(P 391) ATRX LOSS BLOCKS THE DIFFERENTIATION OF MESENCHYMAL STEM CELLS AND ADVANCES UNDIFFERENTIATED SARCOMA DEVELOPMENT

Objective: Undifferentiated sarcoma (US) accounts for over 20% of soft tissue sarcoma and is correlated with high tumor grade and poor prognosis. Recent genomic profiling in US has identified p53 and ATRX among the most frequently mutated or deleted genes. ATRX is a chromatin remodeling factor that functions in the deposition of histone variant H3.3. However, how ATRX loss contributes to US tumorigenesis and whether ATRX-deficient US can be therapeutically targeted remain largely unknown.

Methods: Genetic engineered mouse model was established to target Atrx and Tp53 deletion in mesenchymal cells. Survival and pathological analysis were conducted to assess tumor initiation/progression and subtypes. Bulk RNA-seq and scRNA-seq were conducted to characterize transcriptomic and tumor heterogeneity alterations associated with Atrx loss. MSC with ATRX deletion was generated to evaluate the impact of ATRX loss on the differentiation into multiple lineages. ATAC-seq, ChIP-seq, and CUT&RUN are performed to characterize ATRX-dependent epigenetic alterations during cell differentiation. Genome-wide Cpf1-mediated loss-of-function screens were conducted to identify genetic vulnerabilities in ATRX-deficient US cells.

Results: Deletion of Atrx in mouse MSCs, but not terminally differentiated cells, accelerated p53-loss-induced sarcoma development and significantly shortened the overall survival. Atrx-deficient tumors are highly reminiscent of human high-grade UPS pathologically and transcriptomically. Atrx-null tumor cells exhibit enhanced proliferation and enrichment of cell cycle-related pathways. ATRX loss in MSCs blocks the differentiation into multiple mesenchymal lineages and sustains stemness and proliferation. Epigenetic profiling identified RUNX1 as the potential master regulator for the transcriptional alterations associated with ATRX loss during MSC differentiation. Deletion of Runx1 in MSCs reverses the differentiation defect in ATRX-deficient MSCs. Genome-wide Cpf1-mediated genetic screen in mouse UPS cells further identified Cbfb, the RUNX1-binding partner required for its transcriptional activity, as the top differentially essential gene in ATRX-deficient tumor cells compared to Atrx-wildtype ones. Treatment with RUNX1/CBFB inhibitor preferentially inhibits the tumorigenic activity of ATRX-deficient tumor cells.

Conclusion: ATRX deficiency contributes to UPS development by inhibiting MSC differentiation in RUNX1-dependent manner. RUNX1/CBFB may serve as a therapeutic target for ATRX-deficient UPS.