Loss of myeloid‐specific lamin A/C drives lung metastasis through Gfi‐1 and C/EBPε‐mediated granulocytic differentiation

Abstract The immune‐suppressive tumor microenvironment promotes metastatic spread and outgrowth. One of the major contributors is tumor‐associated myeloid cells. However, the molecular mechanisms regulating their differentiation and function are not well understood. Here we report lamin A/C, a nuclear lamina protein associated with chromatin remodeling, was one of the critical regulators in cellular reprogramming of tumor‐associated myeloid cells. Using myeloid‐specific lamin A/C knockout mice and triple‐negative breast cancer (TNBC) mouse models, we discovered that the loss of lamin A/C drives CD11b+Ly6G+ granulocytic lineage differentiation, alters the production of inflammatory chemokines, decreases host antitumor immunity, and increases metastasis. The underlying mechanisms involve an increased H3K4me3 leading to the upregulation of transcription factors (TFs) Gfi‐1 and C/EBPε. Decreased lamin A/C and increased Gfi‐1 and C/EBPε were also found in the granulocytic subset in the peripheral blood of human cancer patients. Our data provide a mechanistic understanding of myeloid lineage differentiation and function in the immune‐suppressive microenvironment in TNBC metastasis.

tumor-associated neutrophils. [10][11][12] Increased MDSCs in peripheral blood (PB) and tumor sites are recognized as a poor prognosis factor in patients with several cancer types. [13][14][15] There are two major subsets of MDSCs, monocytic (CD11b + Ly6C + ) and granulocytic (CD11b + Ly6G + ) MDSCs that have strong tumor-promoting properties. 16,17 Myeloid transforming growth factor-β signaling is critical for the production of cytokines and chemokines that are important in establishing and maintaining the immunosuppressive microenvironment and premetastatic niche. 18,19 The expansion, differentiation and the functional properties of tumor-associated myeloid cells are transcriptionally and epigenetically regulated by distinct signals from the metastatic microenvironment. [19][20][21][22] Chromatin remodeling alters the accessibility of nucleosomal DNA to enhance transcription factor (TF) binding to gene promoters. The physical interactions between chromatin and nuclear structures are critical in this process. 23,24 In particular, nuclear lamina proteins (ie, lamin A/C and lamin B1) prominently interact with heterochromatin through lamina-associated domains (LADs) to suppress gene transcription. 25,26 Interestingly, mutations or protein deficiency of lamin A/C has been shown to affect in vitro differentiation of neutrophils as well as normal hematopoietic stem or progenitor cells through chromatin remodeling. 27,28 However, the role of lamin A/C in cellular reprogramming of tumor-associated myeloid cells during metastasis remains to be investigated.
Here, we report that the loss of lamin A/C in myeloid cells

| RNA-sequencing and chromatin immunoprecipitation-sequencing analysis
For RNA-sequencing, the sequencing quality of the 51 to 77 million reads per sample was assessed using FastQC (version 0.11.5), Preseq (version 2.0.3), Picard tools (version 1.119), and RSeQC (version 2.6.4). Reads were then trimmed using Cutadapt (version 1.14) to remove sequencing adapters, before mapping to the mm10 mouse genome using STAR (version 2.5.2b) in two-pass mode. Overall expression levels were quantified using RSEM version 1.3.0 with GENCODE annotation M12. DESeq2 (version 1.20.0) was used for differential expression analysis. For differential gene expression, q ≤ 0.05 and absolute fold change greater than or equal to 1.5 were used to define significant changes. (version 1.20.0) were used for differential binding analysis. Significantly differential bound peaks located within promoter regions were annotated using homer (version 4.10.1). Viewer tracks in WashU Epigenome Browser with Cistrome database were used for comparisons of H3K4me3 or H3K27ac peaks between granulocyte and monocyte. To identify the significant genes targeted by lamin A/C in myeloid cells, differentially expressed genes were first listed up from intersections between lamin A/C-deficient and wild-type (wt)-monocytic subsets (#1), between lamin A/C-deficient and wt-granulocytic subsets (#2), and between wt-monocytic and wt-granulocytic subsets (#3). Because there were no differences in lamin A/C expression between lamin A/C-deficient and wt-granulocytic subsets, genes in the list from intersection #2 were identified as background. Thereafter, overlapped genes of intersection #1 with #3, where the genes from intersection #2 were subtracted, were picked up. All high-throughput data were deposited into GEO (GSE GSE141124).    tumor. The whole-cell lysates (100 μg/sample) from these sorted cells were used for assay and a mouse cytokine antibody array was conducted as per manufacturer's protocol. The amounts of various cytokines were detected using SuperSignal West Dura (Thermo Fisher Scientific) and dot density was determined by using the ImageJ software. Heatmaps were made based on the quantification of dots.    Table S3.

