Complex regulatory networks control epithelial-to-mesenchymal transition (EMT) but the underlying epigenetic control is poorly understood. Lysine-specific demethylase 1 (LSD1) is a key histone demethylase that alters the epigenetic landscape. Here we explored the role of LSD1 in global epigenetic regulation of EMT, cancer stem cells (CSCs), the tumour microenvironment, and therapeutic resistance in breast cancer. LSD1 induced pan-genomic gene expression in networks implicated in EMT and selectively elicits gene expression programs in CSCs whilst repressing non-CSC programs. LSD1 phosphorylation at serine-111 (LSD1-s111p) by chromatin anchored protein kinase C-theta (PKC-θ), is critical for its demethylase and EMT promoting activity and LSD1-s111p is enriched in chemoresistant cells in vivo. LSD1 couples to PKC-θ on the mesenchymal gene epigenetic template promotes LSD1-mediated gene induction. In vivo, chemotherapy reduced tumour volume, and when combined with an LSD1 inhibitor, abrogated the mesenchymal signature and promoted an innate, M1 macrophage-like tumouricidal immune response. Circulating tumour cells (CTCs) from metastatic breast cancer (MBC) patients were enriched with LSD1 and pharmacological blockade of LSD1 suppressed the mesenchymal and stem-like signature in these patient-derived CTCs. Overall, LSD1 inhibition may serve as a promising epigenetic adjuvant therapy to subvert its pleiotropic roles in breast cancer progression and treatment resistance.

g Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor ␤ (TGF-␤) and the key inflammatory regulatory protein NF-B. Chromatinized PKC-exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC--sensitive genes that are directly tethered to PKC-in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. Epithelial to mesenchymal transition (EMT) is a key step in cancer progression and the process of metastasis that creates a reservoir for cancer stem cells (CSCs) and is associated with highly aggressive traits (1-4). As well as driving metastasis, these CSCs, or "precursor" metastatic cells, play a pivotal role in therapeutic resistance and relapse in breast cancer patients (5, 6). Breast CSCs are a distinct subpopulation of mesenchymal cells that possess several important features, namely, expression of key surface markers (CD44 high and CD24 low ) (7), a distinct transcriptome (3), the ability to form spherical colonies in suspension cultures (termed mammospheres) (8), and enhanced resistance to chemotherapy (9, 10) and ionizing radiation (11)(12)(13)(14).Eukaryotes utilize the chromatin landscape as its epigenetic template within the nucleus of living cells in order to promote inducible gene transcription in response to environmental signals. Highly compacted chromatin structures are enriched in nucleosomes and are transcriptionally silent, and a net loss of nucleosomes from gene-specific regulatory regions increases chromatin accessibility and initiates context-specific transcriptional programs. In addition to ATP-dependent chromatin remodeling and exchange of histone variants with canonical histones, histone modifications are thought to alter gene expression, by changing chromatin structure and/or by providing a platform that promotes binding of transcriptional regulators (15-23).Novel classes of chromatin-associated enzymes that play critical roles in modulating chromatin structure within the human genome have recently been discovered. In particular, signaling kinases can act as chromatin regulators of inducible gene transcription in both higher and lower eukaryotes by two distinct mechanisms: relaying signals from the cytoplasm to the nucleus and direct association with chromatin-bound transcription complexes at activated tar...

Macrophages play an important role in regulating the tumor microenvironment (TME). Here we show that classical (M1) macrophage polarization reduced expression of LSD1, nuclear REST corepressor 1 (CoREST), and the zinc finger protein SNAIL. The LSD1 inhibitor phenelzine targeted both the flavin adenine dinucleotide (FAD) and CoREST binding domains of LSD1, unlike the LSD1 inhibitor GSK2879552, which only targeted the FAD domain. Phenelzine treatment reduced nuclear demethylase activity and increased transcription and expression of M1-like signatures both in vitro and in a murine triple-negative breast cancer model. Overall, the LSD1 inhibitors phenelzine and GSK2879552 are useful tools for dissecting the contribution of LSD1 demethylase activity and the nuclear LSD1-CoREST complex to switching macrophage polarization programs. These findings suggest that inhibitors must have dual FAD and CoREST targeting abilities to successfully initiate or prime macrophages toward an anti-tumor M1-like phenotype in triple-negative breast cancer.

Lysine specific demethylase 1 (LSD1) is a key epigenetic eraser enzyme implicated in cancer metastases and recurrence. Nuclear LSD1 phosphorylated at serine 111 (nLSD1p) has been shown to be critical for the development of breast cancer stem cells. Here we show that circulating tumor cells isolated from immunotherapy-resistant metastatic melanoma patients express higher levels of nLSD1p compared to responders, which is associated with co-expression of stem-like, mesenchymal genes. Targeting nLSD1p with selective nLSD1 inhibitors better inhibits the stem-like mesenchymal signature than traditional FAD-specific LSD1 catalytic inhibitors such as GSK2879552. We also demonstrate that nLSD1p is enriched in PD-1 + CD8 + T cells from resistant melanoma patients and 4T1 immunotherapy-resistant mice. Targeting the LSD1p nuclear axis induces IFN-γ/TNF-α-expressing CD8 + T cell infiltration into the tumors of 4T1 immunotherapy-resistant mice, which is further augmented by combined immunotherapy. Underpinning these observations, nLSD1p is regulated by the key T cell exhaustion transcription factor EOMES in dysfunctional CD8 + T cells. EOMES co-exists with nLSD1p in PD-1 + CD8 + T cells in resistant patients, and nLSD1p regulates EOMES nuclear dynamics via demethylation/acetylation switching of critical EOMES residues. Using novel antibodies to target these post-translational modifications, we show that EOMES demethylation/acetylation is reciprocally expressed in resistant and responder patients. Overall, we show for the first time that dual inhibition of metastatic cancer cells and re-invigoration of the immune system requires LSD1 inhibitors that target the nLSD1p axis.

A remarkable feature of the adaptive immune system is the speed at which small numbers of antigen-specific lymphocytes can mediate a successful immune response. Rapid expansion of T and B lymphocyte clones that have receptors specific for a particular antigen is one of the primary means by which a swift response is generated. Although much of this clonal expansion is caused by the division of antigenspecific cells, here we demonstrate an additional mechanism by which the pool of effector T cells against a viral infection can quickly enlarge. Our data show that virus-specific CD8 ؉ cytotoxic T lymphocytes (CTL) can transfer their T cell receptors (TCR) to recipient CTL of an unrelated specificity that, as a consequence, gain the antigen specificity of the donor T cell. This process occurs within minutes via membrane exchange and results in the recipient CTL acquiring the ability to recognize and eliminate cells targeted by the donor TCR, while still retaining the antigen specificity of its own TCR. Such receptor sharing allows rapid, proliferation-independent expansion of virus-specific T cell clones of low frequency and plays a highly significant antiviral role that can protect the host from an otherwise lethal infection.antigen receptor transfer ͉ clonal selection theory ͉ poxvirus ͉ trogocytosis ͉ clonal expansion