SNAI2-Mediated Repression of BIM Protects Rhabdomyosarcoma from Ionizing Radiation

Abstract

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. Significance: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

Mitochondrial calcium uptake regulates tumour progression in embryonal rhabdomyosarcoma

“…The decreased expression of such pro-apoptotic genes may underlie the absence of apoptosis in shMCU cells. SNAI2 directly represses the pro-apoptotic gene BIM/BCL2L11 expression in a p53-independent manner in RMS cell lines, and confers protection from ionising radiation [ 64 ]. As SNAI2 levels are also down regulated upon MCU knockdown, it would be interesting to determine the effect of radiation on these cells.…”

Approaches to identifying drug resistance mechanisms to clinically relevant treatments in childhood rhabdomyosarcoma

“…Thus, as in our previous study [17] , there was a relatively poor correlation between the magnitude of tumor volume regression and EFS, suggesting that EFS may be a more reliable metric of tumor response in preclinical models. Clinical data also question the value of early tumor response as a prognostic indicator, as neither computed tomography and/or magnetic resonance imaging predicted outcomes for patients with rhabdomyosarcoma [32,33] .…”

“…This is rarely possible in the context of pediatric RMS, as relapse tumor is rarely biopsied, but also engraftment into mice may select for subclones rather than represent the clonal spectrum in the patient tumor. Modeling intrinsic and acquired resistance in preclinical models has an advantage over clinical studies in that changes in gene expression can be assessed both in parental and resistant tumor, but also in response to therapy [ 34 ] .…”

Sensitization to Ionizing Radiation by MEK inhibition is Dependent on SNAI2 in Fusion-negative Rhabdomyosarcoma

“…Immunofluorescence staining was performed as described in Wang et al ., 2021 18 . Cells were pretreated with DMSO or trametinib (10 nM for 72h or 20 nM for 24h) prior to being plated at 4,000 cells/well (no IR) and 10,000 cells/well (receiving IR), grown in 10% FBS DMEM or RPMI growth media, fixed at 72 hours post IR (hpIR) (0 or 15 Gy) in 4% paraformaldehyde/PBS, permeabilized in 0.5% Triton X-100/PBS, and incubated with rabbit anti-MEF2C (CST; Catalog No.…”