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Hematopoietic stem cells (HSCs) have a unique capacity to undergo self-renewal and multi-lineage differentiation to provide a lifetime supply of mature blood cells. By using conditional knockout technology, we disrupted the c-myb proto-oncogene specifically in adult bone marrow (BM) to demonstrate that this transcription factor is a regulator of self-renewal and multi-lineage differentiation of adult HSCs. Targeted disruption of the c-myb gene resulted in a critical depletion of the HSC pool. In addition, BM hematopoiesis in adult mice was impaired, resulting in profound reductions of various hematopoletic lineages including neutrophilic, monocytic, B lymphoid, erythroid, and, unexpectedly, megakaryocytic cells. Serial BM transplantation into lethally irradiated recipient mice indicated an essential role for c-myb in the self-renewal process. Furthermore, in vitro functional assays demonstrated that deletion of the c-myb gene leads to a slightly reduced proliferative capacity and an aberrant and accelerated differentiation of HSCs. In addition to long-term HSCs, functional studies also show that c-myb plays a critical role in shortterm HSCs and multi-potential progenitors. Collectively, our data indicate a critical role for c-myb in adult BM hematopoiesis and in self-renewal and multi-lineage differentiation of adult HSCs. [ABSTRACT FROM AUTHOR]

Although the role of Jak3 in lymphoid development has been well-characterized, increasing evidence demonstrates that activation of the Jak3 pathway plays an important role in myeloid differentiation as well. Overexpression of Jak3 in murine myeloid 32Dcl3 cells has been shown to result in an acceleration of granulocytic differentiation induced by G-CSF. Early onset of G 1 cell cycle arrest along with upregulation of the cyclin dependent kinase inhibitor p27 Kip1 and downregulation of Cdk2, Cdk4, Cdk6, and Cyclin E has also been observed in Jak3-overexpressing 32Dcl3 cells. In addition, Jak3 overexpression in normal mouse bone marrow cells results in accelerated granulocytic and monocytic differentiation in response to GM-CSF, while pharmacological inhibition of Jak3 results in a block to GM-CSF-induced colony formation in normal mouse bone marrow cells. Jak3 is unique among the members of the Jak kinase family in that it is inducibly expressed and is a target for regulation at the level of transcription. Recent studies have demonstrated that upregulation of Jak3 during myeloid differentiation is achieved through the cooperative action of Sp1 and STAT3, consistent with evidence indicative of a crucial role for STAT3 in myeloid differentiation. These results suggest that cytokine-inducible activation of Jak3 plays a critical role in integrating the processes of growth arrest and differentiation of myeloid cells. [ABSTRACT FROM AUTHOR]

Describes the relationship between Janus protein tyrosine kinases (JAK), signal transducers and activators of transcription and Src kinases in hematopoiesis. Cytokines and their receptors; Tyrosine kinases and signal transduction; Receptors as primary substrates of JAK activation.

Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not the only function performed by this key family of cytoplasmic tyrosine kinases. Emerging evidence indicates that phosphorylation of cytokine and growth factor receptors may be the primary functional attribute of JAK kinases. The JAK-triggered receptor phosphorylation can potentially be a rate-limiting event for a successful culmination of downstream signaling events. In support of this hypothesis, it has been found that JAK kinase function is required for optimal activation of the Src-kinase cascade, the Ras-MAP kinase pathway, the PI3K-AKT pathway and STAT signaling following the interaction of cytokine/interferon receptors with their ligands. Aberrations in JAK kinase activity, that may lead to derailment of one or more of the above mentioned pathways could disrupt normal cellular responses and result in disease states. Thus, over-activation of JAK kinases has been implicated in tumorigenesis. In contrast, loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency. In summary, optimal JAK kinase activity is a critical determinant of normal transmission of cytokine and growth factor signals. Oncogene (2000) 19, 5662–5679. [ABSTRACT FROM AUTHOR]

Hematopoiesis is the cumulative result of intricately regulated signal transduction cascades that are mediated by cytokines and their cognate receptors. Proper culmination of these diverse signaling pathways forms the basis for an orderly generation of different cell types and aberrations in these pathways is an underlying cause for diseases such as cancer. Over the past several years, downstream events initiated upon cytokine/growth factor stimulation have been a major focus of biomedical research. As a result, several key concepts have emerged allowing a better understanding of the complex signaling processes. A group of novel transcription factors, termed signal transducers and activators of transcription (STATs) appear to orchestrate the downstream events propagated by cytokine/growth factor interactions with their cognate receptors. Until recently, the JAK proteins were considered to be the tyrosine kinases, which dictated the levels of phosphorylation and activation of STAT proteins, forming the basis of the JAK-STAT model. However, over the past few years, increasing evidence has accumulated which indicates that at least some of the STAT protein activation may be mediated by members of the Src gene family following cytokine/growth factor stimulation. Studies have demonstrated that the Src-family of tyrosine kinases can phosphorylate and activate certain STAT proteins, in lieu of JAK kinases. In such a scenario, JAK kinases may be more crucial to phosphorylation of the cytokine/growth factor receptors and in the process create docking sites on the receptors for binding of SH2-containing proteins such as STATs, Src-kinases and other signaling intermediates. Tyrosine phosphorylation and activation of STAT proteins can be achieved either by JAKs or Src-kinases depending on the nature of STAT that is being activated. This forms the basis for the JAK-Src-STAT model proposed in this review. The concerted action of JAK kinases, members of the Src-kinase family... [ABSTRACT FROM AUTHOR]

