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.