Emerging roles of histone modifications and HDACs in RNA splicing.

Histone modifications and RNA splicing, two seemingly unrelated gene regulatory processes, greatly increase proteome diversity and profoundly influence normal as well as pathological eukaryotic cellular functions. Like many histone modifying enzymes, histone deacetylases (HDACs) play critical roles in governing cellular behaviors and are indispensable in numerous biological processes. While the association between RNA splicing and histone modifications is beginning to be recognized, a lack of knowledge exists regarding the role of HDACs in splicing. Recent studies however, reveal that HDACs interact with spliceosomal and ribonucleoprotein complexes, actively control the acetylation states of splicing-associated histone marks and splicing factors, and thereby unexpectedly could modulate splicing. Here, we review the role of histone/protein modifications and HDACs in RNA splicing and discuss the convergence of two parallel fields, which supports the argument that HDACs, and perhaps most histone modifying enzymes, are much more versatile and far more complicated than their initially proposed functions. Analogously, an HDAC-RNA splicing connection suggests that splicing is regulated by additional upstream factors and pathways yet to be defined or not fully characterized. Some human diseases share common underlying causes of aberrant HDACs and dysregulated RNA splicing and, thus, further support the potential link between HDACs and RNA splicing.

Loss of the mitochondrial citrate carrier, Slc25a1/CIC disrupts embryogenesis via 2-Hydroxyglutarate.

Biallelic germline mutations in the SLC25A1 gene lead to combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a fatal systemic disease uniquely characterized by the accumulation of both enantiomers of 2-hydroxyglutaric acid (2HG). How SLC25A1 deficiency contributes to D/L-2HGA and the role played by 2HG is unclear and no therapy exists. Both enantiomers act as oncometabolites, but their activities in normal tissues remain understudied. Here we show that mice lacking both SLC25A1 alleles exhibit developmental abnormalities that mirror human D/L-2HGA. SLC25A1 deficient cells undergo premature senescence, suggesting that loss of proliferative capacity underlies the pathogenesis of D/L-2HGA. Remarkably, D- and L-2HG directly induce senescence and treatment of zebrafish embryos with the combination of D- and L-2HG phenocopies SLC25A1 loss, leading to developmental abnormalities in an additive fashion relative to either enantiomer alone. Metabolic analyses further demonstrate that cells with dysfunctional SLC25A1 undergo mitochondrial respiratory deficit and remodeling of the metabolism and we propose several strategies to correct these defects. These results reveal for the first time pathogenic and growth suppressive activities of 2HG in the context of SLC25A1 deficiency and suggest that targeting the 2HG pathway may be beneficial for the treatment of D/L-2HGA.

Stromal Senescence following Treatment with the CDK4/6 Inhibitor Palbociclib Alters the Lung Metastatic Niche and Increases Metastasis of Drug-Resistant Mammary Cancer Cells.

BACKGROUND: CDK4/6 inhibitors (CDKi) have improved disease control in hormone-receptor-positive, HER2-negative metastatic breast cancer, but most patients develop progressive disease. METHODS: We asked whether host stromal senescence after CDK4/6 inhibition affects metastatic seeding and growth of CDKi-resistant mammary cancer cells by using the p16-INK-ATTAC mouse model of inducible senolysis. RESULTS: Palbociclib pretreatment of naïve mice increased lung seeding of CDKi-resistant syngeneic mammary cancer cells, and this effect was reversed by depletion of host senescent cells. RNA sequencing analyses of lungs from non-tumor-bearing p16-INK-ATTAC mice identified that palbociclib downregulates immune-related gene sets and gene expression related to leukocyte migration. Concomitant senolysis reversed a portion of these effects, including pathway-level enrichment of TGF-ß- and senescence-related signaling. CIBERSORTx analysis revealed that palbociclib alters intra-lung macrophage/monocyte populations. Notably, lung metastases from palbociclib-pretreated mice revealed senescent endothelial cells. Palbociclib-treated endothelial cells exhibit hallmark senescent features in vitro, upregulate genes involved with the senescence-associated secretory phenotype, leukocyte migration, and TGF-ß-mediated paracrine senescence and induce tumor cell migration and monocyte trans-endothelial invasion in co-culture. CONCLUSIONS: These studies shed light on how stromal senescence induced by palbociclib affects lung metastasis, and they describe palbociclib-induced gene expression changes in the normal lung and endothelial cell models that correlate with changes in the tumor microenvironment in the lung metastatic niche.

ΔNp63-Regulated Epithelial-to-Mesenchymal Transition State Heterogeneity Confers a Leader-Follower Relationship That Drives Collective Invasion.

