Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.

Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.

Characterization of ductal carcinoma in situ cell lines established from breast tumor of a Singapore Chinese patient.

BACKGROUND: Five cell lines were established from a Singaporean patient of Chinese origin with breast ductal carcinoma in situ (DCIS). These five cell lines express exogenous human telomerase reverse transcriptase (hTERT) which confers the ability to proliferate indefinitely. METHODS: Cells were isolated from the DCIS excision and transfected with a plasmid expressing hTERT, a catalytic subunit of telomerase. Five immortalized colonies were propagated and characterized by karyotyping, array comparative genomic hybridization (CGH), immunostaining and Western blots for biomarkers, in vitro anchorage independent growth, in vivo mouse tumorigenicity, drug sensitivity, species authentication and virology safety testing. RESULTS: Array CGH analysis showed that the cell lines harbored different specific genetic aberrations. Common mutations observed in most breast cancer cell lines such as the general loss of heterozygosity (LOH) throughout chromosome X and chromosome 17 are also observed in our cell lines. The cell lines were further characterized as human breast cells that are estrogen- and progesterone-receptor positive, and sensitive to tamoxifen. The cell lines showed anchorage-independent growth in the soft agar assay and can grow in common culture medium without supplementation with growth factor, therefore demonstrating transformed characteristics. Four of the cell lines can engraft and form measureable tumors after 50 days when injected subcutaneously into immune-deficient (SCID) mice. The weak tumorigenicity of these cell lines corresponded well with their non-malignant growth origin. The cell lines were authenticated to be of human origin based on DNA fingerprint of the cells. The cell lines were free from contamination of 20 viruses and mycoplasma in the virological safety test panel. CONCLUSIONS: Unlike most available breast cell lines, our cell lines are derived from primary breast cancer tissues that represent earlier grades or tumor progression stages. They would be useful as study models for basic and clinical research programs directed at early diagnosis and intervention.

Identification of small-molecule binding sites of a ubiquitin-conjugating enzyme-UBE2T through fragment-based screening.

UBE2T is an attractive target for drug development due to its linkage with several types of cancers. However, the druggability of ubiquitin-conjugating E2 (UBE2T) is low because of the lack of a deep and hydrophobic pocket capable of forming strong binding interactions with drug-like small molecules. Here, we performed fragment screening using 19 F-nuclear magnetic resonance (NMR) and validated the hits with 1 H-15 N-heteronuclear single quantum coherence (HSQC) experiment and X-ray crystallographic studies. The cocrystal structures obtained revealed the binding modes of the hit fragments and allowed for the characterization of the fragment-binding sites. Further screening of structural analogues resulted in the identification of a compound series with inhibitory effect on UBE2T activity. Our current study has identified two new binding pockets in UBE2T, which will be useful for the development of small molecules to regulate the function of this protein. In addition, the compounds identified in this study can serve as chemical starting points for the development of UBE2T modulators.

Combination treatment for myeloproliferative neoplasms using JAK and pan-class I PI3K inhibitors.

Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3'-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs.

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N1G37) Methyltransferase (TrmD) Inhibitors.

Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N1G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based high-throughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD ( PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-l-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAM-noncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.

MicroRNA expression profiling during human cord blood-derived CD34 cell erythropoiesis.

OBJECTIVE: MicroRNA (miRNA) expression profiling was performed on ex vivo differentiating erythroid cultures derived from human umbilical cord blood (UCB) CD34 cells and K562 cells to identify miRNAs involved in erythropoiesis. MATERIALS AND METHODS: Both cell types were subjected to growth factor cocktails stimulating erythroid differentiation and were harvested for small RNA extraction at regular intervals. miRNAs with at least a 1.5-fold expression increase or decrease compared to unstimulated (day 0) cells were identified by array hybridization. Validity of the expression array was confirmed by quantitative real-time polymerase chain reaction on randomly selected miRNAs. RESULTS: Hierarchical clustering analysis and comparison between stimulated UCB-derived CD34 cells and K562 cells revealed miRNAs that are critical for erythroid development and maturation. Correlation analysis on UCB-derived CD34 cells shows that miR-15b, miR-16, miR-22, and miR-185 have strong positive correlation to the appearance of erythroid surface antigens (CD71, CD36, and CD235a) and hemoglobin synthesis, while miR-28 has an inverse relationship to the expression of these markers. Signature miRNAs associated with common myeloid/erythroid progenitor commitment (e.g., miR-181 family, miR-221, miR-154), early erythroid commitment (e.g., miR-32, miR-136, miR-137), and maturation (miR-22, miR-28, miR-185) were also identified by temporal correlation analysis. These miRNAs are predicted to target genes involved in cell development and differentiation. CONCLUSION: Probable signature miRNAs for erythropoiesis are identified. Further experimentations are needed to define the roles of these miRNAs in regulating erythroid commitment.

