MORF4L2 induces immunosuppressive microenvironment and immunotherapy resistance through GRHL2/MORF4L2/H4K12Ac/CSF1 axis in triple-negative breast cancer

Abstract

Background: Although immunotherapy has achieved great progress in advanced triple-negative breast cancer (TNBC), there are still numerous patients who do not benefit from immunotherapy. Therefore, identification of the key molecule that induces immune escape and clarification of its specific mechanism in TNBC are urgently needed.

Methods: In this research, single cell sequencing and bulk sequencing were conducted for biomarker screening. Immunohistochemistry, multiplex immunofluorescence, and orthotopic TNBC tumor model were applied in identifying the key molecule driving immune escape. At the mechanical level, RNA sequencing, in vitro co-culturing system, flow cytometry, Western blotting, ELISA, and real-time qPCR were carried out.

Results: Mortality factor 4 like 2 (MORF4L2) expression is significantly up-regulated among patients who developed anti-PD1 resistance. MORF4L2 enhances anti-PD1 resistance by inducing the chemotaxis of macrophage infiltration and promoting their polarization towards the alternative activation phenotype (M2), thus creating an immunosuppressive microenvironment. Mechanistically, MORF4L2 actes as part of NuA4 histone acetyltransferase (HAT) complex, contributes to to histone 4 lysine 12 acetylation (H4K12Ac) and activates the downstream transcription of macrophage colony-stimulating factor (CSF1). CSF1 is secreted by tumor cells and binds to the macrophage-surface CSF1 receptor (CSF1R), which chemotactically converted and polarized macrophages to the M2 phenotype. Furthermore, we revealed that grainyhead like transcription factor 2 (GRHL2) could promote MORF4L2 transcription by binding to the MORF4L2 enhancer region. Notably, BLZ549, an inhibitor of CSF1R, restored the anti-PD1 sensitivity by blocking the GRHL2/MORF4L2/H4K12Ac/CSF1 axis.

Conclusions: GRHL2/MORF4L2/H4K12Ac/CSF1 axis plays an important role in anti-PD1 resistance. CSF1R inhibitors can reverse GRHL2/MORF4L2-mediated anti-PD1 resistance.

Keywords: Anti-PD1; Combination therapy; Histone 4 lysine 12 acetylation; Mortality factor 4 like 2; Triple-negative breast cancer; Tumor microenvironment.

Fig. 1

DNA repair pathway and MORF4L2 are strongly associated with anti-PD1 treatment resistance in TNBC. A Schematic diagram of patient recruitment and specimen collection. B Volcano plot of DEGs among patients with different efficacy (PD vs CR + PR + SD). C-D Pathway enrichment analysis of DEGs. E Forest plot demonstrating the results of univariate analysis of genes associated with DNA repair pathways. F Analysis of immune cell infiltration among patients with different efficacy. MORF4L2 mortality factor 4 like 2; TNBC triple-negative breast cancer; DEGs differentially expressed genes; PD progressive disease; CR complete response; PR Partial response; SD stable disease

Fig. 2

Characterization of efficacy-associated microenvironmental cells. A, B UMAP plots of tumor tissue from the six patients in this study, colored by major cell type. C Analysis of interactions between lymphocytes and tumor cells. Arrows and edge colors indicate direction. Circle size is proportional to the number of cells in each cell group. Edge thickness indicates the number of interactions between populations. Bar graph indicates the number of cell-to-cell interactions. D UMAP plot of epithelial cells with each cluster displayed in a different color. E Potential trajectory analysis of epithelial cells. F Dot plot of dynamic expression of key genes for DNA repair. G, H Volcano plot and GSVA pathway enrichment analysis of epithelial DEGs among patients with different efficacy (PD vs CR + PR + SD). *P < 0.05, **P < 0.01, ***P < 0.001. UMAP uniform manifold approximation and projection; DEGs differentially expressed genes; GSVA genomic variation analysis; PD progressive disease; CR complete response; PR Partial response; SD stable disease

Fig. 3

MORF4L2 induces suppression of the immune microenvironment. A IHC staining was performed with MORF4L2-specific antibodies to verify the predictive value of MORF4L2 protein expression for anti-PD1 efficacy. B Representative images of MORF4L2 protein detected together with other immune cells by using mIF. Yellow (MORF4L2 cells), pink (CD8 cells), red (CD68 cells), green (CD206 cells). C Expression of MORF4L2 mRNA in normal and tumor tissues was analyzed utilizing FUSCC (left) and TCGA (right) databases. D Dot plot of MORF4L2 mRNA dynamic expression. E UMAP analysis of MORF4L2 expression in tumor and microenvironmental cells. F PFS curve based on MORF4L2 expression was analyzed by log-rank test using the Kaplan–Meier method. G Spider graph depicted the relationship between MORF4L2 expression and percentage change in tumor size at different time points. H, I RT-qPCR and immunoblotting were used to analyze the knockdown efficiency of MORF4L2. J, K Clonogenic cell survival assays and CCK8 were performed with 4T1 cells. MORF4L2 mortality factor 4 like 2; mIF multiple immunofluorescence; FUSCC Fudan University Shanghai Cancer Center; TCGA the Cancer Genome Atlas; UMAP uniform manifold approximation and projection; PFS progression-free survival; CCK8 cell counting kit-8