Defining how interactions between tumor subpopulations contribute to invasion is essential for understanding how tumors metastasize. Here, we find that the heterogeneous expression of the transcription factor ΔNp63 confers distinct proliferative and invasive epithelial-to-mesenchymal transition (EMT) states in subpopulations that establish a leader-follower relationship to collectively invade. A ΔNp63-high EMT program coupled the ability to proliferate with an IL1α- and miR-205-dependent suppression of cellular protrusions that are required to initiate collective invasion. An alternative ΔNp63-low EMT program conferred cells with the ability to initiate and lead collective invasion. However, this ΔNp63-low EMT state triggered a collateral loss of fitness. Importantly, rare growth-suppressed ΔNp63-low EMT cells influenced tumor progression by leading the invasion of proliferative ΔNp63-high EMT cells in heterogeneous primary tumors. Thus, heterogeneous activation of distinct EMT programs promotes a mode of collective invasion that overcomes cell intrinsic phenotypic deficiencies to induce the dissemination of proliferative tumor cells. SIGNIFICANCE: These findings reveal how an interaction between cells in different EMT states confers properties that are not induced by either EMT program alone.

Histone Deacetylase SIRT1 Targets Plk2 to Regulate Centriole Duplication.

The protein deacetylase SIRT1 (Sirtuin 1) regulates many cellular processes, including cell-cycle progression, DNA damage response, and metabolism. Although the centrosome is a key regulator of cell-cycle progression and genome stability, little is known concerning SIRT1 controlled centrosome-associated events. Here we report that the centrosome protein Plk2 is acetylated and undergoes deacetylation by SIRT1. Acetylation protects Plk2 from ubiquitination, and SIRT1-mediated deacetylation promotes ubiquitin-dependent degradation of Plk2. SIRT1 controls centriole duplication by temporally modulating centrosomal Plk2 levels. AURKA phosphorylates SIRT1 and promotes the SIRT1-Plk2 interaction in mitosis. In early-mid G1, phosphorylated SIRT1 deacetylates and promotes Plk2 degradation. In late G1, SIRT1 is hypophosphorylated and its affinity to Plk2 is decreased, resulting in a rapid accumulation of centrosomal Plk2, which contributes to the timely initiation of centriole duplication. Collectively, our findings uncover a critical role of SIRT1 in centriole duplication and provide a mechanistic insight into SIRT1-mediated centrosome-associated functions.

Therapeutic Effects of XPO1 Inhibition in Thymic Epithelial Tumors.

Exportin 1 (XPO1) mediates nuclear export of many cellular factors known to play critical roles in malignant processes, and selinexor (KPT-330) is the first XPO1-selective inhibitor of nuclear export compound in advanced clinical development phase for cancer treatment. We demonstrated here that inhibition of XPO1 drives nuclear accumulation of important cargo tumor suppressor proteins, including transcription factor FOXO3a and p53 in thymic epithelial tumor (TET) cells, and induces p53-dependent and -independent antitumor activity in vitro Selinexor suppressed the growth of TET xenograft tumors in athymic nude mice via inhibition of cell proliferation and induction of apoptosis. Loss of p53 activity or amplification of XPO1 may contribute to resistance to XPO1 inhibitor in TET. Using mass spectrometry-based proteomics analysis, we identified a number of proteins whose abundances in the nucleus and cytoplasm shifted significantly following selinexor treatment in the TET cells. Furthermore, we found that XPO1 was highly expressed in aggressive histotypes and advanced stages of human TET, and high XPO1 expression was associated with poorer patient survival. These results underscore an important role of XPO1 in the pathogenesis of TET and support clinical development of the XPO1 inhibitor for the treatment of patients with this type of tumors. Cancer Res; 77(20); 5614-27. ©2017 AACR.

PI3K as a Potential Therapeutic Target in Thymic Epithelial Tumors.

INTRODUCTION: Thymic epithelial tumors (TETs) are rare tumors originating from the epithelium of the thymus with limited therapeutic options beyond surgery. The pathogenesis of TETs is poorly understood, and the scarcity of model systems for these rare tumors makes the study of their biology very challenging. METHODS: A new cell line (MP57) was established from a thymic carcinoma specimen and characterized using standard biomarker analysis, as well as next-generation sequencing (NGS) and functional assays. Sanger sequencing was used to confirm the mutations identified by NGS. RESULTS: MP57 possesses all the tested thymic epithelial markers and is deemed a bona fide thymic carcinoma cell line. NGS analysis of MP57 identified a mutation in the gene PIK3R2, which encodes a regulatory subunit of PI3K. Further analysis identified different mutations in multiple PI3K subunit genes in another cell line and several primary thymic carcinoma samples, including two catalytic subunits (PIK3CA and PIK3CG) and another regulatory subunit (PIK3R4). Inhibiting PI3K with GDC-0941 resulted in in vitro antitumor activity in TET cells carrying mutant PI3K subunits. CONCLUSIONS: Alterations of PI3K due to mutations in its catalytic or regulatory subunits are observed in a subgroup of TETs, in particular, thymic carcinomas. Targeting PI3K may be an effective strategy to treat these tumors.

Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands.

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10µL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.