Novel Acetamide Indirectly Targets Mycobacterial Transporter MmpL3 by Proton Motive Force Disruption.

To identify novel inhibitors of Mycobacterium tuberculosis cell envelope biosynthesis, we employed a two-step approach. First, we screened the diverse synthetic small molecule 71,544-compound Enamine library for growth inhibitors using the non-pathogenic surrogate Mycobacterium bovis BCG as screening strain and turbidity as readout. Second, 16 confirmed hits were tested for their ability to induce the cell envelope stress responsive promoter piniBAC controlling expression of red fluorescent protein in an M. bovis BCG reporter strain. Using a fluorescence readout, the acetamide E11 was identified. Resistant mutant selection and whole genome sequencing revealed the mycolic acid transporter Mmpl3 as a candidate target of E11. Biochemical analysis using mycobacterial spheroplasts and various membrane assays suggest that E11 indirectly inhibits MmpL3-facilitated translocation of trehalose monomycolates by proton motive force disruption. E11 showed potent bactericidal activity against growing and non-growing M. tuberculosis, low cytotoxic, and hemolytic activity and a dynamic structure activity relationship. In addition to activity against M. tuberculosis, E11 was active against the non-tuberculous mycobacterium M. abscessus, an emerging opportunistic pathogen. In conclusion, we identified a novel bactericidal anti-mycobacterial lead compound targeting MmpL3 providing an attractive starting point for optimization.

Phenotypic Screening for Inhibitors of a Mutant Thrombopoietin Receptor.

An inhibitor for the thrombopoietin receptor (TpoR) would be more specific for the treatment of myeloproliferative neoplasms (MPNs) due to constitutively active mutant TpoR compared to the current treatment approach of inhibiting Janus kinase 2 (JAK2). We describe a cell-based high-throughput phenotypic screening approach to identify inhibitors for constitutively active mutant TpoR. A stepwise elimination process is used to differentiate generally cytotoxic compounds from compounds that specifically inhibit growth of cells expressing wild-type TpoR and/or mutant TpoR. We have systematically optimized the phenotypic screening assay and documented in this chapter critical parameters for a successful phenotypic screen, such as cell growth and seeding optimization, plate reproducibility and uniformity studies, and an assay robustness analysis with a pilot screen.

Optimization of Selective Mitogen-Activated Protein Kinase Interacting Kinases 1 and 2 Inhibitors for the Treatment of Blast Crisis Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.

A Phenotypic Screen for Small-Molecule Inhibitors of Constitutively Active Mutant Thrombopoietin Receptor Implicated in Myeloproliferative Neoplasms.

BACKGROUND: Rather than a Janus Kinase 2 inhibitor (ruxolitinib), a specific thrombopoietin receptor (TpoR) inhibitor would be more specific for the treatment of myeloproliferative neoplasms due to TpoR mutations. OBJECTIVE: A cell-based phenotypic approach to identify specific TpoR inhibitors was implemented and a library of 505,483 small molecules was screened for inhibitory effects on cells transformed by TpoR mutants. RESULTS: Among the identified hits are two analogs of 3-(4-piperidinyl) indole. The analogs showed about five-fold preferential inhibition of cell viability towards Ba/F3 cells expressing the TpoR W515L mutation compared to the parental cells. There was no significant difference in inhibition of cell viability between the TpoR wild type and the TpoR W515L mutant cells. Preferential inhibition of viability was observed in Ba/F3 cells expressing erythropoietin receptor (EpoR) when stimulated with Epo compared to stimulation with interleukin-3 (IL3). The indole analog inhibited ex vivo colony formations of primary bone marrow cells from heterozygous JAK2 V617F knock-in mice. Drug combination treatment study was performed using ruxolitinib and the indole analog. Drug synergistic effects were observed when cells were stimulated to proliferate through both the IL3 and TpoR pathways. Our compound specifically targets monoamine receptors in the rhodopsin-like receptor family of G protein-coupled receptor. CONCLUSION: This screen has identified a monoamine receptor inhibitor that can inhibit viability of cells with active TpoR or EpoR signalings. Drug synergism with ruxolitib is demonstrated.

Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.

A novel role for proliferin-2 in the ex vivo expansion of hematopoietic stem cells.