v-myc is the viral homolog of c-myc transduced by several acute transforming retroviruses, many of which encode this gene as a Gag-Myc fusion protein. The v-myc oncogene can transform several lineages of mammalian and avian cells either alone or in co-operation with other oncogenes. While the Gag portion of the Gag-Myc fusion protein and the nuclear localization signal each appear to be dispensable for transformation, the N- and C-termini of the Myc sequence have been found to be essential for transformation. All v-myc genes contain point mutations which seem to confer a greater potency to v-myc in the process of transformation, proliferation, and apoptosis. In v-myc-transformed myelomonocytic cells, secondary events occur, such as the expression of colony stimulating factor-1 (CSF-1) which play a critical role in immortalization and subsequent tumor progression. Inhibition of the autocrine loop of CSF-1 was found to induce apoptosis in the immortalized cells. While overexpression of v-Myc blocks terminal differentiation of hematopoietic cells, this is not sufficient to block the differentiation of certain neural and skeletal muscle cells. Recent developments on the effects of v-myc on cell growth, transformation, differentiation and apoptosis are discussed in this review. [ABSTRACT FROM AUTHOR]

The myb gene family consists of three members, named A, B and c-myb which encode nuclear proteins that function as transcriptional transactivators. Proteins encoded by these three genes exhibit a tripartate structure with an N-terminal DNA-binding domain, a central transactivation domain and a C-terminal regulatory domain. These proteins exhibit highest homology in their DNA binding domains and appear to bind DNA with overlapping sequence specificities. Transactivation by myb gene family varies considerably depending on cell type and promoter context suggesting a dependence on interaction with other cell type specific co-factors. While the C-terminal domains of A-Myb and c-Myb proteins exert a negative regulatory effect on their transcriptional transactivation function, the C-terminal domain of B-Myb appears to function as a positive regulator of this activity. One or more of these proteins interact with other transcription factors such as Ets-2, CEBP and NF-M. In addition, expression of these genes is cell cycle-regulated and inhibition of their expression with anti-sense oligonucleotides has been found to affect cell cycle-progression, cell division and/or differentiation. Members of the myb gene family exhibit different temporal and spatial expression patterns suggesting a distinctive function for each of these genes. Gene knockout experiments show that these genes play an essential role in development. Loss of c-myb function results in embryonic lethality due to failure of fetal hepatic hematopoiesis. A-myb null mutant mice, on the other hand are viable but exhibit growth abnormalities, and defects in spermatogenesis and female breast development. While the role of c-myb in oncogenesis is well established, future experiments are likely to provide further clues regarding the role of A-myb and B-myb in tumorigenesis. [ABSTRACT FROM AUTHOR]

The tumor necrosis factor receptor (TNFR) superfamily represents a growing family, with over 20 members having been identified thus far in mammalian cells. These proteins share significant homologies in their extracellular ligand binding domains and intracellular effector (death) domains. These receptors appear to transmit their signals via protein-protein interactions, which convey either a death or survival signal. Isolation and characterization of death domain containing proteins (TRADD, FADD/MORT-1, RIP), TRAF domain containing proteins (TRAF1-6) as well as new members and adaptor proteins such as DAXX have provided new insights to our understanding of signaling mechanisms associated with this family of receptors. While the death signals seem to be associated with the activation of both the caspase and JUN kinase pathways, the survival signals are mediated via the activation of the NF-κB pathway. [ABSTRACT FROM AUTHOR]

Dual specificity kinases that phosphorylate the Thr- and Tyr-residues within the TXY motif of MAP-kinases of play a central role in the regulation of various processes of cell growth. These dual specificity kinases also known as MAP kinase kinases are constituents of the sequential kinase signaling modules. Seven distinct mammalian MAP kinases kinases have been identified. Some of the unique signaling properties of these kinases are discussed here. [ABSTRACT FROM AUTHOR]

The' retroviral long -erminal repeat (LTR) contains transcriptional control elements that affect viral gene expression. By deletion mutagenesis of the genome of the cloned Abelson murine leukemia virus, regulatory signals could be mapped to at least three domains within the LTR. A defective 5' LTR that did not sustain transforming gene function was complemented by an intact LTR positioned at the 3' end of the genotne. This versatility of the retroviral genome with respect to its transcriptional control elements appears to provide a strong selective advantage for viral gene expression. [ABSTRACT FROM AUTHOR]

The benzyl styryl sulfone, ON 01910.Na, is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell lines. We examined the effect of ON 01910.Na on DNA damage-signaling molecules upstream of Cdc25C (Chk1, Chk2, and H2AX), as well as on Ran GTPase-activating protein 1 conjugated to small ubiquitin-related modifier 1 (RanGAP1·SUMO1), a mitosis coordinator. Prostate cancer, lymphoma, and leukemic cells were incubated with the drug for 4, 16, or 24 hours. Cell lysates were resolved on SDS-PAGE and analyzed by Western blot. Camptothecin and doxorubicin treatment caused activation/phosphorylation of DNA damage-responsive molecules by 4 hours, whereas ON 01910.Na did not do so. ON 01910.Na caused hyperphosphorylation of RanGAP1·SUMO1 within 4 hours that was sustained for more than 24 hours. Mild phosphorylation of Chk2 was observed only after 24-hour exposure, indicating that DNA damage response was not an initial effect of ON 01910.Na. MOLT-3 cells, synchronized by double-thymidine block, when released into a medium containing ON 01910.Na, accumulated mitotic cell number with a peak from 10 to 14 hours and remained near plateau for 20 hours, which corresponded with the time of RanGAP phosphorylation. ON 01910.Na had minimal effects on tubulin polymerization. These findings imply that ON 01910.Na neither induces DNA damage directly nor acts as a tubulin toxin. Its biological activity appears to rely on prolonged phosphorylation/hyperphosphorylation of RanGAP1·SUMO1. M-phase arrest and the consequent induction of apoptosis that follows could possibly be attributed to it. ON 01910.Na may act as an inhibitor of a RanGAP1·SUMO1 phosphatase or a stimulant of a new kinase. RanGAP1·SUMO1 appears to be a new target pathway for cancer chemotherapy. Cancer Res; 71(14); 4968-76. ©2011 AACR. [ABSTRACT FROM AUTHOR]

Background: While immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma, it is effective for ~ 52% of patients and has dangerous side effects. The objective here was to identify the feasibility and mechanism of RAS/RAF/PI3K pathway inhibition in melanoma to sensitize tumors to ICB therapy. Methods: Rigosertib (RGS) is a non-ATP-competitive small molecule RAS mimetic. RGS monotherapy or in combination therapy with ICB were investigated using immunocompetent mouse models of BRAFwt and BRAFmut melanoma and analyzed in reference to patient data. Results: RGS treatment (300 mg/kg) was well tolerated in mice and resulted in ~ 50% inhibition of tumor growth as monotherapy and ~ 70% inhibition in combination with αPD1 + αCTLA4. RGS-induced tumor growth inhibition depends on CD40 upregulation in melanoma cells followed by immunogenic cell death, leading to enriched dendritic cells and activated T cells in the tumor microenvironment. The RGS-initiated tumor suppression was partially reversed by either knockdown of CD40 expression in melanoma cells or depletion of CD8+ cytotoxic T cells. Treatment with either dabrafenib and trametinib or with RGS, increased CD40+SOX10+ melanoma cells in the tumors of melanoma patients and patient-derived xenografts. High CD40 expression level correlates with beneficial T-cell responses and better survival in a TCGA dataset from melanoma patients. Expression of CD40 by melanoma cells is associated with therapeutic response to RAF/MEK inhibition and ICB. Conclusions: Our data support the therapeutic use of RGS + αPD1 + αCTLA4 in RAS/RAF/PI3K pathway-activated melanomas and point to the need for clinical trials of RGS + ICB for melanoma patients who do not respond to ICB alone. Trial registration: NCT01205815 (Sept 17, 2010). [ABSTRACT FROM AUTHOR]

Studies the mechanism of action and the nature of target genes through which c-Myb mediates the block in differentiation of 32Dcl3 murine myeloid cells. Central role played by c-myb proto-oncogene in hematopoiesis; Effects of the expression of c-Myc; Target gene of c-Myb.

Examines two isolates of the Abelson Murine Leukemia Virus (A-MuLV) the acute transforming retrovirus encoding the v-Abl oncogene. History and virological studies of A-MuLV; Discussion on v-Abl-mediated transformation; Signal transduction pathways activated by v-Abl.

Background: While immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma, it is effective for ~ 52% of patients and has dangerous side effects. The objective here was to identify the feasibility and mechanism of RAS/RAF/PI3K pathway inhibition in melanoma to sensitize tumors to ICB therapy. Methods: Rigosertib (RGS) is a non-ATP-competitive small molecule RAS mimetic. RGS monotherapy or in combination therapy with ICB were investigated using immunocompetent mouse models of BRAFwt and BRAFmut melanoma and analyzed in reference to patient data. Results: RGS treatment (300 mg/kg) was well tolerated in mice and resulted in ~ 50% inhibition of tumor growth as monotherapy and ~ 70% inhibition in combination with aPD1 + aCTLA4. RGS-induced tumor growth inhibition depends on CD40 upregulation in melanoma cells followed by immunogenic cell death, leading to enriched dendritic cells and activated T cells in the tumor microenvironment. The RGS-initiated tumor suppression was partially reversed by either knockdown of CD40 expression in melanoma cells or depletion of CD8+ cytotoxic T cells. Treatment with either dabrafenib and trametinib or with RGS, increased CD40+SOX10+ melanoma cells in the tumors of melanoma patients and patient-derived xenografts. High CD40 expression level correlates with beneficial T-cell responses and better survival in a TCGA dataset from melanoma patients. Expression of CD40 by melanoma cells is associated with therapeutic response to RAF/MEK inhibition and ICB. Conclusions: Our data support the therapeutic use of RGS + aPD1 + aCTLA4 in RAS/RAF/PI3K pathway-activated melanomas and point to the need for clinical trials of RGS + ICB for melanoma patients who do not respond to ICB alone. [ABSTRACT FROM AUTHOR]

Interaction of IL-3 with its receptor is known to activate STAT-3 via phosphorylation of Tyrosine 701, which facilitates its dimerization and translocation to the nucleus, leading to the transcription of its target genes. In this communication, we have investigated the nature of tyrosine kinases that mediate STAT-3 phosphorylation during IL-3-mediated activation of myeloid cell proliferation. Our results show that interaction of IL-3 with its receptor leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing this transcription factor to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA. On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no effect on IL-3-mediated activation of STAT-3. Our results also show that AMSrc does not affect the phosphorylation of JAK2, suggesting that JAK and STAT phosphorylation events are mediated by two independent pathways. Inhibition of c-Src activation by AMSrc, which leads to a block to STAT-3 activation, results in a dramatic inhibition of cell proliferation mediated by IL-3. However, expression of AMSrc does not activate apoptotic pathways. In contrast, expression of JAK2KE results in accelerated apoptosis of 32Dcl3 cells grown in the absence of IL-3 with concomitant down-regulation of Erk-2 kinase activity. These results suggest that Src family kinases mediate the phosphorylation of STATs and play a critical role in signal transduction pathways associated with myeloid cell proliferation while JAK kinases mediate the activation of Erk-2 pathway which appears to provide anti-apoptotic signals. Thus the activation of JAKs and STATs appear to be two independent but related events, which dictate two separate biological outcomes, the combination of... [ABSTRACT FROM AUTHOR]

