Abstract PR010: Elucidating epigenetic regulators of GD2 in Ewing sarcoma
Xiaochen Zhang, Nathaniel W. MabeAbstract
Background:
Ewing sarcoma (EWS) is the second most common solid bone and soft tissue malignancies in children and young adults. Patients receive multimodal therapy including chemotherapy and surgery, however prognosis remains poor in relapsed and metastatic disease. There are no FDA-approved immunotherapies for EWS. The disialoganglioside GD2 (GD2) is highly expressed in some pediatric cancers, such as neuroblastoma, where anti-GD2 antibody and engineered chimeric antigen receptor (CAR) T cells have shown significant promise. GD2 is also expressed in EWS, albeit heterogeneously and too low to achieve a clinical response. We hypothesize that interventions that increase GD2 expression could enable combination with anti-GD2 therapy.
Results:
To identify regulators of GD2 expression in EWS, we performed a FACS-based genome-wide CRISPR/Cas9 screen. This screen revealed that three members in the transcriptional intermediary family 1 (TIF1) chromatin-binding proteins, TRIM28, TRIM24, and TRIM33, were significantly enriched in a GD2-high population. Single gene knockout (KO) for each TIF1 member gene increased GD2 expression, with TRIM28 KO performing the strongest. Consistent with our prior findings showing that the ganglioside biosynthetic enzyme ST8SIA1 is a rate-limiting enzyme in GD2 synthesis in neuroblastoma, we also observed a significantly higher ST8SIA1 mRNA expression in TRIM28 KO cells. The resulting increase of ST8SIA1 and GD2 expression enhanced the sensitivity of EWS cells to anti-GD2 CAR T cells. There are currently no known TRIM28 inhibitors, therefore we engineered EWS cells with an exogenous TRIM28-WEIS construct that enables degradation with nanomolar concentrations of an IMiD-based molecular glue compound, C01. Induced degradation of TRIM28-WEIS increased GD2 expression, which was reversible with compound withdrawal. To define TRIM28’s mechanism of action, RNA-sequencing revealed that TRIM28 KO increased the expression of multiple Krüppel-associated box zinc finger proteins (KRAB-ZNFs), known interacting partners of TRIM28. Consistent with this, exogenous expression of TRIM28 lacking the KRAB-ZNF-binding TRIM domain ablated KRAB-ZNF binding and failed to rescue GD2 in cells with TRIM28 KO. Intersection of proteomics with the CRISPR/Cas9 screen identified several putative KRAB-ZNF regulators of GD2 expression, suggesting TRIM28 utilizes a network of KRAB-ZNFs as a regulatory mechanism underlying GD2 expression.
Conclusion:
Our findings identify TRIM28 as a regulator of GD2 expression in EWS, warranting further investigation into small molecule inhibitors as promising mechanism in combination with anti-GD2 therapy. Future studies dissecting epigenetic mechanisms governing GD2 expression and in vivo validation with anti-GD2 CAR T cell treatment will be required to fully establish TRIM28 perturbation and anti-GD2 immunotherapy in EWS.
Citation Format:
Xiaochen Zhang, Nathaniel W. Mabe. Elucidating epigenetic regulators of GD2 in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Bridging Discovery and Clinical Impact in Pediatric Cancer; 2026 Sep 22-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(18_Suppl_1):Abstract nr PR010.