A family of proliferin genes was discovered on a microarray analysis of hematopoiesis supportive stromal cell lines. Proliferin-2 (PLF2) increased the frequency of long-term culture-initiating cells (LTC-IC) from 1 in 340 to 1 in 256 of the primary hematopoietic stem cell (HSC)-enriched bone marrow cells grown on MS5.1 feeder layer. A repeat using AFT024 feeder layer also showed a similar increase in LTC-IC (from 1 in 386 cells to 1 in 260 cells). The clonogenic output of the LTC-ICs was also increased significantly. The growth of various hematopoietic and stromal cell lines treated with PLF2 was found to increase by 4-27%, as measured by cell count and DNA synthesis assay. These findings open up the possibility of using PLF2 as a new member of the growth factor cocktails for the ex vivo expansion of HSC.

Establishment and characterization of a singaporean chinese lung adenocarcinoma cell line with four copies of the epidermal growth factor receptor gene.

We have established a lung adenocarcinoma cell line, ETCC016, from lung pleural effusion of a male Singaporean Chinese with advanced lung adenocarcinoma. The subject smoked 20 cigarettes per day for more than 30 years. The cell line arose from spontaneous transformation of cells grown in a collagen-coated culture dish. Transformed characteristics of the cell line include the ability to reach high confluency in a culture dish, low cell doubling time, ability to form colonies in soft agar, and ability to form solid tumor in immune-compromised SCID mice. Immunostaining showed that the cells originated from lung epithelial cells. Genomic analysis revealed a large amount of chromosomal aberrations (gain and loss of genetic materials, and loss of heterozygosity [LOH]), indicative of a long history of smoking. The cells have four copies of epidermal growth factor receptor (EGFR) and three copies of MYC, but have lost one copy of the RB1 gene. LOH was detected in TP53 and BRAF genes. There is no anaplastic lymphoma kinase (ALK) gene rearrangement. The ETCC016 lung adenocarcinoma cell line has demonstrated susceptibility towards inhibitors specific for EGFR/HER2 and ALK targets, but resistance to MYC-specific inhibitor. This cell line will be a useful model for further understanding of lung adenocarcinoma.

Molecular integrative clustering of Asian gastric cell lines revealed two distinct chemosensitivity clusters.

Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy.

Specific activation of the p53 pathway by low dose actinomycin D: a new route to p53 based cyclotherapy.

The activation of p53 has been proposed as a novel anti-cancer treatment in two distinct contexts. In the first activation of p53 in tumor cells can promote apoptosis and senescence and enhance the anti-tumor activity of cytotoxic chemotherapeutic drugs. In the second application activation of p53 in normal tissues can cause a reversible cell cycle arrest that can be used to protect normal cells from the action of anti-mitotics. In this cyclotherapy role p53 mutant tumor cells are not arrested and remain sensitive to anti-mitotics. The advent of specific p53 activating molecules such as nutlin-3 has encouraged both approaches. We have sought for a clinically approved drug that can mimic nutlin-3. We show here that low doses of actinomycin D mimic nutlin-3 in the highly specific activation of p53 dependant transcription, in the induction of a reversible protective growth arrest in normal cells and in the enhancement of the activity of chemotherapeutic drug induced killing of p53 positive human tumor cells. While high doses of actinomycin D reveal its more non-specific activities, low doses of the drug will allow exploration of the value of p53 activation in preclinical and clinical models before nutlin-3 like drugs are approved.

LIX: a chemokine with a role in hematopoietic stem cells maintenance.

LIX is a chemokine usually associated with cell migration and activation in neutrophil. While using a microarray approach to dissect the hematopoietic microenvironment, we have discovered that LIX is also expressed in the hematopoietic stromal cells and its expression is associated with hematopoietic supportive phenotypes. LIX microarray profiles were verified using reverse transcription and semi-quantitative polymerase chain reaction. We subsequently tested the effects of LIX on the primary bone marrow derived lineage depleted (Lin(-)) cells. LIX was found to increase the number of long-term culture-initiating cells by 34% (from 1 in 342 to 1 in 255 of Lin(-) cells). LIX also increased the clonogenicity of the long-term culture Lin(-) cells by 2-fold (p=0.029 ). When compared to untreated EML hematopoietic progenitor cell line, LIX was found to increase the level of DNA synthesis in EML cells significantly (1.63-fold; 0.010p=), with a corresponding increase in viable cell number by 1.11-fold 96 h after seeding. Similar effect was not observed with the M1 mature myeloid leukemia cell line or with the MS5.1 and AFT024 stromal cell lines. This suggested that the proliferative effect of LIX is specific towards the primitive hematopoietic cells.