During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-κB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-κB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of c-myb. The cAMP-induced NF-κB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-κB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-κB. We conclude that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-κB p50/RelB heterodimers. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of the region of Gardner-Rasheed feline sarcoma virus (GR-FeSV) encoding its primary translation product, P70gag-fgr, has been determined. From the nucleotide sequence, the amino acid sequence of this transforming protein was deduced. Computer analysis indicates that a portion of P70gag-fgr has extensive amino acid sequence homology with actin, a eukaryotic cytoskeletal protein. A second region Of p70ms-fv is closely related to the tyrosinespecific kinase gene family. Thus, the v-fgr oncogene appears to have arisen as a result of recombinational events involving two distinct cellular genes, one coding for a structural protein and the other for a protein kinase. [ABSTRACT FROM AUTHOR]

Summary Oncogenic activation of RAS genes via point mutations occurs in 20%–30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling. [ABSTRACT FROM AUTHOR]

Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma. [ABSTRACT FROM AUTHOR]

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao , was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao . [ABSTRACT FROM AUTHOR]

JLP (JNK-associated leucine zipper protein) is a scaffolding protein that interacts with various signaling proteins associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation, and apoptosis. Here we identified PLK1 (Polo-like kinase 1) as a novel interaction partner of JLP through mass spectrometric approaches. Our results indicate that JLP is phospho-primed by PLK1 on Thr-351, which is recognized by the Polo box domain of PLK1 leading to phosphorylation of JLP at additional sites. Stable isotope labeling by amino acids in cell culture and quantitative LC-MS/MS analysis was performed to identify PLK1-dependent JLP-interacting proteins. Treatment of cells with the PLK1 kinase inhibitor BI2536 suppressed binding of the Forkhead box protein K1 (FOXK1) transcriptional repressor to JLP. JLP was found to interact with PLK1 and FOXK1 during mitosis. Moreover, knockdown of PLK1 affected the interaction between JLP and FOXK1. FOXK1 is a known transcriptional repressor of the CDK inhibitor p21/WAF1, and knockdown of JLP resulted in increased FOXK1 protein levels and a reduction of p21 transcript levels. Our results suggest a novel mechanism by which FOXK1 protein levels and activity are regulated by associating with JLP and PLK1. [ABSTRACT FROM AUTHOR]

We used a proteomics approach to identify the binding partners of Trypanosoma brucei 14-3-3 (Tb14-3-3) which led to the identification of a novel kinase, AKB1. The binding between these two proteins was mediated by an amphipathic groove structure in Tb14- 3-3 and 1-438 amino acid sequence of AKB1. Recombinant AKB1 but not its ATP-binding-deficient mutant (DFG to NFG) possessed an auto-phosphorylation activity as well as a kinase activity towards a peptide substrate in vitro. However, the autophosphorylation was not required for the binding of AKB1 to Tb14-3-3. Interestingly, the kinase activity of AKB1 was inhibited by calcium, and the kinase was found to utilize GTP, and dATP in addition to ATP as phospho-donors. AKB1 formed homodimers through a leucine-zipper structure. Either knockdown of AKB1 or overexpression of AKB1, but not kinase-dead AKB1 mutant, deregulated cytokinesis and cell division, suggesting that kinase activity of AKB1 is crucial for its function. Furthermore, we showed that AKB1 exists in a detergent insoluble fraction. Laser confocal microscopy revealed that the majority of AKB1 is co-localized with a-tubulin. Taken together, these findings suggest that AKB1 might regulate cytokinesis and cell division by phosphorylating cytoskeleton-associated proteins. [ABSTRACT FROM AUTHOR]

Abstract: Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81±0.17. Six Hsp90 inhibitory compounds identified by this screening with IC50 values between 1.0 and 6μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI50 values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9–Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy. [Copyright &y& Elsevier]

Purpose: To compare rigosertib versus cisplatin as an effective radiosensitizing agent for cervical malignancies. Methods and Materials: Rigosertib and cisplatin were tested in cervical cancer cell lines, HeLa and C33A. A 24-hour incubation with rigosertib and cisplatin, before irradiation (2-8 Gy), was used for clonogenic survival assays. Cell cycle analysis (propidium iodide staining) and DNA damage (γ-H2AX expression) were evaluated by fluorescence-activated cell sorter cytometry. Rigosertib was also tested in vivo in tumor growth experiments on cervical cancer xenografts. Results: Rigosertib was demonstrated to induce a G2/M block in cancer cells. Survival curve comparison revealed a dose modification factor, as index of radiosensitization effect, of 1.1-1.3 for cisplatin and 1.4-2.2 for rigosertib. With 6-Gy irradiation, an increase in DNA damage of 15%-25% was achieved in both HeLa and C33A cells with cisplatin pretreatment, and a 71-108% increase with rigosertib pretreatment. In vivo tumor growth studies demonstrated higher performance of rigosertib when compared with cisplatin, with 53% longer tumor growth delay. Conclusions: Rigosertib was more effective than cisplatin when combined with radiation and caused minimal toxicity. These data support the need for clinical trials with rigosertib in combination therapy for patients with cervical carcinoma. [Copyright &y& Elsevier]

Thesuccess of imatinib, a BCR-ABL inhibitor for the treatmentof chronic myelogenous leukemia, has created a great impetus for thedevelopment of additional kinase inhibitors as therapeutic agents.However, the complexity of cancer has led to recent interest in polypharmacologicalapproaches for developing multikinase inhibitors with low toxicityprofiles. With this goal in mind, we analyzed more than 150 novelcyano pyridopyrimidine compounds and identified structure–activityrelationship trends that can be exploited in the design of potentkinase inhibitors. One compound, 8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x), was foundto be the most active, inducing apoptosis of tumor cells at a concentrationof approximately 30–100 nM. In vitro kinase profiling revealedthat 7xis a multikinase inhibitor with potent inhibitoryactivity against the CDK4/CYCLIN D1 and ARK5 kinases. Here, we reportthe synthesis, structure–activity relationship, kinase inhibitoryprofile, in vitro cytotoxicity, and in vivo tumor regression studiesby this lead compound. [ABSTRACT FROM AUTHOR]

A seriesof novel (E)-N-aryl-2-arylethenesulfonamides(6) were synthesized and evaluated for their anticanceractivity. Some of the compounds in this series showed potent cytotoxicityagainst a wide spectrum of cancer cell-lines (IC50valuesranging from 5 to 10 nM) including all drug resistant cell-lines.Nude mice xenograft assays with compound (E)-N-(3-amino-4-methoxyphenyl)-2-(2′,4′,6′-trimethoxyphenyl)ethenesulfonamide(6t) showed dramatic reduction in tumor size, indicatingtheir in vivo potential as anticancer agents. A preliminary drug developmentstudy with compound 6tis predicted to have increasedblood–brain barrier permeability relative to many clinicallyused antimitotic agents. Mechanistic studies indicate that 6tand some other analogues disrupted microtubule formation, formationof mitotic spindles, and arrest of cells in mitotic phase. Compound 6tinhibited purified tubulin polymerization in vitro andin vivo and circumvented drug resistance mediated by P-glycoprotein.Compound 6tspecifically competed with colchicine bindingto tubulin and with similar avidity as podophylltoxin, indicatingits binding site on tubulin. [ABSTRACT FROM AUTHOR]

Abstract: Receptor internalization is a common mechanism underlying surface receptor down-regulation (and thus receptor signaling) upon its engagement with the cognate ligand. Tight regulation of surface CD40 expression is critical in regulating different functional properties of dendritic cell (DC). Engagement of CD40 on mature DC and the cognate CD40 ligand on T cell activates c-Jun N-terminal MAPK, p38 and ERK1/2 MAPK pathways in mature DC. JNK-associated leucine zipper protein (JLP) is a scaffolding protein that interacted with p38 and JNK. The molecular mechanism underlying CD40 internalization and its physiological impact on DC functions remained unclear. Here we reported that the engagement of CD40 on the LPS-activated DC down-regulated the surface expression of CD40. We examined the role of the JLP protein in DC differentiation, and in the regulation of DC function(s) in vitro. In contrast to the abundant JLP expression observed in immortal cell lines, primary immature DC expressed low levels of the JLP proteins. The induction of the JLP protein expression was observed in the LPS-mature DC that were activated by CD40 ligation, and also in the poly I:C stimulated DC. JLP-silenced DC was impaired in regulating CD40 surface expression upon LPS stimulation and CD40 induced receptor internalization. Such aberrant change in the regulation of surface CD40 expression was associated with an augmented capacity of the JLP-silenced DC in IL-12 production. Collectively, our data identified a novel role of a scaffolding protein JLP in the regulation of surface CD40 expression and fine-tuning of DC function. [Copyright &y& Elsevier]

Abstract: ON 013100, (E)-2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzyl sulfone, is a potent kinase inhibitor whose phosphate form is in Phase I clinical trials in lymphoma and acute lymphoid leukemia. The objectives were to: (a) investigate the possible presence of the glucuronide metabolite of the drug in two representative colon cancer cell lines, a drug resistant (colo-205) and a drug sensitive (colo-320); (b) quantify the glucuronide metabolite and the unchanged drug in the cells after treatment with ON 013100. The glucuronide was synthesized and a selective LC/MS/MS method was developed and validated for the characterization and quantification of the metabolite. The glucuronide metabolite (570.6Da) was found in the drug-resistant cells upon a 1h incubation with ON 013100 (20μg/ml). After treatment with the drug, the concentration of the metabolite gradually decreased from 0.84μg/ml at 0h through 0.21μg/ml at 6h to below detection limit of 8.0ng/ml at 9h. No glucuronide metabolite was detected in the drug-sensitive cells. The concentrations of intact ON 013100 in the drug-resistant cells gradually decreased from 0.41μg/ml (0h) to 0.06μg/ml (9h). The corresponding concentrations of the intact drug in the drug-sensitive cells were from 2.88μg/ml to 0.94μg/ml. [Copyright &y& Elsevier]