| Proliferation and CTL assay of CD8 T cells
Percent input method was used for evaluation: %input = H3K4me3 or IgG control , which were compared between WT and Lmna Myeko mice.

| Immunofluorescence
Cells were fixed in 100% cold methanol for 10

| Statistical analysis
GraphPad Prism was used for graphs and for statistics. Unless otherwise indicated, all data were analyzed using the one-tailed Student t test and are expressed as mean ± standard error of the mean. Differences were considered statistically significant when the p-value was <.05.

| Lamin A/C expression in granulocytic or monocytic myeloid cell differentiation
We first noticed a gradual decrease of lamin A/C in immature myeloid cells or Gr-1 + CD11b + cells during mammary tumor progression in both E0771-M1-and 4T1 tumor models ( Figure 1A). This result was also observed in genetically engineered MMTV-PyMT and MMTV-Neu transgenic mice ( Figure S1A). Gr-1 + CD11b + cells are composed of monocytic and granulocytic myeloid subsets. 17 The granulocytic subset showed a clear lack of lamin A/C expression compared with that of a monocytic subset ( Figure 1B), with no difference in lamin B1 expression ( Figure S1B). This difference in lamin A/C expression between the two myeloid subsets was further verified in sorted CD11b + Ly6G + cells from healthy control and E0771-M1 or 4T1 tumor-bearing mice ( Figures 1C,D and S1C). Notably, the CD11b + Ly6G + granulocytic subset but not the CD11b + Ly6C + monocytic subset was expanded during metastatic progression in both E0771-M1 and 4T1 tumor models ( Figure 1E). Thus, we speculate that lamin A/C may have a role in myeloid cell lineage differentiation under tumor conditions. In addition, lamin A/C expression was decreased in PBMCs from breast cancer patients compared to those from healthy donors ( Figure 1F; GSE27567 data set).

| Myeloid-specific lamin A/C knockout promotes a granulocytic lineage differentiation that enhances tumor metastasis
Lamin A/C has been shown to modulate human hematopoietic differentiation programs. 28  Moreover, lamin A/C knockout enhanced trans-differentiation to granulocytic CD11b + Ly6G + cells in ex vivo culture compared to those from wt mice ( Figures 2F and S2G). In contrast, CD11b + Ly6G + cells were not able to trans-differentiate into CD11b + Ly6C + cells ( Figure 2F). These data suggest that loss of lamin A/C promotes and accumulates granulocytic lineage differentiation in tumor-bearing mice. To examine whether the increased granulocytic subset due to loss of lamin A/C is responsible for enhanced lung metastasis, CD11b + Ly6G + cells were depleted with Ly6G neutralizing antibody ( Figure S2H), which showed a decreased number of metastatic nodules in E0771-M1 tumor-bearing Lmna MyeKO mice ( Figure 2G). In contrast, depletion of CD11b + Ly6C + cells did not significantly affect metastatic phenotype in E0771-M1 tumor-bearing Lmna MyeKO mice ( Figure S2I). These data demonstrate that myeloid-specific lamin A/C knockout increases lung metastasis via increased granulocytic myeloid cells. lamin A/C ( Figure 3B). H3K4m3-high and lamin A/C-low cells also showed hyperlobular nuclear shape ( Figure 3B). In contrast, there was no difference in H3K9me3 ( Figure 3B). The higher H3K4me3 level was further validated in lamin A/C-deficient CD11b + Ly6C + cells compared with wt control, which is comparable to that of wt-CD11b + Ly6G + cells showing decreased lamin A/C ( Figure 3C). These data indicate that loss of lamin A/C causes epigenetic activation in nuclei of immature myeloid cells.

