A pre-B acute lymphoblastic leukemia cell line model reveals the mechanism of thalidomide therapy-related B-cell leukemogenesis.

Lenalidomide, a thalidomide derivative, is prescribed as maintenance therapy for multiple myeloma (MM). Patients with MM receiving lenalidomide were found to develop a distinct therapy-related B cell acute lymphoblastic leukemia (B-ALL). However, the molecular mechanism by which lenalidomide drives B-ALL is unknown. We show that thalidomide treatment of B cell lines increased CD34 expression and fibronectin adhesion. This resembled the effects of Ikzf1 loss of function mutations in B-ALL. IKZF1 is a transcription factor that can act as both a transcriptional activator and a repressor depending upon the target loci. In our experiments, thalidomide-induced degradation of IKZF1 increased the expression of its transcriptional repression targets Itga5 and CD34 explaining the increased adhesion and stemness. Strikingly, withdrawal of thalidomide lead to the mis-localization of IKZF1 to the cytoplasm. Moreover, chromatin immunoprecipitation data showed a long-term effect of thalidomide treatment on IKZF1 target loci. This included decreased chromatin occupancy at early B cell factor 1 (EBF1) and Spi1 (PU.1). Consequently, B-cell lineage specifying transcription factors including Pax5, Spi1 and EBF1 were downregulated even after 7 days of thalidomide withdrawal. Our study thus provides a molecular mechanism of thalidomide-induced B-ALL whereby thalidomide alters the chromatin occupancy of IKZF1 at key B-cell lineage transcription factors leading to a persistent block in B-cell differentiation.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

In and out: Traffic and dynamics of thrombopoietin receptor.

Thrombopoiesis had long been a challenging area of study due to the rarity of megakaryocyte precursors in the bone marrow and the incomplete understanding of its regulatory cytokines. A breakthrough was achieved in the early 1990s with the discovery of the thrombopoietin receptor (TpoR) and its ligand thrombopoietin (TPO). This accelerated research in thrombopoiesis, including the uncovering of the molecular basis of myeloproliferative neoplasms (MPN) and the advent of drugs to treat thrombocytopenic purpura. TpoR mutations affecting its membrane dynamics or transport were increasingly associated with pathologies such as MPN and thrombocytosis. It also became apparent that TpoR affected hematopoietic stem cell (HSC) quiescence while priming hematopoietic stem cells (HSCs) towards the megakaryocyte lineage. Thorough knowledge of TpoR surface localization, dimerization, dynamics and stability is therefore crucial to understanding thrombopoiesis and related pathologies. In this review, we will discuss the mechanisms of TpoR traffic. We will focus on the recent progress in TpoR membrane dynamics and highlight the areas that remain unexplored.

Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants.

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.

Activity of nonmuscle myosin II isoforms determines localization at the cleavage furrow of megakaryocytes.

Megakaryocyte polyploidy is characterized by cytokinesis failure resulting from defects in contractile forces at the cleavage furrow. Although immature megakaryocytes express 2 nonmuscle myosin II isoforms (MYH9 [NMIIA] and MYH10 [NMIIB]), only NMIIB localizes at the cleavage furrow, and its subsequent absence contributes to polyploidy. In this study, we tried to understand why the abundant NMIIA does not localize at the furrow by focusing on the RhoA/ROCK pathway that has a low activity in polyploid megakaryocytes. We observed that under low RhoA activity, NMII isoforms presented different activity that determined their localization. Inhibition of RhoA/ROCK signaling abolished the localization of NMIIB, whereas constitutively active RhoA induced NMIIA at the cleavage furrow. Thus, although high RhoA activity favored the localization of both the isoforms, only NMIIB could localize at the furrow at low RhoA activity. This was further confirmed in erythroblasts that have a higher basal RhoA activity than megakaryocytes and express both NMIIA and NMIIB at the cleavage furrow. Decreased RhoA activity in erythroblasts abolished localization of NMIIA but not of NMIIB from the furrow. This differential localization was related to differences in actin turnover. Megakaryocytes had a higher actin turnover compared with erythroblasts. Strikingly, inhibition of actin polymerization was found to be sufficient to recapitulate the effects of inhibition of RhoA/ROCK pathway on NMII isoform localization; thus, cytokinesis failure in megakaryocytes is the consequence of both the absence of NMIIB and a low RhoA activity that impairs NMIIA localization at the cleavage furrow through increased actin turnover.

Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization.

Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint.

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.

Neonatal Persistent Exposure to 6-Propyl-2-thiouracil, a Thyroid-Disrupting Chemical, Differentially Modulates Expression of Hepatic Catalase and C/EBP-ß in Adult Rats.

Persistent exposure of rats to 6-propyl-2-thiouracil (PTU) from birth resulted in decreases in plasma thyroid hormone (TH) levels and hepatic expression of catalase and CCAAT enhancer binding protein ß (C/EBP-ß). Catalase promoter region (-185 to +52) that contains binding sites for C/EBP-ß showed an augmentation in the methylation level along with a change in methylation pattern of CpG islands in response to PTU treatment. PTU withdrawal on 30 days of birth restored TH levels and C/EBP-ß to control rats in adulthood. Although catalase expression was restored to some extent in adult rats in response to PTU withdrawal, a permanent change in its promoter CpG methylation pattern was recorded. The results suggest that downregulation of adult hepatic catalase gene in response to persistent neonatal PTU exposure may not solely be attributed to thyroid-disrupting properties of PTU. It is possible that besides thyroid-disrupting behavior, PTU may impair expression of hepatic catalase by altering methylation pattern of its promoter.

Alteration of mitochondrial proteome due to activation of Notch1 signaling pathway.

The Notch signaling pathway, a known regulator of cell fate decisions, proliferation, and apoptosis, has recently been implicated in the regulation of glycolysis, which affects tumor progression. However, the impact of Notch on other metabolic pathways remains to be elucidated. To gain more insights into the Notch signaling and its role in regulation of metabolism, we studied the mitochondrial proteome in Notch1-activated K562 cells using a comparative proteomics approach. The proteomic study led to the identification of 10 unique proteins that were altered due to Notch1 activation. Eight of these proteins belonged to mitochondria-localized metabolic pathways like oxidative phosphorylation, glutamine metabolism, Krebs cycle, and fatty acid oxidation. Validation of some of these findings showed that constitutive activation of Notch1 deregulated glutamine metabolism and Complex 1 of the respiratory chain. Furthermore, the deregulation of glutamine metabolism involved the canonical Notch signaling and its downstream effectors. The study also reports the effect of Notch signaling on mitochondrial function and status of high energy intermediates ATP, NADH, and NADPH. Thus our study shows the effect of Notch signaling on mitochondrial proteome, which in turn affects the functioning of key metabolic pathways, thereby connecting an important signaling pathway to the regulation of cellular metabolism.

p27 and leukemia: cell cycle and beyond.

Cell division is the foundation to development and the regulation of cell cycle progression is therefore of paramount importance to the living organisms. Primary control of cell cycle is achieved by an array of cyclins and cyclin dependent kinases (CDKs). The functions of these cyclin-CDK complexes are again regulated by a host of cyclin dependent kinase inhibitors (CDKI). Till date CDKIs are broadly classified into two groups-INK4 family (p15, p16, p18, and p19) and the cip/kip family (p21, p27, and p57). Collectively these CDKIs regulate the progression from G1 to S phase of cell cycle. This review summarizes the functions of p27 while highlighting its emerging roles in leukemia.

Molecular cross talk between Notch1, Shh and Akt pathways during erythroid differentiation of K562 and HEL cell lines.

Erythropoiesis is a tightly regulated process dependent on extrinsic signals conveyed by the bone marrow niche. The signalling pathways thus activated or repressed do not act in isolation; rather an intricate cross talk among these pathways ensues homoeostasis within the erythroid compartment. In this study, we describe the effects of two such signalling pathways namely the Notch1 and the Shh pathway on erythropoiesis in immortalised K562 and HEL cell lines as well as the cross talk that ensues between them. We show that while activation of the Notch1 pathway inhibits differentiation of erythroid lineage cell lines as well as in in-vitro primary erythroid cultures from the human CD34(+) cells; Shh pathway favours erythroid differentiation. Further, the Notch1 pathway activates the Akt pathway and constitutively active Akt partially mimics the effect of Notch1 activation on erythropoiesis. Moreover, the Notch1, Akt and Shh pathways were found to cross talk with each other. In this process, activation of Notch1 was found to down regulate the Shh pathway independent of Akt activation. Significantly, Notch1 not only down regulated the Shh pathway, but also inhibited recombinant Shh mediated erythropoiesis. Our study thus reveals an intricate crosstalk among the Notch1, Shh and Akt pathways wherein Notch1 emerges as a key regulator of erythropoiesis.