Fig. 4

MORF4L2 induces macrophage recruitment and M2 polarization. A TNBC cells were injected into BALB/c mice, tumor volume was measured at regular intervals and tumor weight was measured at the end point. B, C Flow cytometry detected the expression of CD86 and CD206 in tumor tissues. D, E Macrophage chemotaxis and migration assay (F) RT-qPCR detection of Tnf-a, Cd86, Inos, Arg2, Cd206, Il10 mRNA levels in macrophages. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance. MORF4L2 mortality factor 4 like 2; TNBC triple-negative breast cancer

Fig. 5

MORF4L2 promotes CSF1 transcription and regulates macrophages by constituting NuA4 HAT complex. A Critical gene analysis by RNA sequencing in control and MORF4L2 knockdown group. B RNA sequencing revealed downregulation of CSF1 mRNA expression after MORF4L2 knockdown. C Overexpression efficiency of MORF4L2 was analyzed using immunoblotting. D, E Detection of CSF1 expression by RT-qPCR and ELISA. F Macrophage chemotactic migration assays were performed on the control, MORF4L2 and MORF4L2 + siCSF1 groups. G RT-qPCR detection of CD86 and CD206 mRNA levels in macrophages. H Immunoprecipitation and Western blot analyses were performed to verify the interaction between MORF4L2, TIP60/KAT5, and TRRAP. I Immunoblotting identifies histone H4K12 acetylation modifications. J The visualization of enrichment of H4K12Ac at CSF1 gene locus. Both of data were normalized using reads per genomic content (RPGC). The peak at transcript start site of CSF1 were enclosed with dash line. K, L ChIP-qPCR analysis of H4K12Ac occupancy of the CSF1 promoter region in cells knocking down or overexpressing MORF4L2. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance. CSF1 colony stimulating factor; MORF4L2 mortality factor 4 like 2; H4K12Ac histone 4 lysine 12 acetylation; ELISA eznyme linked immunosorbent assay; TIP60/KAT5 lysine acetyltransferase 5; TRRAP transformation/transcription domain-associated protein; ChIP chromatin immunoprecipitation

Fig. 6

GRHL2 regulates transcription of MORF4L2. A Correlation analysis of MORF4L2 and GRHL2 expression in the TCGA TNBC transcriptome databases. B ChIP-seq peaks of GRHL2, H3K4me1 and H3K4me3 in the MORF4L2 enhancer region. C Schematic representation of the molecular mechanism by which GRHL2 drives MORF4L2 transcription. D Protein expression levels of GRHL2 and MORF4L2 after knockdown of GRHL2. E ChIP assay with GRHL2 antibody followed by RT-qPCR for MORF4L2 enhancer. F Detection and analysis of luciferase activity in HEK293T and SUM159 co-transfected with GRHL2 and pGL3-MORF4L2 enhancer plasmids. G RT-qPCR detection of CD86 and CD206 mRNA levels in macrophages. H Macrophage chemotactic migration assays were performed on the control, siGRHL2, MORF4L2 and MORF4L2 + siGRHL2 groups. I PFS curve based on GRHL2 expression was analyzed by log-rank test using the Kaplan–Meier method. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance. MORF4L2 mortality factor 4 like 2; TNBC triple-negative breast cancer; GRHL2 grainyhead like transcription factor 2; PFS progression-free survival; ChIP chromatin immunoprecipitation

Fig. 7

BLZ945 improves anti-PD1 efficacy by inhibiting the GRHL2/MORF4L2/H4K12A/CSF1 axis. Study design for each treatment group. B-D 4T1 cells were injected into BALB/c mice. Mice were treated with anti-PD1, BLZ945 or combination therapy. Tumor volume and weight were measured at the indicated times. E–G Flow cytometric analysis of microenvironmental cells in tumor tissues. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance. MORF4L2 mortality factor 4 like 2; GRHL2 grainyhead like transcription factor 2; CSF1 colony stimulating factor; H4K12Ac histone 4 lysine 12 acetylation

Fig. 8

The model demonstrates the multifaceted role of MORF4L2 in breast cancer-macrophage communication, macrophage recruitment and polarization. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance