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Abstract Osteosarcoma (OS), a mesenchymal bone tumor affecting mainly children and adolescents is characterized by a particularly aggressive behavior, with 20% of patients showing lung metastasis already at diagnosis. Current OS treatment strategies rely on multi-drug chemotherapy, and the prognosis for metastatic cases remains grim, underscoring the pressing need for novel therapeutic approaches. OS is considered a differentiation-related disorder, stemming from the inability of mesenchymal stem cells or osteoblastic progenitors to progress towards terminal differentiation. In our study we explored the impact of targeting ZEB1, a transcription factor known to play a pivotal role in maintaining mesenchymal and stemness characteristics in cancer, on OS cells. We employed CRISPR-Cas9 and shRNA interference techniques to inhibit ZEB1 expression in mouse immune competent OS models. In vitro, ZEB1 deficiency significantly diminished the stemness potential of OS cells, as assessed by spheroid formation assays, while promoting osteogenic differentiation. In vivo, ZEB1 deletion in OS cells led to a significant reduction in primary tumor growth. Tumors originating from ZEB1 KO cells exhibited a more differentiated morphology and higher extracellular matrix deposition compared to controls. Intriguingly, the absence of ZEB1 in tumor cells also influenced the immune infiltrate, with ZEB1-deficient tumors showing significantly fewer pro-tumoral CD206+ M2-like macrophages. This observation is in line with the significant down-modulation of CCL2 expression we detected in ZEB1 KO cells. When injected intravenously, ZEB1-deficient cells produced fewer and smaller lung metastases than control cells. Transcriptional changes induced by ZEB1 deletion were investigated by gene expression profile (GEP) and identified 849 down-regulated and 1093 up-regulated genes in ZEB1 knockout (KO) clones versus ZEB1-competent controls. Gene set enrichment analysis showed a down-modulation of pathways related to cellular proliferation and survival, such as MTORC1 signaling, MYC targets, GM2 checkpoint, E2F targets and oxidative phosphorylation in the absence of ZEB1. Single gene analysis identified among the most up-regulated genes in ZEB1-deficient cells Sfrp1, a negative regulator of the WNT/b-CAT pathway, which in turn regulates MYC, whose pathway is indeed repressed in ZEB1 KO cells. We hypothesize that a high expression of SFRP1 could vicariate the effects of ZEB1 inhibition on OS cell stemness, differentiation state, and in vivo aggressiveness, and therefore could act as a tumor suppressor, which could be potentially exploited for therapeutic strategies. Citation Format: Caterina Cascini, Daniele Lecis, Valeria Cancila, Katia Scotlandi, Claudio Tripodo, Mario P. Colombo, Claudia Chiodoni. Identification of new potential vulnerabilities for differentiation-based therapeutic strategies against osteosarcoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3349.
Cascini et al. (Fri,) studied this question.