Development of radio-protective agents that are non-toxic is critical in light of ever increasing threats associated with proliferation of nuclear materials, terrorism and occupational risks associated with medical and space exploration. In this communication, we describe the discovery, characterization and mechanism of action of ON01210.Na, which effectively protects mouse and human bone marrow cells from radiation-induced damage both in vitro and in vivo. Our results show that treatment of normal fibroblasts with ON01210.Na before and after exposure to ionizing radiation provides dose dependent protection against radiation-induced damage. Treatment of mice with ON01210.Na prior to radiation exposure was found to result in a more rapid recovery of their hematopoietic system. The mechanistic studies described here show that ON01210.Na manifests its protective effects through the up-regulation of PI3-Kinase/AKT pathways in cells exposed to radiation. These results suggest that ON 01210.Na is a safe and effective radioprotectant and could be a novel agent for use in radiobiological disasters. [ABSTRACT FROM AUTHOR]

Tubulin, the major structural component of microtubules,is a targetfor the development of anticancer agents. A series of (Z)-1-aryl-3-arylamino-2-propen-1-one (10) were synthesizedand evaluated for antiproliferative activity in cell-based assay.The most active compound (Z)-1-(2-bromo-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)prop-2-en-1-one(10ae) was tested in 20 tumor cell lines including multidrugresistant phenotype and was found to induce apoptosis in all thesecell lines with similar GI50values. Flow cytometry studiesshowed that 10aearrested the cells in G2/M phase ofcell cycle. In addition to G2/M block, these compounds caused microtubulestabilization like paclitaxel and induced apoptosis via activationof the caspase family. The observations made in this investigationdemonstrate that (Z)-1-Aryl-3-arylamino-2-propen-1-one(10) represents a new class of microtubule-stabilizingagents. [ABSTRACT FROM AUTHOR]

Abstract: ON01910 is a small molecular weight benzyl styryl sulfone currently under investigation as a novel anticancer agent. The purpose of the investigation was to develop a sensitive and reproducible liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to quantitate levels of ON01910 in small amounts of five biological matrices; mouse plasma, feces, urine, normal brain and brain tumor. For all matrices, protein precipitation sample preparation was used that led to linear calibration curves with coefficients of determination greater than 0.99. The lower limit of quantitation (LLOQ) for all matrices was 5ng/ml except that for mouse urine which was 10ng/ml. The calibration standard curves were reproducible for all matrices with inter- and intra-day variability in precision and accuracy being less than 15% at all quality control concentrations except for the LLOQ in mouse plasma for which the accuracy was within 17%. The assay was successfully applied to characterize the systemic pharmacokinetics of ON01910 as well as its disposition in brain and brain tumor in mice. ON01910 exhibited a clearance of 3.61±0.85l/h/kg and a half life of 8.66±3.30h at 50mg/kg dose given I.V. [Copyright &y& Elsevier]

Cyclin D proteins are elevated in many cancer cells, and targeted deletion of cyclin D1 gene in the mammary tissues protects mice from breast cancer. Accordingly, there is an increasing awareness of this novel nonenzymatic target for cancer therapeutics. We have developed novel, nonalkylating styrylbenzylsulfones that induce cell death in wide variety of cancer cells without affecting the proliferation and survival of normal cells. The development of derivatized styrylbenzylsulfones followed logically from a tumor cell cytotoxicity screen performed in our laboratory that did not have an a priori target profile. Modifications of some of the precursor molecules led to lead optimization with regard to tumor cell cytotoxicity. In this report we describe the synthesis and structure–activity relationships of novel, nonalkylating (E)-styrylbenzylsulfones and the development of the novel anticancer agent sodium (E)-2-{2-methoxy-5-[(2′,4′,6′-trimethoxystyrylsulfonyl)methyl]phenylamino}acetate (ON 01910.Na), which is in phase III trials for myelodysplastic syndromes (MDS) associated with aberrant expression of cyclin D proteins. [ABSTRACT FROM AUTHOR]

The activated mutants of the α-subunits of G proteins G12 and G13 have been designated as the gep oncogenes owing to their ability to stimulate diverse oncogenic signaling pathways that lead to neoplastic transformation of fibroblast cell lines and tumorigenesis in nude mice models. Studies from our laboratory as well as others have shown that the growth-promoting activities of Gα12 and Gα13 involve potent activation of c-Jun N-terminal kinases (JNKs). Our previous studies have indicated that the JNK-interacting leucine zipper protein (JLP), a scaffold protein involved in the structural and functional organization of the JNK/p38 mitogen-activated protein kinase module, tethers Gα12 and Gα13 to the JNK signaling module. In the present study, in addition to demonstrating the physical association between JLP and Gα12, we show that this interaction is enhanced by the receptor- or mutation-mediated activation of Gα12. We also establish that JLP interacts with Gα12 through the C-terminal domain that has been previously identified to be involved in binding to Gα13. Furthermore, using this C-terminal domain as a competitively inhibitor of JLP that can disrupt G12-JLP interaction, we demonstrate that JLP is required for the stimulation of JNK by Gα12. Our results also indicate that such JLP interaction is required for Gα12 as well as Gα13-mediated neoplastic transformation of JLP. These studies demonstrate for the first time a functional role for JLP in the oncogene-regulated neoplastic signaling pathway. [ABSTRACT FROM AUTHOR]

