Quantitative DIA-MS Uncovers Functional Impact of SMARCA4 Knockdown in Group 3 Medulloblastoma

Medulloblastoma, a pediatric brain tumor, frequently features chromatin modifier mutations, including SMARCA4 loss in the aggressive Group 3 subgroup. While SMARCA4 is considered a tumor suppressor, the functional impact of its loss on the oncogenic programs in Group 3 MB remains poorly understood. Using doxycycline-inducible shRNA constructs in HD-MB03 cells (a MYC-amplified Group MB model) to achieve SMARCA4 knockdown, we applied quantitative mass spectrometry to profile the resulting proteomic changes. DIA-MS with an in-house library achieved superior proteome depth over DDA and proved optimal for detecting subtle chromatin remodeler effects. Key findings include dysregulation of multiple subunits of the SWI/SNF complex including SMARCA2 overexpression, upregulation of histones, and PRMT5 disrupting chromatin architecture. GSEA revealed cell cycle, spindle and kinetochore organization, DNA replication/repair, and amino acid catabolism to be suppressed. SMARCA4 loss also led to a striking lipid metabolism reprogramming, with steroid biosynthesis, fatty acid biosynthesis, and other peroxisomal lipid pathways being enriched. The overexpression of top candidates like SMARCA2, CRABP2, FABP5, TAGLN2, CYP27A1, and SCP2 was validated in a separate validatory set. Our study reveals the proteomic landscape of Group 3 medulloblastoma following SMARCA4 loss, highlighting novel therapeutic targets for functional validation and exploitation.