EGFR Inhibition Overcomes Resistance to FGFR4 Inhibition and Potentiates FGFR4 Inhibitor Therapy in Hepatocellular Carcinoma.

Aberrant activation of the FGF19-FGFR4 signaling pathway plays an essential role in the tumorigenesis of hepatocellular carcinoma (HCC). As such, FGFR4 inhibition has emerged as a novel therapeutic option for the treatment of HCC and has shown preliminary efficacy in recent clinical trials for patients exhibiting aberrant FGF19 expression. Resistance to kinase inhibitors is common in oncology, presenting a major challenge in the clinical treatment process. Hence, we investigated the potential mechanisms mediating and causing resistance to FGFR4 inhibition in HCC. Upon the successful establishment of a battery of cellular models developing resistance to FGFR4 inhibitors, we have identified the activation of EGFR, MAPK, and AKT signaling as the primary mechanisms mediating the acquired resistance. Combination of inhibitors against EGFR or its downstream components restored sensitivity to FGFR4 inhibitors. In parental HCC cell lines, EGF treatment also resulted in resistance to FGFR4 inhibitors. This resistance was effectively reverted by inhibitors of the EGFR signaling pathway, suggesting that EGFR activation is a potential cause of intrinsic resistance. We further confirmed the above findings in vivo in mouse xenograft tumor models. Genomic analysis of patient samples from The Cancer Genome Atlas confirmed that a segment of patients with HCC harboring FGF19 overexpression indeed exhibited increased activation of EGFR signaling. These findings conclusively indicate that both induced and innate activation of EGFR could mediate resistance to FGFR4 inhibition, suggesting that dual blockade of EGFR and FGFR4 may be a promising future therapeutic strategy for the treatment of FGF19-FGFR4 altered HCC.

TCGA RNA-Seq and Tumor-Infiltrating Lymphocyte Imaging Data Reveal Cold Tumor Signatures of Invasive Ductal Carcinomas and Estrogen Receptor-Positive Human Breast Tumors.

Comparative studies of immune-active hot and immune-deserted cold tumors are critical for identifying therapeutic targets and strategies to improve immunotherapy outcomes in cancer patients. Tumors with high tumor-infiltrating lymphocytes (TILs) are likely to respond to immunotherapy. We used the human breast cancer RNA-seq data from the cancer genome atlas (TCGA) and classified them into hot and cold tumors based on their lymphocyte infiltration scores. We compared the immune profiles of hot and cold tumors, their corresponding normal tissue adjacent to the tumor (NAT), and normal breast tissues from healthy individuals from the Genotype-Tissue Expression (GTEx) database. Cold tumors showed a significantly lower effector T cells, lower levels of antigen presentation, higher pro-tumorigenic M2 macrophages, and higher expression of extracellular matrix (ECM) stiffness-associated genes. Hot/cold dichotomy was further tested using TIL maps and H&E whole-slide pathology images from the cancer imaging archive (TCIA). Analysis of both datasets revealed that infiltrating ductal carcinoma and estrogen receptor ER-positive tumors were significantly associated with cold features. However, only TIL map analysis indicated lobular carcinomas as cold tumors and triple-negative breast cancers (TNBC) as hot tumors. Thus, RNA-seq data may be clinically relevant to tumor immune signatures when the results are supported by pathological evidence.

Upregulation of CD11b and CD86 through LSD1 inhibition promotes myeloid differentiation and suppresses cell proliferation in human monocytic leukemia cells.