Hereditary cancer syndromes provide powerful insights into dysfunctional signaling pathways that lead to sporadic cancers. Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome currently linked to deregulated imprinting at chromosome 11p15 and uniparental disomy. However, causal molecular defects and genetic models have remained elusive to date in the majority of cases. The non-pleckstrin homology domain β-spectrin (β2SP) (the official name for human is Spectrin, beta, nonerythrocytic 1 (SPTBN1), isoform 2; the official name for mouse is Spectrin beta 2 (Spnb2), isoform 2), a scaffolding protein, functions as a potent TGF-β signaling member adaptor in tumor suppression and development. Yet, the role of the β2SP in human tumor syndromes remains unclear. Here, we report that β2SP+/- mice are born with many phenotypic characteristics observed in BWS patients, suggesting that β2SP mutant mice phenocopy BWS, and β2SP loss could be one of the mechanisms associated with BWS. Our results also suggest that epigenetic silencing of β2SP is a new potential causal factor in human BWS patients. Furthermore, β2SP+/- mice provide an important animal model for BWS, as well as sporadic cancers associated with it, including lethal gastrointestinal and pancreatic cancer. Thus, these studies could lead to further insight into defects generated by dysfunctional stem cells and identification of new treatment strategies and functional markers for the early detection of these lethal cancers that otherwise cannot be detected at an early stage. [ABSTRACT FROM AUTHOR]

Abstract: Novel (E)-α-benzylthio chalcones are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phosphorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leukemic cells. [Copyright &y& Elsevier]

JLP (JNK-associated leucine zipper protein) is a novel scaffolding protein involved in JNK signaling. Although it is known that JLP is highly expressed in brain, the biological function of JLP in neuronal systems remains unknown. Here, we report a novel interaction between JLP and SCG10 (superior cervical ganglia clone 10), which is a microtubule-destabilizing factor that is essential for neurite outgrowth. Inhibition of endogenous JLP expression using small interference RNA methodology strongly enhanced nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Our results show that JLP negatively regulates NGF-induced neurite outgrowth by decreasing the level of phosphorylated SCG10. Furthermore, inhibition of JNK phosphorylation by a small molecule inhibitor, SP600125, resulted in inhibition of SCG 10 phosphorylation and inhibition of neurite growth. Taken together, our results suggest that JLP negatively regulates NGF-induced neurite outgrowth through a sequestering mechanism that results in an attenuation of NGF-induced SCG10 phosphorylation. [ABSTRACT FROM AUTHOR]

Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Posiphen's safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington's disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5′-untranslated regions (5′-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT. [ABSTRACT FROM AUTHOR]

Abstract: A series of 20 novel 1-(4-sulfamylphenyl)-3-trifluoromethyl-5-indolyl pyrazolines were designed, synthesized, and screened in vitro for anti-inflammatory activity. These compounds were designed for evaluation as dual inhibitors of cyclooxygenases (COX-1 and COX-2) and lipoxygenases (LOX-5, LOX-12, and LOX-15) that are responsible for inflammation and pain. All pyrazoline molecules prepared are optically active and compounds that are more potent in COX-2 inhibitory activity (5a and 5f) were resolved by chiral column and each enantiomer was tested for cyclooxygenase inhibitory activity. Molecular modeling and comparison of molecular models of 5a enantiomers with that of celecoxib model shows that 5a (enantiomer-1) and 5a (enantiomer-2) have more hydrogen bonding interactions in the catalytic domain of COX-2 enzyme than celecoxib. Compounds 5a, 5e, and 5f showed moderate to good LOX-5 and LOX-15 inhibitory activity and this is comparable to that of celecoxib and more potent than rofecoxib. [Copyright &y& Elsevier]

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cell differentiation, but the signaling mechanisms by which it is activated during this process are largely unknown. Cdo is an immunoglobulin superfamily member that functions as a component of multiprotein cell surface complexes to promote myogenesis. In this study, we report that the Cdo intracellular region interacts with JLP, a scaffold protein for the p38α/β MAPK pathway. Cdo, JLP, and p38α/β form complexes in differentiating myoblasts, and Cdo and JLP cooperate to enhance levels of active p38α/β in transfectants. Primary myoblasts from Cdo-/- mice, which display a defective differentiation program, are deficient in p38α/β activity, and the expression of an activated form of MKK6 (an immediate upstream activator of p38) rescues the ability of Cdo-/- cells to differentiate. These results document a novel mechanism of signaling during cell differentiation: the interaction of a MAPK scaffold protein with a cell surface receptor. [ABSTRACT FROM AUTHOR]

Scaffolding proteins play a critical role in conferring specificity and fidelity to signaling pathways. The JNK-interacting leucine zipper protein (JLP) has been identified as a scaffolding protein involved in linking components of the JNK signaling module. Gα12 and Gα13, the α-subunits of heterotrimteric G proteins G12 and G13, respectively, stimulate the JNK module in diverse cell types. Here, we report that Gα13 physically interacts with JLP, and this interaction enhances Gα13-mediated JNK activation. We also demonstrate endogenous interaction between JLP and Gα13 in MCF-7 cells. JLP interaction is specific to the G12 family of α-subunits via its C-terminal domain (termed GID-JLP), spanning amino acids 1165–1307, and this interaction is more pronounced with the mutationally or functionally activated form of Gα13 compared to that of wild-type Gα13. The presence of a ternary complex consisting of Gα13, JLP, and JNK suggests a role for JLP in tethering Gα13 to the signaling components involved in JNK activation. Coexpression of GID-JLP disrupts ternary complex formation in addition to attenuating Gα13-stimulated JNK activity. These findings identify JLP as a novel scaffolding protein in the Gα13- mediated JNK signaling pathway. [ABSTRACT FROM AUTHOR]

