PROTAC EZH2 degrader-1 overcomes the resistance of podophyllotoxin derivatives in refractory small cell lung cancer with leptomeningeal metastasis

Abstract

Background: Leptomeningeal metastasis (LM) of small cell lung cancer (SCLC) is a highly detrimental occurrence associated with severe neurological disorders, lacking effective treatment currently. Proteolysis-targeting chimeric molecules (PROTACs) may provide new therapeutic avenues for treatment of podophyllotoxin derivatives-resistant SCLC with LM, warranting further exploration.

Methods: The SCLC cell line H128 expressing luciferase were mutated by MNNG to generate H128-Mut cell line. After subcutaneous inoculation of H128-Mut into nude mice, H128-LM and H128-BPM (brain parenchymal metastasis) cell lines were primarily cultured from LM and BPM tissues individually, and employed to in vitro drug testing. The SCLC-LM mouse model was established by inoculating H128-LM into nude mice via carotid artery and subjected to in vivo drug testing. RNA-seq and immunoblotting were conducted to uncover the molecular targets for LM.

Results: The SCLC-LM mouse model was successfully established, confirmed by in vivo live imaging and histological examination. The upregulated genes included EZH2, SLC44A4, VEGFA, etc. in both BPM and LM cells, while SLC44A4 was particularly upregulated in LM cells. When combined with PROTAC EZH2 degrader-1, the drug sensitivity of cisplatin, etoposide (VP16), and teniposide (VM26) for H128-LM was significantly increased in vitro. The in vivo drug trials with SCLC-LM mouse model demonstrated that PROTAC EZH2 degrader-1 plus VM26 or cisplatin/ VP16 inhibited H128-LM tumour significantly compared to VM26 or cisplatin/ VP16 alone (P < 0.01).

Conclusion: The SCLC-LM model effectively simulates the pathophysiological process of SCLC metastasis to the leptomeninges. PROTAC EZH2 degrader-1 overcomes chemoresistance in SCLC, suggesting its potential therapeutic value for SCLC LM.

Keywords: Chemoresistance; Leptomeningeal metastases; Mouse model; PROTAC EZH2 degrader-1; Small cell lung cancer.

Fig. 1

A: Timeline of in vivo drug testing in this study. B: In vivo imaging of leptomeningeal metastases in mice. The colour scale indicates the photon flux (photon/s) emitted from each group. C: Quantitative bar graph representing the total photon flux calculated from the region of interest. The data are presented as the means ± SDs, “*” indicates P < 0.05. D: Following the euthanasia of the mice, LM tumour tissues were extracted and subjected to weight measurements. E: The mean tumour weight is depicted in the bar graphs; error bars represent standard deviation. “*” represents P < 0.05

Fig. 2

A: Study flow chart. B: Live imaging of 12 mice subcutaneously transplanted with = tumour cells and subcutaneous tumour tissue extracted after resection. C: 5 live brain tumour metastasis images. D: Mouse brain tissue sections subjected to H&E staining

Fig. 3

A: A CCK-8 assay was used to determine tumour cell growth. B: Transwell assay; tumour cells in the lower chamber were viewed at 10× magnification, “*” indicates P < 0.05. C: Immunohistochemical grading control D-G: Immunohistochemistry of subcutaneous tumour tissue (control), brain parenchyma metastatic tissue, and leptomeningeal metastatic tissue at 10x objective and 63x objective, respectively: (D) CD56 primary antibody (P = 0.88); (E) SYP primary antibody; (F) CHGA primary antibody (P = 0.58); (G) INSM1 primary antibody (P = 0.33)

Fig. 4

A: H128-LM cells were injected via the right carotid artery. B: In vivo imaging of leptomeningeal metastasis in mice. C: Leptomeningeal metastasis tumour tissue, H&E staining. D-G: Volcano plot demonstrating differential gene expression, especially genes with upregulated expression, with a twofold threshold and P value < 0.05. (D): GEO database; (E): H128 cells and H128-BPM cells; (F): H128 cells and H128-LM cells; (G): H128-BPM cells and H128-LM cells. In Panels E, F, and G, there were 2 samples per group. H, I: The H128-LM and H128-BPM cell lines were passaged for three generations. Immunoblot analysis of SCL44A4, VEGFA, OCLN, IGF2, EZH2, CDH1, CDH2 and MMP9 expression in four H128 cell lines (H128, H128-Mut, H128-LM, H128-BPM); beta-actin was used as a loading control

Fig. 5

A-C: The sensitivity of H128, H128-Mut, H128-BPM or H128-LM cells to carboplatin (A), etoposide (B) and teniposide (C) through the EZH2 degrader. H128 cells were pretreated with PROTAC EZH2 degrader-1 (2 nM) followed by carboplatin (A), etoposide (B) or teniposide (C) treatment for 72 h prior to cell viability assays. D: The viability of cells treated with anlotinib, both alone and in combination with the EZH2i regimen, was examined. E-H: Cell viability was measured 72 h after treatment with PROTAC EZH2 degrader-1 alone (E) or in combination with three regimens (F: carboplatin + etoposide, G: VM26, H: anlotinib)