LSD1 (Lysine Specific Demethylase1)/KDM1A (Lysine Demethylase 1A), a flavin adenine dinucleotide (FAD)-dependent histone H3K4/K9 demethylase, sustains oncogenic potential of leukemia stem cells in primary human leukemia cells. However, the pro-differentiation and anti-proliferation effects of LSD1 inhibition in acute myeloid leukemia (AML) are not yet fully understood. Here, we report that small hairpin RNA (shRNA) mediated LSD1 inhibition causes a remarkable transcriptional activation of myeloid lineage marker genes (CD11b/ITGAM and CD86), reduction of cell proliferation and decrease of clonogenic ability of human AML cells. Cell surface expression of CD11b and CD86 is significantly and dynamically increased in human AML cells upon sustained LSD1 inhibition. Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) analyses of histone marks revealed that there is a specific increase of H3K4me2 modification and an accompanied increase of H3K4me3 modification at the respective CD11b and CD86 promoter region, whereas the global H3K4me2 level remains constant. Consistently, inhibition of LSD1 in vivo significantly blocks tumor growth and induces a prominent increase of CD11b and CD86. Taken together, our results demonstrate the anti-tumor properties of LSD1 inhibition on human AML cell line and mouse xenograft model. Our findings provide mechanistic insights into the LSD1 functions in controlling both differentiation and proliferation in AML.

Histone Demethylase LSD1 Promotes Adipocyte Differentiation through Repressing Wnt Signaling.

Adipose tissue plays important roles in animals. White fat stores energy in lipids, while brown fat is responsible for nonshivering thermogenesis through UCP1-mediated energy dissipation. Although epigenetic mechanisms modulate differentiation in multiple lineages, the epigenetic regulation of brown adipocyte differentiation is poorly understood. By screening a collection of epigenetic compounds, we found that Lysine-Specific Demethylase 1 (LSD1) inhibitors repress brown adipocyte differentiation. RNAi-mediated Lsd1 knockdown causes a similar effect, which can be rescued by expression of wild-type but not catalytic-inactive LSD1. Mechanistically, LSD1 promotes brown adipogenesis by demethylating H3K4 on promoter regions of Wnt signaling components and repressing the Wnt pathway. Furthermore, deletion of Lsd1 in mice leads to inhibition of brown adipogenesis, validating the pivotal role of LSD1 in brown fat development in vivo. Our work identifies LSD1 as a key epigenetic regulator in brown adipogenesis. The link between LSD1 and the Wnt pathway provides potential opportunities to modulate brown fat differentiation.

Histone methyltransferase SETDB1 regulates liver cancer cell growth through methylation of p53.

SETDB1 is a histone H3K9 methyltransferase that has a critical role in early development. It is located within a melanoma susceptibility locus and facilitates melanoma formation. However, the mechanism by which SETDB1 regulates tumorigenesis remains unknown. Here we report the molecular interplay between SETDB1 and the well-known hotspot gain-of-function (GOF) TP53 R249S mutation. We show that in hepatocellular carcinoma (HCC) SETDB1 is overexpressed with moderate copy number gain, and GOF TP53 mutations including R249S associate with this overexpression. Inactivation of SETDB1 in HCC cell lines bearing the R249S mutation suppresses cell growth. The TP53 mutation status renders cancer cells dependent on SETDB1. Moreover, SETDB1 forms a complex with p53 and catalyses p53K370 di-methylation. SETDB1 attenuation reduces the p53K370me2 level, which subsequently leads to increased recognition and degradation of p53 by MDM2. Together, we provide both genetic and biochemical evidence for a mechanism by which SETDB1 regulates cancer cell growth via methylation of p53.

Rapid identification of anti-inflammatory compounds from Tongmai Yangxin Pills by liquid chromatography with high-resolution mass spectrometry and chemometric analysis.

We present an integrated approach to rapidly identify anti-inflammatory compounds of TongmaiYangxin Pills (TMYXP), a botanical drug for the treatment of cardiovascular disease. Liquid chromatography coupled with high-resolution mass spectrometry was used to analyze the chemical composition of TMYXP. Eighty compounds of TMYXP including flavonoids, coumarins, iridoid glycosides, saponins, and lignans, were identified unambiguously or tentatively. After the rapid isolation and bioassay, 18 fractions of TMYXP were obtained and their anti-inflammatory activities were evaluated in lipopolysaccharide-stimulated RAW 264.7 macrophages. We performed chemometric analysis to reveal the correlation between the chemical and pharmacological information of the fractions to facilitate the identification of active compounds. To verify the reliability of the proposed method in discovering active components from a complex mixture, activities of seven compounds, which were positively or negatively related to bioactivity according to calculation, were validated in vitro. Results indicated that six active compounds with high R values exerted certain anti-inflammatory effects in a dose-dependent manner with IC50 values of 53.6-204.1 µM. Our findings suggest that the integrated use of identification based on high-resolution mass spectrometry and chemometric methods could rapidly identify active compounds from complex mixture of natural products.

