity. B, NIH-3T3 cells were incubated with the SAG (100 nmol/L) and the indicated concentrations of SSTC3. The expression of GLI1 was determined at the indicated timepoints and normalized to that of the housekeeping gene GAPDH. Data were normalized to a vehicle control. C, LIGHT2 cells were transduced with lentivirus expressing the indicated shRNA to generate stable polyclonal cell lines. These cells were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 48 hours. The expression of GLI1 was determined and normalized to that of GAPDH. D, GPCs were incubated in the presence of DMSO, SAG (100 nmol/L), or SAG (100 nmol/L) and SSTC3 (200 nmol/L), for 24 hours prior to quantification of BrdU incorporation. E, Representative images from similarly treated GPC are shown. F, GPC were treated with vehicle, vismodegib (100 nmol/L), or SAG (100 nmol/L) and SSTC3 (200 nmol/L), and expression of the indicated genes determined 6 hours later. Data were normalized to a SAG-treated vehicle control. G, SUFU−/− MEFs were treated for 48 hours with vehicle, the indicated concentrations of SSTC3, or vismodegib (200 nmol/L). The expression of GLI1 was then determined and normalized to that of GAPDH. Data were normalized to a vehicle control. H, LIGHT2 cells expressing WT SMO, the vismodegib-resistant oncogenic SMO mutant M2-D473, or GLI1, were treated with vehicle, vismodegib (200 nmol/L), or SSTC3 (200 nmol/L). Firefly luciferase activity was then determined and normalized to Renilla luciferase activity. Data were normalized to a SMO WT vehicle control. I, The indicated MEFs were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 24 hours, and the levels of PTCH1 expression determined and normalized to that of housekeeping gene GAPDH by qRT-PCR. Data were normalized to a SAG-treated vehicle control. J, The indicated MEFs were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 24 hours, and the levels of GLI1 expression determined and normalized to that of housekeeping gene GAPDH by qRT-PCR. Data were normalized to a SAG-treated vehicle control.
Figure 2.
Figure 2.
SSTC3 inhibits the growth of SHH subgroup medulloblastoma. A, MSCs were isolated from a PTCH1+/− medulloblastoma. The MSCs were incubated for 3 days with the indicated concentrations of vismodegib and cell viability determined using a MTT reduction assay. Data were normalized to a vehicle control. B, PTCH1-mutant MSCs were transduced with the indicated shRNA and cell viability determined 5 days later using a MTT assay. Data were normalized to control shRNA (Scramble)-transduced cells. C, PTCH1-mutant MSC were treated with the indicated concentrations of SSTC3 or SSTC111, and cell viability determined 3 days later. Data were normalized to a vehicle control. D, Expression of the indicated genes in similarly treated cells was determined 24 hours after treatment. Data were normalized to a vehicle control. E, MSCs were treated with SSTC3 (400 nmol/L) or vehicle control at the indicated time points. The levels of the indicated proteins were then determined by immunoblotting. Representative blots are shown. F, MSC were stably transduced with the indicated shRNA and CK1α expression determined by qRT-PCR. Data were normalized to control shRNA (Scramble)-transduced cells. G, MSCs were stably transduced with the indicated shRNA and then treated with SSTC3 (2,000 nmol/L) or vehicle control. Cell viability was determined 3 days later using a MTT assay. Data were normalized to a vehicle control. H, SSTC3 distribution was determined in mouse plasma and brain tissue at the indicated times (N = 3). I, Mice carrying orthotopically implanted PTCH1-mutant medulloblastoma tumors were treated every other day with SSTC3 (10 mg/kg i.p. in DMSO) or vehicle for 30 days. The residual tumors from these mice were immunostained for the proliferation biomarker PCNA and the numbers of PCNA+ cells per field in 4 fields from 3 independent mice quantified. J, Representative images of PCNA-immunostained cells are shown. K, These residual tumors were immunostained for the apoptosis biomarker Cleaved CASPASE-3 and the numbers of Cleaved CASPASE-3+ cells per field in 4 fields from 3 independent mice quantified. L, Representative images of Cleaved CASPASE-3 immunostaining are shown.
Figure 3.
Figure 3.
