Int J Stem Cells.  2024 Aug;17(3):319-329. 10.15283/ijsc24032.

Suppression of Glioblastoma Stem Cell Potency and Tumor Growth via LRRK2 Inhibition

• Park S
• Kim KH
• Bae YH
• Oh YT
• Shin H
• Kwon HJ
• Kim CI
• Kim SS
• Choi HG
• Park JB
• Lee BD

• KMID: 2558609
• DOI: http://doi.org/10.15283/ijsc24032

Abstract

Leucine-rich repeat kinase 2 (LRRK2), a large GTP-regulated serine/threonine kinase, is well-known for its mutations causing late-onset Parkinson’s disease. However, the role of LRRK2 in glioblastoma (GBM) carcinogenesis has not yet been fully elucidated. Here, we discovered that LRRK2 was overexpressed in 40% of GBM patients, according to tissue microarray analysis, and high LRRK2 expression correlated with poor prognosis in GBM patients. LRRK2 and stemness factors were highly expressed in various patient-derived GBM stem cells, which are responsible for GBM initiation. Canonical serum-induced differentiation decreased the expression of both LRRK2 and stemness factors. Given that LRRK2 is a key regulator of glioma stem cell (GSC) stemness, we developed DNK72, a novel LRRK2 kinase inhibitor that penetrates the blood-brain barrier. DNK72 binds to the phosphorylation sites of active LRRK2 and dramatically reduced cell proliferation and stemness factors expression in in vitro studies. Orthotopic patient-derived xenograft mouse models demonstrated that LRRK2 inhibition with DNK72 effectively reduced tumor growth and increased survival time. We propose that LRRK2 plays a significant role in regulating the stemness of GSCs and that suppression of LRRK2 kinase activity leads to reduced GBM malignancy and proliferation. In the near future, targeting LRRK2 in patients with high LRRK2-expressing GBM could offer a superior therapeutic strategy and potentially replace current clinical treatment methods.

Keyword

Leucine-rich repeat serine-threonine protein kinase-2; Glioblastoma; Glioma; Stem cells; Phosphorylation; Proteomics

Figure

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  Fig. 1 Leucine-rich repeat kinase 2 (LRRK2) is highly expressed in glioblastoma (GBM) and GBM stem cells. (A) LRRK2 staining for various brain tumor types, including oligodendroglioma, astrocytoma, and GBM. GBM showed higher LRRK2 expression than less malignant tumors like oligodendroglioma and astrocytoma. Scale bar=50 μm. (B, C) Kaplan–Meier survival plots for all glioma patients and GBM patients with high and low LRRK2 expression. Patients with high LRRK2 expression had shorter survivals, as demonstrated in the tissue microarray (TMA) data of all 161 glioma and 101 GBM patients. (D) Kaplan–Meier survival plots for all glioma patients with high and low LRRK2 expression. Data were obtained from the REMBRANDT of the National Cancer Institute (log-rank test). High LRRK2 expression in 165 of 329 glioma cases from the REMBRANDT database indicated poor prognosis. (E, F) Upon serum-induced differentiation of 0317 and 448T patient-derived cancer cells, a decrease in phospho-LRRK2 (pLRRK2) and total LRRK2 levels was noted, along with reduced cancer stemness factors (CD133, NESTIN, OLIG2, SOX2), while the differentiation marker glial fibrillary acidic protein (GFAP) increased.

