Abstract 3124: SNAI2 function in embryonal RMS
Authors: Pomelo Silvia , Sreenivas Prethish , Gryder Berkley , Wang Long , Kunal Baxi , Hensch Nicole , Chen Eleanor , Houghton Peter , Rota Rossella , Khan Javed , Ignatius Myron S. 
Published: August 2019

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle and a key feature of the histology is muscle cells blocked in differentiation despite robust expression of diagnostic muscle differentiation factors MYOD1 and Myogenin. Thus, there are mechanisms operating in tumors that block myogenic differentiation. We previously defined roles in differentiation, self-renewal and growth for a NOTCH1/SNAI1/MEF2C pathway in Embryonal RMS, the major RMS subtype driven predominantly by Ras signaling. However, we observed that SNAI1 knockdown did not result in as robust differentiation as in NOTCH1 shRNA knockdown cells. We hypothesized that SNAI1 and SNAI2 function might be redundant in ERMS. Analysis of SNAI2 expression in primary tumors and cell lines finds that indeed SNAI2 is highly expressed in RMS and ERMS tumors typically express higher SNAI2 compared to SNAI1. To address SNAI2 function, we knocked down SNAI2 using 2 independent shRNAs and assessed effects on differentiation, self-renewal and growth in ERMS RD, SMS-CTR and JR1 cells. Knockdown of SNAI2 both in stable and transient experiments resulted in robust differentiation (10 fold increase) as assessed by differentiated myosin MF20 expression in RD, JR1 and SMS-CTR cells p&lt;0.001). This increase in differentiation was associated with increased expression differentiation genes including MYOD1, MYOGENIN, MEF2C, MEF2D and differentiated myosins and a loss of precursor gene PAX7 as assessed by quantitative RT-PCR and protein expression. SNAI2 knockdown RD and JR1 cells also formed significantly fewer rhabdospheres (p&lt;0.01). Finally, SNAI2 knockdown with 2 independent shRNAs resulted in significantly smaller and more differentiated tumors when xenografted subcutaneously in vivo in SCID mice. Since SNAI2 is a known DNA binding transcriptional repressor, we performed ChIPseq for SNAI2 and H3K27acetyl in SMS-CTR and RD cells coupled with RNAseq to define direct and indirect SNAI2 regulated genes. Our ChIPseq results identified the known SNAI2 DNA binding motif, however additionally we find that SNAI2 chromatin binding significantly enriched for myogenic E box elements bound by MYOD1, E2A and Myogenin. Additionally, SNAI2 binding was more significantly associated with the muscle differentiation program. Given the differential roles of MYOD1 and SNAI2 on gene activation vs. gene repression, we hypothesized that SNAI2 by competing with MYOD1 at terminally differentiated genes maintains early cell cycle effects of MYOD1 but blocks terminal differentiation. Analysis of MYOD1 expression in SNAI2 knockdown cells finds a redistribution of MYOD1 binding from cell cycle to more differentiated muscle genes and is associated with a concomitant exit from the cell cycle and robust differentiation. In summary, SNAI2 is a robust driver of ERMS differentiation and in vivo growth. High SNAI2 expression competes with MYOD1 at terminally differentiated genes blocking differentiation and exit from the cell cycle in ERMS.

Citation Format: Silvia Pomelo, Prethish Sreenivas, Berkley Gryder, Long Wang, Baxi Kunal, Nicole Hensch, Eleanor Chen, Peter Houghton, Rossella Rota, Javed Khan, Myron S. Ignatius. SNAI2 function in embryonal RMS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3124.