SSTC3 increases the symptom-free survival of a GEMM of SHH subgroup medulloblastoma. A, SAG (100 nmol/L)-induced WT- or ND2:SMOA1-derived GPC were treated with the indicated concentrations of vismodegib or SSTC3. GPC proliferation was subsequently determined by BrdU incorporation 24 hours later. Data were normalized to a vehicle control. B, Two-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (20 mg/kg i.p. in DMSO) daily for 1 month. Representative IVIS images from vehicle or SSTC3-treated mice are shown. C, Similar mice were treated every other day with SSTC3 (10 mg/kg i.p. in DMSO) for a month. Representative images of brains and H&E staining from WT vehicle, ND2:SMOA1 vehicle, or SSTC3-treated mice are shown. D, The area of tumor from the vehicle and SSTC3-treated mice was determined (N = 6). E, Metastatic lesions were quantified from similarly treated mice (N = 6). F, Tumors from these mice were immunostained for the proliferation biomarker PCNA and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. G, Representative images of PCNA immunostaining are shown. H, Orthotopic tumors were immunostained for the apoptosis biomarker Cleaved CASPASE-3 and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. I, Representative images of Cleaved CASPASE-3 immunostaining are shown. J, Four-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (10 mg/kg i.p. in DMSO) for 2 consecutive days. The mice were then sacrificed and their cerebella harvested 6 hours after the last injection. The expression of the indicated genes was then determined and normalized to the expression of GAPDH. Data was normalized to a vehicle control (N = 5). K, The levels of indicated proteins in similarly treated mice were determined by immunoblotting from a cohort of vehicle (1–5) or SSTC3 (6–10)-treated mice. L, Two-month-old ND2:SMOA1 mice were treated with SSTC3 (10 mg/kg i.p. in DMSO) every other day for 1 month and medulloblastoma symptom-free survival monitored for 9 additional months (N = 10).
Figure 4.
Figure 4.
SSTC3 inhibits the growth of a patient-derived TRP53-mutant, MYCN-amplified medulloblastoma model. A, Mice carrying orthotopically implanted SHH subgroup medulloblastoma PDXs (TB-14–7196) for 3 weeks were treated for 2 consecutive days with vehicle or SSTC3 (10 mg/kg i.p in DMSO). Residual tumor tissue was harvested 6 hours after the last injection and expression of the indicated genes determined and normalized to that of GAPDH. Data were normalized to a vehicle control (N = 10). B, Mice carrying subcutaneous medulloblastoma PDX, TB-14–7196, were similarly treated with vehicle (1,2) or SSTC3 (3,4). GLI proteins were enriched using a DNA pull-down assay, containing either GLI binding site or scramble (SC) oligonucleotide beads. The GLI affinity-bead enriched proteins were subsequently immunoblotted with antibodies to GLI1, GLI2, or phospho-serine/threonine. Phospho-GLI1 and Phospho-GLI2 were identified on the basis of their similar molecular size to GLI1 or GLI2, respectively. A representative blot is shown. C, Mice carrying an orthotopically implanted medulloblastoma PDX, TB-14–7196, were treated with vehicle or SSTC3 (10 mg/kg i.p. in DMSO) every other day for 30 days. The size of the tumors from these mice was then determined (N = 10). D, Representative H&E staining of tumors from these mice. E, Mice carrying an orthotopically implanted medulloblastoma PDX (TB-14–7196) for 15 days, and engineered to express luciferase, were treated daily with vehicle or SSTC3 (10 mg/kg i.p. in DMSO) for an additional 20 days. Tumor growth was determined by IVIS imaging on day 15 (before dosing) and 35 (after dosing). Representative IVIS imaging is shown. F, Distant metastasis in similarly treated mice was detected by IVIS imaging on day 35 after implantation. Representative IVIS imaging is shown. G, The number of cranial lesions were quantified in similar mice after 30 days treatment (N = 10). H, Representative images of cerebrum and leptomeningeal tumor spread in these mice. I, Residual tumors from these mice were immunostained for the proliferation biomarker PCNA and the number of positive cells per field, in 4 fields from 3 independent mice, quantified. J, Representative images of PCNA immunostaining are shown. K, Residual tumors from these mice were immunostained for the apoptosis biomarker Cleaved CASPASE-3 and the numbers of positive cells per field, in 4 fields from 3 independent mice, quantified. L, Representative images of Cleaved CASPASE-3 immunostaining are shown. M, Mice carrying an orthotopically implanted medulloblastoma PDX, TB-14–7196, were treated with vehicle or SSTC3 (10 mg/kg i.p in DMSO) every other day for 30 days and medulloblastoma symptom-free survival monitored (N = 10).
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