LSD1: Expanding Functions in Stem Cells and Differentiation

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) provide a powerful model system to uncover fundamental mechanisms that control cellular identity during mammalian development. Histone methylation governs gene expression programs that play a key role in the regulation of the balance between self-renewal and differentiation of ESCs. Lysine-specific demethylase 1 (LSD1, also known as KDM1A), the first identified histone lysine demethylase, demethylates H3K4me1/2 and H3K9me1/2 at target loci in a context-dependent manner. Moreover, it has also been shown to demethylate non-histone substrates playing a central role in the regulation of numerous cellular processes. In this review, we summarize current knowledge about LSD1 and the molecular mechanism by which LSD1 influences the stem cells state, including the regulatory circuitry underlying self-renewal and pluripotency.

Human Sex Matters: Y-linked lysine demethylase 5D drives accelerated male osteogenic differentiation

Female sex is increasingly associated to a loss of bone mass during aging and an increased risk for fractures developing nonunion. Hormonal factors and cell-intrinsic mechanisms are suggested to drive these sexual dimorphisms, although underlying molecular mechanisms are still a matter of debate. Here, we observed a decreased capacity of calvarial bone recovery in female rats and a profound sexually dimorphic osteogenic differentiation human adult neural crest-derived stem cells (NCSCs). Next to an elevated expression of pro-osteogenic regulators, global trancriptomics revealed Lysine Demethylase 5D (KDM5D) to be highly upregulated in differentiating male NCSCs. Loss of function by siRNA or pharmacological inhibition of KDM5D significantly reduced the osteogenic differentiation capacity of male NCSCs. In summary, we demonstrate craniofacial osteogenic differentiation to be sexually dimorphic with the expression of KDM5D as a prerequisite for accelerated male osteogenic differentiation, emphasizing the analysis of sex-specific differences as a crucial parameter for treating bone defects.

Therapeutic Targeting of Histone Lysine Demethylase KDM4B Blocks the Growth of Castration-Resistant Prostate Cancer

Background: Accumulating evidence points to epigenetic mechanisms as essential in tumorigenesis. Treatment that targets epigenetic regulators is becoming an attractive strategy for cancer therapy. The role of epigenetic therapy in prostate cancer (PCa) remains elusive. Previously we demonstrated a correlation of levels of histone lysine demethylase KDM4B with the appearance of castration resistant prostate cancer (CRPC) and identified a small molecular inhibitor of KDM4B, B3. In this study, we aim to define the role of KDM4B in promoting PCa progression and test the efficacy of B3 using clinically relevant PCa models. Methods: KDM4B was overexpressed in LNCaP cells or knocked down (KD) in 22Rv1 cells. The specificity of B3 was determined in vitro using recombinant KDM proteins and in vivo using 22Rv1 cell lysates. The efficacy of B3 monotherapy or in combination with androgen receptor (AR) antagonist enzalutamide or the mTOR inhibitor rapamycin was tested using xenograft models in castrated mice. Comparative transcriptomic analysis was performed on KDM4B KD and B3-treated 22Rv1 cells to determine the on-target (KDM4B-dependent) and off-target (non-KDM4B-associated) effects of B3.Results: Overexpression of KDM4B in LNCaP cells enhanced its tumorigenicity whereas knockdown of KDM4B in 22Rv1 cells reduced tumor growth in castrated mice. B3 suppressed the growth of both 22Rv1 and VCaP xenografts and sensitized 22Rv1 cells to enzalutamide inhibition. B3 also inhibited 22Rv1 tumor growth synergistically with rapamycin that resulted in cell apoptosis. Mechanistically, B3 inhibited expression of AR-V7 and genes involved in epithelial-to-mesenchymal transition. DNA replication stress marker gH2A.X was upregulated by B3, which is further increased when combined with rapamycin. Based on transcriptomic and biochemical analyses, B3 inhibits both H3K9me3 and H3K27me3 demethylase activity, which is believed to underlie its anti-tumor action. Conclusions: Our studies establish KDM4B as a potent target for CRPC and B3 as a potential therapeutic agent. B3 as monotherapy or in combination with other anti-PCa therapeutics offers proof of principle for the clinical translation of epigenetic therapy targeting KDMSs for CRPC patients.

Inhibiting Lysine Demethylase 1A Improves L1CAM-Specific CAR T Cell Therapy by Unleashing Antigen-Independent Killing via the FAS-FASL Axis

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy.

Therapeutic Targeting of Histone Lysine Demethylase KDM4B Blocks the Growth of Castration-resistant Prostate Cancer

Background: Accumulating evidence points to epigenetic mechanisms as essential in tumorigenesis. Treatment that targets epigenetic regulators is becoming an attractive strategy for cancer therapy. The role of epigenetic therapy in prostate cancer (PCa) remains elusive. Previously we demonstrated a correlation of levels of histone lysine demethylase KDM4B with the appearance of castration resistant prostate cancer (CRPC) and identified a small molecular inhibitor of KDM4B, B3. In this study, we aim to define the role of KDM4B in promoting PCa progression and test the efficacy of B3 using clinically relevant PCa models. Methods: KDM4B was overexpressed in LNCaP cells or knocked down (KD) in 22Rv1 cells. The specificity of B3 was determined in vitro using recombinant KDM proteins and in vivo using 22Rv1 cell lysates. The efficacy of B3 monotherapy or in combination with androgen receptor (AR) antagonist enzalutamide or the mTOR inhibitor rapamycin was tested using xenograft models in castrated mice. Comparative transcriptomic analysis was performed on KDM4B KD and B3-treated 22Rv1 cells to determine the on-target (KDM4B-dependent) and off-target (non-KDM4B-associated) effects of B3.Results: Overexpression of KDM4B in LNCaP cells enhanced its tumorigenicity whereas knockdown of KDM4B in 22Rv1 cells reduced tumor growth in castrated mice. B3 suppressed the growth of both 22Rv1 and VCaP xenografts and sensitized 22Rv1 cells to enzalutamide inhibition. B3 also inhibited 22Rv1 tumor growth synergistically with rapamycin that resulted in cell apoptosis. Mechanistically, B3 inhibited expression of AR-V7 and genes involved in epithelial-to-mesenchymal transition. DNA replication stress marker γH2A.X was upregulated by B3, which is further increased when combined with rapamycin. Based on transcriptomic and biochemical analyses, B3 inhibits both H3K9me3 and H3K27me3 demethylase activity, which is believed to underlie its anti-tumor action.Conclusions: Our studies establish KDM4B as a potent target for CRPC and B3 as a potential therapeutic agent. B3 as monotherapy or in combination with other anti-PCa therapeutics offers proof of principle for the clinical translation of epigenetic therapy targeting KDMSs for CRPC patients.