JAK2V617F drives Mcl-1 expression and sensitizes hematologic cell lines to dual inhibition of JAK2 and Bcl-xL.

Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in JAK2V617F-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating JAK2 mutations.

Immobilized magnetic beads-based multi-target affinity selection coupled with HPLC-MS for screening active compounds from traditional Chinese medicine and natural products.

Screening and identifying active compounds from traditional Chinese medicine (TCM) and other natural products plays an important role in drug discovery. Here, we describe a magnetic beads-based multi-target affinity selection-mass spectrometry approach for screening bioactive compounds from natural products. Key steps and parameters including activation of magnetic beads, enzyme/protein immobilization, characterization of functional magnetic beads, screening and identifying active compounds from a complex mixture by LC/MS, are illustrated. The proposed approach is rapid and efficient in screening and identification of bioactive compounds from complex natural products.

Immobilized magnetic beads based multi-target affinity selection coupled with high performance liquid chromatography-mass spectrometry for screening anti-diabetic compounds from a Chinese medicine "Tang-Zhi-Qing".

We developed an approach for screening bioactive compounds from botanical drug using multiple target-immobilized magnetic beads coupled with high performance liquid chromatography-mass spectrometry. This novel approach was called magnetic beads based multi-target affinity selection-mass spectrometry (MT-ASMS). It can enrich and identify different types of ligands from mixture extracts. Multiple targets (maltase, invertase, lipase) were immobilized on the magnetic beads by covalent linkage using 1-(3-dimethyl-aminopropyl)-3-ethyl-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as reaction reagents, respectively. The properties of enzyme conjugated magnetic beads were characterized using transmission electron microscopy, X-ray diffractometer and vibration sample magnetometer. Several factors including pH, ion strength, incubation time and temperature were optimized using three known ligands (caffeic acid, ferulic acid, and hesperidin). The established MT-ASMS approach was applied to screening for ligands from a Chinese medicine "Tang-Zhi-Qing", which was used to treat type II diabetes in China. Seven bound compounds were identified via liquid chromatography-mass spectrometry (LC/MS). Five active compounds including 2,3,4,6-tetra-O-galloyl-D-glucose, 1,2,3,4-tetra-O-galloyl-D-glucose, 1,2,3,4,6-penta-O-galloyl-d-glucose, quercetin-3-O-ß-D-glucuronide and quercetin-3-O-ß-D-glucoside were identified and their activities were validated by conventional inhibitory assay. Our findings suggested that the proposed approach is efficient in screening compounds with multiple activities from extracts of botanical drugs.

Activation of MEKK1 by Rho GTPases.

Mammalian MAP/ERK kinase kinase 1 (MEKK1) is MAP kinase kinase kinase (MAP3K) that is a crucial regulator of many cellular signaling cascades. One of the most important physiological functions of MEKK1 is its ability to regulate cell migration, because MEKK1 null mice are defective in eyelid closure. MEKK1 exhibits its signaling activity through interaction with a large array of cellular factors, including several proteins that are known to play central roles in controlling cell movement and motility. We have recently identified an interaction between MEKK1 and RhoA. This interaction occurs between the GTP-bound, active form of RhoA and the amino terminal region of MEKK1 that harbors a PHD domain with E3 ubiquitin ligase activity. RhoA-GTP activates MEKK1 in vitro and in cells. Here we describe in detail the assay methods for RhoA activation of MEKK1, including preparation of recombinant proteins and proteins immunoprecipitated from cells, pretreatment of proteins, and assay conditions. We also briefly explain the methods and conditions we use to identify the interaction between MEKK1 and RhoA in yeast and in mammalian cells.

High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors.

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.