Cytotoxic Impact of Costunolide Isolated from Costus speciosus on Breast Cancer via Differential Regulation of Cell Cycle-An In-vitro and In-silico Approach.

Costunolide, a sesquiterpene lactone, is a biologically active molecule found in most of the medicinally valuable plants. The present study aims to evaluate the anticancer property of costunolide isolated from Costus speciosus against breast cancer cell lines (MCF-7 and MDA-MB-231). Costunolide effectively reduced the viability of both MCF-7 and MDA-MB-231 cell lines at an IC50 value of 40 µM. Flow cytometric analysis revealed costunolide mediated cell cycle arrest at G2/M phase in both the cell types. Western blotting results confirmed the alterations in the expression of cell cycle regulators (cyclin D1, D3, CDK-4, CDK-6, p18 INK4c, p21 CIP1/Waf-1 and p27 KIP1) and apoptosis inducers (caspase-3 and caspase-9) upon costunolide treatment in comparison with their expressions in normal breast cell line (MCF-10A). Costunolide mediated downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators were related to the induction of apoptosis in cancer cells. The above results were validated with in-silico results that predicted stable interactions between costunolide and cancer targets. Thus costunolide effectively induced breast cancer cell apoptosis targeting cell cycle regulation, and the compound can be used as an effective herbal therapeutic molecule to treat breast cancer with further explorations.

Differential regulation of MCM7 and its intronic miRNA cluster miR-106b-25 during megakaryopoiesis induced polyploidy.

Megakaryocytes exit from mitotic cell cycle and enter a phase of repeated DNA replication without undergoing cell division, in a process termed as endomitosis of which little is known. We studied the expression of a DNA replication licensing factor mini chromosome maintenance protein 7 (MCM7) and its intronic miR-106b-25 cluster during mitotic and endo-mitotic cycles in megakaryocytic cell lines and in vitro cultured megakaryocytes obtained from human cord blood derived CD34(+) cells. Our results show that contrary to mitotic cell cycle, endomitosis proceeds with an un-coupling of the expression of MCM7 and miR-106b-25. This was attributed to the presence of a transcript variant of MCM7 which undergoes nonsense mediated decay (NMD). Additionally, miR-25 which was up regulated during endomitosis was found to promote megakaryopoiesis by inhibiting the expression of PTEN. Our study thus highlights the importance of a transcript variant of MCM7 destined for NMD in the modulation of megakaryopoiesis.

In vitro and in silico evaluation of NF-κB targeted costunolide action on estrogen receptor-negative breast cancer cells--a comparison with normal breast cells.

Costunolide, a sesquiterpene lactone is a plant-derived secondary metabolite found to be present in most of the pharmacologically active herbs, being the cause for their medicinal values. The present study aims to evaluate the cytotoxic effect of costunolide isolated from Costus speciosus rhizome extract on MDA-MB-231 cells and explore its targeted action in comparison with its action on the normal breast cells (MCF 10A). The effect of costunolide on cell viability of the cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay. The targeted action of the compound was analyzed comparing the effectiveness of the compound to alter the protein expression levels of NF-κB subunits in the normal and the cancer cells using western blotting analysis. In silico studies were performed to predict the targeted interaction of costunolide with the NF-κB subunit proteins. Costunolide inhibited the cell viability of MDA-MB-231 cells in a dose-dependent manner leaving no significant change in the viability of the normal breast cells. The over expressed NF-κB subunits - p65, 52 and 100 in the cancer cells were found to be downregulated when treated with costunolide at an effective dose of 20 and 40 µM costunolide. In silico results provided stable interactions between costunolide and the target proteins, supporting the in vitro results in addition. Thus, costunolide derived from C. speciosus plant source elevates a fresh conviction for its use in breast cancer therapy for its cytotoxic efficacy and non-toxic nature.

Exploring Anticancer Properties of Medicinal Plants against Breast Cancer by Downregulating Human Epidermal Growth Factor Receptor 2.