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  Fig. 2 Leucine-rich repeat kinase 2 (LRRK2) regulates the stemness and tumorigenesis of glioma stem cells (GSCs). (A) Representative western blot images of phospho-LRRK2 (pLRRK2), LRRK2, stemness factors (CD133, NESTIN, OLIG2, SOX2), and differentiation marker glial fibrillary acidic protein (GFAP) in control 0317 cells (1, 0317-shControl), and two different LRRK2-knockdown 0317 cell lines (2, 0317-shLRRK2-1; 3, 0317-shLRRK2-2). (B) Limiting dilution assays (LDAs) performed using 0317-shControl, 0317-shLRRK2-1, and 0317-shLRRK2-2 cells (**p<0.01, t-test). (C) Cell proliferation assays performed using 0317-shControl, 0317-shLRRK2-1, and 0317-shLRRK2-2 cells. All error bars represent mean±SEM (n=3). **p<0.01, t-test. (D) Representative western blot images of pLRRK2, LRRK2, stemness factors (CD133, NESTIN, OLIG2, SOX2), and differentiation marker GFAP in control 448T cells (1, 448T-shControl), and two different LRRK2-knockdown 448T cell lines (2, 448T-shLRRK2-1; 3, 448T-shLRRK2-2). (E) LDAs performed using 448T-shControl, 448T-shLRRK2-1, and 448T-shLRRK2-2 cells (**p<0.01, t-test). (F) Cell proliferation assays performed using 448T-shControl, 448T-shLRRK2-1, and 448T-shLRRK2-2 cells. All error bars represent mean±SEM (n=3). **p<0.01, t-test. (G) Magnetic resonance imaging images of the whole brains from mice implanted with 448T-shControl, 448T-shLRRK2-1, or 448T-shLRRK2-2 cells. (H) H&E staining of the whole brains from mice implanted with 448T-shControl, 448T-shLRRK2-1, or 448T-shLRRK2-2 cells. Scale bars=100 μm. (I) Kaplan–Meier survival plots for the orthotopic xenograft mouse model (log-rank test). The shLRRK2 treated orthotopic mouse models showed dramatically decrease of tumor size and increase of overall survivals.

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  Fig. 3 The effect of phospho-leucine-rich repeat kinase 2 (pLRRK2) targeting drugs including DNK72 in 0317, 448T glioma stem cells (GSCs) and orthotopic mouse models. (A, B) Representative western blot images of pLRRK2, LRRK2 in two GSCs with different concentrations of DNK72. The pLRRK2 level gradually decreased as the concentration of DNK72 increased. (C, D) Cell proliferation assays performed using two GSCs with different concentrations of DNK72. All error bars represent mean±SEM (n=3). **p<0.01, t-test. (E, F) Representative western blot images of pLRRK2, LRRK2 in two GSCs with different inhibitors of pLRRK2. pLRRK2 targeting drugs in a clinical trial for Parkinson’s disease treatment, PF-06447475, GNE7915, MLi-2, and LRRK2-IN-1, and we compared the effect of these drugs with DNK72 for the inhibition of pLRRK2 level. (G, H) Cell proliferation assays performed using two GSCs with different inhibitors of pLRRK2. All error bars represent mean±SEM (n=3). **p<0.01, t-test. The treatment of 1,000 nM of these compounds gave similar pLRRK2 inhibition capability (E, F), while DNK72 reduced cell proliferation slightly better than other compounds (G, H). (I) Kaplan–Meier survival plots for the orthotopic xenograft mouse model (log-rank test). The shLRRK2 treated orthotopic mouse models showed dramatically decrease of tumor size and increase of overall survivals. (J) H&E staining of the whole brains from mice implanted with 448 cells with different inhibitors of pLRRK2. Scale bars=1 mm.

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  Fig. 4 The immunohistochemical experiment has shown a dramatic decrease in the expression of leucine-rich repeat kinase 2 (LRRK2) and ste-mness factors and increased differentiation marker glial fibrillary acidic protein (GFAP) in DNK72 treated orthotopic mouse models. Scale bars=100 μm.

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  Fig. 5 Analysis of changes in the proteome and phosphoproteome according to DNK72 treatment in patient derived glioblastoma cells. (A) In the GSEA EnrichmentMap, leucine-rich repeat kinase 2 (LRRK2) protein expression was positively correlated with the cancer and cell cycle pathway, the RNA-processing associated pathway, and the mitochondria-associated pathway. On the other hand, LRRK2 protein expression was negatively correlated with the FGFR/TP53 pathway. (B) The protein pathway analysis showed that treatment of LRRK2-positive 448T cells with DNK72 resulted in a significant decrease in phosphorylation sites involved in various cellular functions, suggesting that LRRK2 may play a role in regulation stemness through multiple pathways. FDR: false discovery rate, NES: normalized enrichment score, CNS: central nervous system.

Reference

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