Fig 1.
LSD1 expression in tumors and SP-2577 toxicity in SWI/SNF mutated tumors.
(A) TCGA analysis for LSD1 expression in different human cancers. The cancer type acronym to full cancer type key from TCGA PanCancer Atlas is in S2 Table. (B) TCGA analysis for LSD1 expression in SWI/SNF-mutated tumors.
Table 1.
Cytotoxicity of SP-2577 and SP-2513 in SWI/SNF-mutated cancer cell lines.
Cell viability was evaluated after 72 h treatment with the drugs by Cell Titer Glow assay.
Table 2.
Inhibition of LSD1 enzymatic activity of SP-2577 and SP-2513 measured by Resorufin assay in a cell-free system.
Fig 2.
SP-2577 promotes ERVs expression and activation of IFNβ pathway in SCCOHT cell lines.
(A) qPCR analysis of SCCOHT cell lines SCCOHT-1, BIN67, and COV434 after 72 h of SP-2577 treatment showing increased expression of ERVs and IFN pathway cytokines. (B) MSD panel of chemokines and cytokines from SCCOHT COV434 cell conditioned media in the presence or absence of SP-2577 for 72 h. * = p<0.05, ** = p<0.01.
Fig 3.
SP-2577 promotes lymphocyte infiltration in SCCOHT tumor organoids.
(A) Immune infiltration assay in SCCOHT organoids imaging analysis. COV434, BIN67 and SCCOHT-1 derived-organoids were incubated with conditioned medium pretreated with 3 μM SP-2577, SP-2513, or DMSO in the presence of RFP-tagged PBMCs. After 48 h, the levels of lymphocyte infiltration were measured by z-stack analysis by Cytation 5 imaging. P values for COV434 = <0.0001, BIN67 = 0.0016, and SCCOHT-1 = 0.0196. Right panel: Experimental design (B) IF analysis: RFP-tagged lymphocyte infiltration in SCCOHT organoid microsections was assessed by analysis of RFP levels on confocal scope after 48 h co-culture in presence of SP-2577 or SP-2513 conditioned medium. (C) Immune infiltration assay of COV434 cells treated with increased concentration of SP-2577 for 48 h. The amount of lymphocyte infiltration correlates with SP-2577 dose.
Fig 4.
Flow cytometry analysis of the COV434 organoids after lymphocyte infiltration assay.
COV434 organoids were dissociated and the infiltrated lymphocytes were stained with different markers and analyzed by flow cytometry as described in ‘Materials and Methods’. Panel A and B are showing the status of lymphocytes infiltrated in DMSO control organoids and SP-2577 treated organoids, respectively, in the representative dot plots. (Ai and Bi) The dot plots showed the CD3+ population in CD45+ gated lymphocytes. (Aii and Bii) The status of CD4+ and CD8+ T cells gated on CD3+ cells are presented in different quadrants. (Aiii and Biii) Percentage of CD56+ NK in CD3+ cells are presented with quadrant statistics. (Aiv and Biv) The status of CD19+ B cells are shown in CD3+ cells.
Fig 5.
SP-2577 promotes PD-L1 expression and rescues checkpoint inhibition sensitivity in SCCOHT COV 434 pIND 20 BRG1-2.7 cell line.
(A) RT-PCR analysis of COV434 pIND 20 BRG1-2.7 cells after SP-2577 treatment shows increase of PD-L1 expression levels, which are lost after doxycycline treatment. (B) Co-treatment of COV434 pIND 20 BRG1-2.7 organoids with SP-2577 and anti-PD-L1 antibodies or lymphocytes treated with anti-CTLA-4 antibodies showed an increase in lymphocyte infiltration as checked after 48 h.
Fig 6.
SMARCA4 re-expression in SCCOHT cell lines blocks lymphocyte infiltration.
(A) Western blot and densitometry analysis for SMARCA4 expression levels in COV 434 pIND20 BRG1-2.7 after 1 μM doxycycline daily treatment (D = days). Total protein load was verified by LSD1 expression analysis. (B) RT-PCR for BRG1 and BRM expression levels in COV434 pIND20 BRG1-2.7. Both gene expression increased after SP-2577 treatment. (C) The levels of lymphocyte infiltration are significantly reduced after SMARCA4 re-expression in COV434 pIND20 BRG1-2.7 (p-value = 0.038). (D) RT-PCR analysis of SMARCA4-induced COV434 pIND20 BRG1-2.7 after 72 h SP2577 treatment shows decrease of ERVs activity and INF expression. (E) MSD analysis of SMARCA4-induced COV434 pIND20 BRG1-2.7 after 72 h SP-2577 treatment shows significant decrease of cyto/chemokines released in medium after treatment. (F) Treatment of SMARCA4-induced COV434 pIND20 BRG1-2.7 organoids with conditioned medium (CM) from SMARCA4-deficient COV434 pIND20 BRG1-2.7 does not affect lymphocyte infiltration suggesting SMARCA4 plays role in immune response. Left panel: Experimental design.
Fig 7.
SP-2577 promotes lymphocyte infiltration in ARID1A deficient cells.
(A) Amount of lymphocyte infiltration is dependent on SP-2577 treatment in TOV21G pIND20-ARID1A cell lines. (B) Lymphocyte infiltration is significantly reduced in TOV21G pIND20-ARID1A cells after ARID1A re-expression upon doxycycline treatment (p-value = 0.004). (C) Co-treatment of SP-2577 and CTLA-4 antibodies in the immune infiltration assay showed an increase in lymphocyte infiltration in TOV21G-pIND20-ARID1A after 48 h.