RNA-seq for monocytic and granulocytic myeloid cells from wt
and Lmna MyeKO mice revealed 608 differentially expressed genes associated with the presence of lamin A/C (Figures 3D and S3A).
The intersection of H3K4me3 peaks with 180 increased genes in lamin A/C-deficient CD11b + Ly6C + cells revealed 28 genes ( Figure 3D and Table S2). Among them, there were two TFs, growth factor independent 1 (Gfi-1) and CCAAT/enhancer-binding protein (C/EBPε), which are critical for granulocytic lineage differentiation ( Figure 3D). Upregulation of these TFs was confirmed by H3K4me3-ChIP-seq analysis (Cistrome database) and For human studies, monocytic (CD11b + CD14 + HLA-DR − ) and granulocytic (CD11b + CD14 − CD15 + ) subsets from PB of advanced GI cancer patients were isolated and evaluated. RT-qPCR analyses revealed a lower level of lamin A/C expression in the granulocytic subset than that in the monocytic subset, and the lamin A/C expression was inversely correlated with C/EBPε and Gfi-1 levels ( Figure 3J), which is consistent with the observation from mouse models, suggesting the correlation of decreased lamin A/C with increased C/EBPε and Gfi-1.
3.4 | Loss of lamin A/C increased immunesuppressive function of CD11b + Ly6C + cells RNA-seq analysis indicated that loss of lamin A/C decreased the expression of genes that are critical for antigen-presenting pathways such as MHC (major histocompatibility complex) class II (H2-Aa, H2-Ab1, and H2-Eb1), CD74, CD86, and Ciita in CD11b + Ly6C + cells, which was confirmed by RT-qPCR ( Figure 4A). The monocytic myeloid cell subset is already known to be immunosuppressive. 30,31 Loss of lamin A/C apparently further decreased the antigenpresenting capacity of these monocytic myeloid cells ( Figure 4A).
Cytokine protein array analysis showed a decreased immunestimulatory chemokine profile in lamin A/C-deficient CD11b + Ly6C + cells compared to wt control ( Figure 4B). This is likely critical in trafficking of antigen-presenting cells into lymph node and cancer tissues as antigen presentation to effector cells is strictly regulated by secretory cytokines and chemokines. 32 Unexpectedly, there was no obvious difference in M1/M2 cytokine profile between wt and lamin A/C-deficient CD11b + Ly6C + cells as both IL-12 (M1 cytokine) and IL-4 (M2 cytokine) were decreased in lamin A/C-deficient cells ( Figure S4A). In functional assays, lamin A/C-deficient CD11b + Ly6C + cells inhibited CD8 T-cell proliferation ( Figure 4C) and antigen-specific cytotoxicity of CTLs ( Figure 4D). Consistently,  wt control ( Figure 4E and S4B). These data suggest that loss of lamin A/C not only promotes the granulocytic lineage differentiation but also negatively regulates monocytic myeloid cell function, leading to attenuating antitumor immunity ( Figure 4F).

| DISCUSSION
We report for the first time that myeloid-specific lamin A/C attenuation promotes tumor metastasis through increased granulocytic Our data provide insight to the molecular mechanisms of wellnoted immune suppression, dysregulation of myelopoiesis under tumor conditions. 22,30,31 Previous report showed a role of lamin B receptor in all trans-retinoic acid induced granulopoiesis, which was accompanied by downregulation of lamin A/C. 33 In addition, overexpression of lamin A/C impaired nucleus shape transition which is important for granulopoiesis. 34 However, the detailed mechanism of loss of lamin A/C in granulocytic differentiation was largely unknown.
In our studies, loss of lamin A/C is a causal factor in driving granulocytic lineage differentiation. Lamin A/C is a nuclear lamina component that retains nuclear shape and makes physical connections with chromatin and loss of lamin A/C causes chromatin remodeling and altered gene transcription profiles. 25,26 Intersection of the transcriptome from Lmna-deficient myeloid cell subsets with public H3K4me3-ChIP-seq 35 provided creditable profiles of genes that were epigenetically regulated by lamin A/C. We found lamin A/C is associated with chromatin remodeling in myeloid cells through H3K4me3-mediated expression of C/EBPε and Gfi-1 which are important in granulocytic lineage differentiation of immature myeloid cells. 36,37 In fact, the abnormal differentiation of HS/PCs in BM leads to an expansion of immune-suppressive granulocytic subset by several transcrption factors, such as STAT3, IRF8, C/EBPβ, and RB1. 38 We identify two TFs C/EBPε and Gfi-1 in promoting granulocytic lineage differentiation and in driving a prometastatic microenvironment.
In addition to the granulocytic lineage differentiation, we revealed that loss of lamin A/C also contributes to decreased immune surveillance. Lamin A/C deficiency affects two major aspects of host antitumor immunity: attenuation of the antigen-presenting pathway and decreased chemoattractant for interaction with effector T cells.
For the first aspect, the monocytic subset under tumor condition shows impaired antigen presentation which is consistent with published reports. 30,31 Loss of lamin A/C negatively regulates monocytic myeloid cell function, leading to attenuated antitumor immunity. The second aspect is particularly interesting. Our data suggest that the critical chemokines that bring the APC and effector T cells into approximate interactions are at stake when lamin A/C expression is reduced ( Figure 4B). Our observations are supported by previous studies reporting that lamina-associated domains contain genes important in host immune response and loss of lamin-mediated chromatin remodeling affects the regulation of those genes. 26 In conclusion, our studies provide mechanistic insight for lamin A/C mediated epigenetic regulation of myeloid lineage differentiation and immune-suppressive function, suggesting that proper epigenetic inhibitors could be utilized to redirect myeloid lineage differentiation and to enhance host antitumor immunity. This systemic correction of the immune-suppressive microenvironment provides additional options to enhance the efficacy of cancer immunotherapy.