Plants have a history of being employed in managing breast cancer. However, no scientific evidence supports the idea that these plants can effectively reduce the level of HER2 expression. In this study, extracts from 10 medicinal plants were evaluated for their anticancer properties against HER2-positive breast cancer cells through various methods, including the SRB assay, comet assay, annexin V-FITC dual staining, and immunoblotting. All extracts exerted antiproliferative activity against HER2-positive breast cancer cells. Furthermore, Terminalia chebula (T. chebula), Berberis aristata (B. aristata), and Mucuna pruriens (M. pruriens) reduced HER2 expression in tested cell lines. In addition, an increased Bax/Bcl-2 ratio was observed after the treatment. A comparative proteomics study showed modulation in the proteome profile of breast cancer cells after treatment with T. chebula, B. aristata, Punica granatum, M. pruriens, and Acorus calamus. Metabolic profiling of lead plants revealed the existence of multiple anticancer compounds. Our study demonstrates the considerable potential of the mentioned plants as innovative therapies for HER2-positive breast cancer.

Thiol redox status critically influences mitochondrial response to thyroid hormone-induced hepatic oxidative injury: A temporal analysis.

Liver is a major target organ for thyroid hormone. The objective of the present study was to investigate temporal regulation of mitochondrial glutathione and protein-bound thiol redox status in hyperthyroid liver. Mitochondria were isolated from control and hyperthyroid rat liver tissues at different time intervals, i.e., 24, 72, and 120 h following treatment, and sub-fractionated into sub-mitochondrial particles (SMPs) and matrix fractions. Increased prooxidant levels were indicative of oxidative stress in hyperthyroid mitochondria. Sensitivity to membrane lipid peroxidation (LPx) was maximal after 24 h, which subsided with time. Oxidative damage to proteins was evident as high carbonylation after 72 h; thiol residue damage was an early phenomenon. Reduced and oxidized glutathione (GSH and GSSG) pools of mitochondria were progressively depleted, thereby, impairing matrix antioxidant capacity. However, adaptations to withstand oxidative challenge were elicited in both SMPs and matrix fractions over the long term. It is concluded that maintenance of appropriate intra-mitochondrial glutathione and protein-bound thiol redox status could be instrumental in attenuating thyroid hormone-induced oxidative stress.

T3 fails to restore mitochondrial thiol redox status altered by experimental hypothyroidism in rat testis.

Oxidative stress impaired sperm function might lead to infertility. The objective of this study was to evaluate the effects of altered thyroid hormone levels on regulation of mitochondrial glutathione redox status and its dependent antioxidant defense system in adult rat testis and their correlation with testicular function. Adult male Wistar rats were rendered hypothyroid by administration of 6-n-propyl-2-thiouracil in drinking water for six weeks. At the end of the treatment period, a subset of the hypothyroid rats was treated with T(3) (20 µg/100g body weight/day for 3 days). Mitochondria were isolated from euthyroid, hypothyroid and hypothyroid+T(3)-treated rat testes, and sub-fractionated into sub-mitochondrial particles and matrix fractions. Mitochondrial respiration, oxidative stress indices and antioxidant defenses were assayed. The results were correlated with daily testicular sperm production and epididymal sperm viability. Increased pro-oxidant level and reduced antioxidant capacity rendered the hypothyroid mitochondria susceptible to oxidative injury. The extent of damage was more evident in the membrane fraction. This was reflected in higher degree of oxidative damages inflicted upon membrane lipids and proteins. While membrane proteins were more susceptible to carbonylation, thiol residue damage was evident in matrix fraction. Reduced levels of glutathione and ascorbate further weakened the antioxidant defenses and impaired testicular function. Hypothyroid condition disturbed intra-mitochondrial thiol redox status leading to testicular dysfunction. Hypothyroidism-induced oxidative stress condition could not be reversed with T(3) treatment.

DaiCee: A database for anti-cancer compounds with targets and side effect profiles.

Identification of the toxicity of compounds is more crucial before entering clinical trials. Awareness of physiochemical properties, possible targets and side effects has become a major public health issue to reduce risks. Experimental determination of analyzing the physiochemical properties of a drug, their interaction with specific receptors and identifying their side-effects remain challenging is time consuming and costly. We describe a manually compiled database named DaiCee database, which contains 2100 anticancer drugs with information on their physiochemical properties, targets of action and side effects. It includes both synthetic and herbal anti-cancer compounds. It allows the search for SMILES notation, Lipinski's and ADME/T properties, targets and side effect profiles of the drugs. This helps to identify drugs with effective anticancer properties, their toxic nature, drug-likeness for in-vitro and in-vivo experiments. It also used for comparative analysis and screening of effective anticancer drugs using available data for compounds in the database. The database will be updated regularly to provide the users with latest information. The database is available at the URL http://www.hccbif.org/usersearch.php.