Scaffolding proteins exist in eukaryotes to properly assemble signaling proteins into specific multimeric functional complexes. JLP is a novel leucine zipper protein belonging to a family of scaffolding proteins that assemble JNK signaling modules. JLP is a proline-rich protein that contains two leucine zipper domains and a highly conserved C-terminal domain. We have identified kinesin light chain 1 (KLC1) as a binding partner for the second leucine zipper domain of JLP using yeast two-hybrid screening. The interaction domain of KLC1 was mapped to its tetratripeptide repeat, which contains a novel leucine zipper-like domain that is crucial for the interaction with JLP. Mutations of Leu-280, Leu-287, Val-294, and Leu-301 within this domain of KLC1 disrupted its ability to associate with JLP. Immunofluorescence studies showed that JLP and KLC1 co-localized in the cytoplasm and that the localization of JLP was dependent on its second leucine zipper. Ectopic expression of a dominant negative form of KLC1 resulted in the mislocalization of endogenous JLP. Moreover, the association between JLP and KLC1 occurred in vivo and was important in the formation of ternary complex with JNK1. These results identify a novel protein-protein interaction between KLC1 and JLP that involves leucine zipper-like domains and support the role of motor proteins in the spatial regulation of signaling modules. [ABSTRACT FROM AUTHOR]

Abstract: A series of novel coumarin carboxamides were synthesized, and their tumor cell cytotoxic activity was investigated. These compounds specifically inhibited the growth of cancer cells that have a high level of ErbB-2 expression. Immunoprecipitation analysis of the cell lysates prepared from carboxamide treated cancer cells showed the inhibition of ErbB-2 phosphorylation suggesting the interaction of these compounds with ErbB-2 receptor. The down regulation of the kinase activity was further confirmed by performing in vitro kinase assay with recombinant ErbB-2 incubated with carboxamides. The inhibition of ErbB-2 phosphorylation correlated with down-regulation of ERK1 MAP kinase activation that is involved in proliferative signaling pathway. Furthermore, the cell-killing activity of many of these inhibitors is restricted to tumor cells with no demonstrable cytotoxicity against normal human fibroblasts suggesting that these compounds are tumor-specific. [Copyright &y& Elsevier]

Abstract: A new series of styryl acetoxyphenyl sulfides and sulfones possessing (E)- and (Z)-configurations were designed and prepared by stereospecific syntheses. All these compounds were evaluated for their ability to inhibit COX-2 enzyme in vitro. Structure–activity relationship studies on these compounds revealed that only sulfides with (Z)-configuration have potential COX-2 inhibitory activity. This inactivation of the enzyme is believed to be due to the selective covalent modification of COX-2 by the inhibitors. [Copyright &y& Elsevier]

Summary: Elevated expression of polo-like kinase1 (Plk1) has been reported in many human tumors, and inhibition of Plk1 activity results in their mitotic arrest and apoptosis. Here we describe the profile of ON01910, a small molecule inhibitor of Plk1 activity, which induces mitotic arrest of tumor cells characterized by spindle abnormalities leading to their apoptosis. This compound was not ATP-competitive, but competed for the substrate binding site of the enzyme. In vivo, this compound did not exhibit hematotoxicity, liver damage, or neurotoxicity, and was a potent inhibitor of tumor growth in a variety of xenograft nude mouse models. ON01910 showed strong synergy with several chemotherapeutic agents, often inducing complete regression of tumors. [Copyright &y& Elsevier]

Imatinib, which is an inhibitor of the BCR-ABL tyrosine kinase, has been a remarkable success for the treatment of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemias (CMLs). However, a significant proportion of patients chronically treated with imatinib develop resistance because of the acquisition of mutations in the kinase domain of BCR-ABL. Mutations occur at residues directly implicated in imatinib binding or, more commonly, at residues important for the ability of the kinase to adopt the specific closed (inactive) conformation to which imatinib binds. In our quest to develop new BCR-ABL inhibitors, we chose to target regions outside the ATP-binding site of this enzyme because these compounds offer the potential to be unaffected by mutations that make CML cells resistant to imatinib. Here we describe the activity of one compound, ON012380, that can specifically inhibit BCR-ABL and induce cell death of Ph+ CML cells at a concentration of <10 nM. Kinetic studies demonstrate that this compound is not ATP-competitive but is substrate-competitive and works synergistically with imatinib in wild-type BCR-ABL inhibition. More importantly, ON012380 was found to induce apoptosis of all of the known imatinib-resistant mutants at concentrations of <10 nM concentration in vitro and cause regression of leukemias induced by i.v. injection of 32Dcl3 cells expressing the imatinib-resistant BCR-ABL isoform T3151. Daily i.v. dosing for up to 3 weeks with a >100 mg/kg concentration of this agent is well tolerated in rodents, without any hematotoxicity. [ABSTRACT FROM AUTHOR]

Previous reports have suggested that the protooncogene c-myb participates in T cell development in the thymus and mature T cell proliferation. We have generated two T cell-specific c-myb knockout mouse models, myb/Lckcre and myb/CD4Cre. We have demonstrated that c-myb is required for the development of thymocytes at the DN3 stage, for survival and proliferation of double-positive thymocytes, for differentiation of single-positive CD4 and CD8 T cells, and for the proliferative responses of mature T cells. In addition, our data show that c-myb is directly involved in the formation of double-positive CD4+CD8+CD25+, CD4+CD25+, and CD8+CD25+ T cells, developmental processes that may imply a role for c-myb in autoimmune dysfunction. [ABSTRACT FROM AUTHOR]