SMARCA4 biology in alveolar rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.

Figure 1.. CRISPR epigenome screen for mouse sarcoma cell cultures.

(A) Comparison of Day12/Day2 raw depletion values (as log2 fold change values) between NIH-3T3 (mouse fibroblasts) and U66788 (mouse ARMS line), NIH3T3 and U37125 (mouse ERMS line) and U37125 and U66788 cell lines. Fold change of sgRNA is calculated between d2 and d12 after transduction with CRISPR sgRNA library. Rosa and Rpa3 depletions are represented as negative and positive controls, respectively. (B) Comparison of U66788 ARMS cell line and U37125 ERMS cell line. Cell survival of U66788, but not U37125, was dependent on SMARCA4. (C) Comparison of NIH-3T3 cell line and U37125 ERMS cell line.

Figure 2.. SMARCA2/4 expression in ARMS, ERMS primary tumors and cell lines.

(A) SMARCA2/SMARCA4 expression in ARMS/ERMS tumors accessed by immunohistochemistry (see Table 1 for expanded results and description). Scale bar, 100 μm. (B) Graphical representation of percentage of ARMS tumor samples expressing SMARCA4 and SMARCA2 (strong vs weak expression). (C) Expression of SMARCA4 and SMARCA2 in murine ARMS and ERMS cell lines assessed via western blotting. Proliferating and differentiated C2C12 cell line used as control. (D) Expression of SMARCA4 and SMARCA2 in human ARMS and ERMS cell line assessed via western blotting. Proliferating HSMM and 3T3 cell lines were used as controls. (E) Analysis of the components of SWI-SNF complexes in ARMS cell lines. (F) Immunoprecipitation studies in Rh30 ARMS cell line to analyze the association of SMARCA4 with SWI-SNF BAF complex component ARID1A, SWI-SNF PBAF complex component PBRM1, and SWI-SNF ncBAF complex component BRD9. (G) Schematic of BAF complexes adapted from .

Figure 3:. Short-term knockdown of SMARCA4 using siRNA only partially compromises cell viability of human ARMS cell lines.

(A-D) Relative cell viability at 72 hours as measured by CellTiterGlo assay of Rh30, Rh41, Rh5 and CF-1. Non-targeting siRNA (Nts) served as a control. (E) Relative cell viability at 72 hours of murine ARMS U667788 primary cell culture. Paired immunoblots show efficiency of SMARCA4 knockdown in each of the cultures. Error bars indicates mean± SE of biological replicates (n=3, p*≤ 0.05, p**≤ 0.01, p***≤ 0.001)

Figure 4.. Suppression of SMARCA4 inhibits both anchorage-independent and dependent colony forming ability of ARMS cells.

(A) Representative photographic images of anchorage-independent colony formation assay for Rh30 cells (B-D) Quantitative analysis of anchorage-independent soft agar colony formation assay for Rh30, Rh5 and CF-1 cells after 21 days of growth (CFU: Colony forming units). (E) Representative photographic images of anchorage-dependent colony formation assay for Rh30, Rh41 and Rh5 cells after staining with crystal violet at the end of 15 days of growth. Nts: Non-targeting siRNA. Error bars indicates mean± SE of biological replicates (n>=2, p*=< 0.05, p= 0.05–0.15). (F) Analysis of tumor growth in murine orthotopic allograft after a long-term DOX-inducible SMARCA4 knockdown. (G) Pharmacodynamic western blot analysis of SMARCA4 (BRG1) & SMARCA2 (BRM1).

Figure 5.. A SMARCA4/A2 protein degrader requires long term exposure for cellular effect in RMS.

(A) Treatment of ARMS cell line Rh30 with ACBI1 for 24–48 hours followed by analysis of biochemical degradation of SMARCA4 and SMARCA2 by western blot analysis. (B) Analysis of tumor cell viability in the short term (3 days) and long-term SMARCA4/A2 depletion (8 or 11 days). (C) In vivo studies for which 10⁶ Rh30 cells were injected into mouse gastrocnemius 24h after cardiotoxin pre-injury. Cells had been pre-treated for 3 days with 50 nM ACBI-1 SMARCA4/A2 PROTAC or cis- ACBI-1 (negative control) prior to injection and tumor initiation analyzed.