Peter Worland - Academia.edu (original) (raw)
Papers by Peter Worland
Radiation Oncology Investigations, 1993
We have previously reported the radiation-induced malignant transformation of immortalized human ... more We have previously reported the radiation-induced malignant transformation of immortalized human epidermal keratinocytes. Two sets of tumorigenic transformants (treated with either 2 Gy or 4 Gy twice) and their respective clonal derivatives were characterized [Thraves et al.: Proc Natl Acad Sci USA 87:117&1177, 19901. We have tested the hypothesis that the multiple changes which take place during cellular transformation are reflected at the level of protein expression. Toward this end, we have compared protein expression in the non-tumorigenic, parental keratinocytes and their radiationtransformed derivatives during the various stages of progression to tumorigenicity. We employed two-dimensional gel electrophoresis (2D-PAGE) coupled with silver staining and computer-assisted data analyses. Eighteen differentially expressed proteins were identified in the various transformed cultures. Changes in 8 of these proteins exhibited a similar pattern in both the 2 and 4 Gy transformants, while the expression of the other 10 varied. Two of the eight proteins that showed identical changes in both sets of transformed keratinocytes have been identified as tropomyosin and proliferating cell nuclear antigen (PCNA) by their characteristic 2D-map locations and reactivity with specific antisera. The identification of these differentially expressed polypeptides in the tumorigenic keratinocytes constitutes a preliminary step in the study of the complex process of radiationinduced transformation and its regulation by specific gene products.
Journal of Medicinal Chemistry, 2015
We report here the synthesis and structure-activity relationship (SAR) of a novel series of triaz... more We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC 115 for clinical development.
Journal of Medicinal Chemistry, 2015
We report here the synthesis and structure-activity relationship (SAR) of a novel series of mamma... more We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development.
Molecular cancer therapeutics, Jan 8, 2015
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth,... more The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, proliferation and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K/AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. While rapamycin analogs, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity; it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 (pAKT(S473)) in cellular systems. Growth inhibitory activity was demonstrated in hematological and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of th...
Cancer research, Jan 15, 1999
Flavopiridol is a novel flavonoid that induces cell cycle arrest at different stages of the cell ... more Flavopiridol is a novel flavonoid that induces cell cycle arrest at different stages of the cell cycle because of the inhibition of cyclin-dependent kinases (cdks). In previous studies from our laboratory, (B. A. Carlson et al., Cancer Res., 56: 2973-2978, 1996), we observed that exposure of the MCF-7 breast carcinoma cell line to flavopiridol resulted in G1-S arrest, which was associated with the loss of cdk4 and cdk2 activity by 24 h of exposure. Along with this inhibition, flavopiridol decreased total cyclin-D protein levels in this cell line. In this work, we demonstrate that using isoform-specific antibodies, flavopiridol induces an early (by 6 h) decrease in cyclin D1 protein levels. This decline is followed by a decline in cyclin D3 with no effect on cyclin D2 or cyclin E levels by 10 h. Furthermore, at early time points (up to 8 h), the activity of cdk4 and the expression of endogenous phosphorylated retinoblastoma species from intact cells exposed to flavopiridol are unchan...
Cancer research, 1997
Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result o... more Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result of screening. Because traditional prognostic parameters are either lacking (tumor size) or rare (nodal metastases), a marker(s) is needed to identify the subset of patients who could benefit from adjuvant therapy. A retrospective series of 202 patients with stage T1a,b invasive breast carcinomas was evaluated. The clinicopathological features (age, histological grade, extensive in situ carcinoma, hormone receptor status, and nodal metastasis) as well as microvessel density and the expression of c-erb-B2, p53, MIB-1/Ki-67, and cdc25B were assessed. In addition, expression of the cell cycle inhibitor p27 was evaluated. Nineteen patients (18% of patients who had axillary dissection) had locoregional lymph node metastases. Forty-two % of them died of disease (median survival, 112 months), whereas mortality was 11% in node-negative patients (median survival, 168 months; P = 0.0055). Patients w...
Cancer research, 1989
To characterize differences in gene expression between hormone-dependent and hormone-independent ... more To characterize differences in gene expression between hormone-dependent and hormone-independent mammary carcinoma, we cloned complementary DNAs of genes expressed in a hormone-independent breast carcinoma cell line that were not expressed in a hormone-dependent line. One clone, which was isolated in many copies, coded for the intermediate filament protein vimentin. A complementary DNA clone 1.8 kilobases long included the entire protein-coding region for vimentin. Vimentin was expressed by more than one-half of the hormone-independent breast carcinoma cell lines tested but not by the hormone-dependent cell lines. The cell lines which expressed vimentin expressed only low levels of cytokeratins. The correlation between vimentin expression and more advanced stages of mammary cell transformation was tested in a model system in which immortal, nontumorigenic human mammary epithelial cells or derivative lines transformed with v-ras-H or SV40 T-antigen were found not to express vimentin,...
Flavopiridol (L86-8275), a JV-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle pro... more Flavopiridol (L86-8275), a JV-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G, or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cycIin Dl and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack
The polypeptide patterns of MCF-7 human breast cancer cells (MCF- 7gpt) and a stably v-H-roi-tran... more The polypeptide patterns of MCF-7 human breast cancer cells (MCF- 7gpt) and a stably v-H-roi-transfected subclone (MCF-7ras) have been analyzed following estradili! treatment. Since both estradili! and \-H-ras transfection increase tumorigenicity of MCF-7 cells, this study was designed to ascertain if specific changes in polypeptides were common in both treatments. Separation of cellular and secreted polypeptides was accomplished by 2-dimensional
Somatic Cell and Molecular Genetics, 1997
O, of l)NA monoadducts normalO:. Our results are compatible with repair o[" DNA crosslinks being ... more O, of l)NA monoadducts normalO:. Our results are compatible with repair o[" DNA crosslinks being slower in leA than in nornud cells and FA cells having normal cell cycle ('heckl.~oints.
Oncogene, 2000
Replicative senescence may be an important tumor suppressive mechanism for human cells. We invest... more Replicative senescence may be an important tumor suppressive mechanism for human cells. We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of`self-selection', and as a consequence exhibit diminished p16 INK4A levels. As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age. In contrast to observations in ®broblast, p21 Cip1 was not increased at senescence in HMECs. There was no increase in p27 Kip1 levels nor in KIP association with targets cdks. While p15 INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15 INK4B . The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A. Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence. Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase. Oncogene 19, 5314 ± 5323.
JNCI Journal of the National Cancer Institute, 1996
Background: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viabilit... more Background: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G 2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G 2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. Purpose: We studied the effect of UCN-01 on G 2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 response to DNA damage have been characterized. We also assessed the ability of UCN-01 to enhance the cytotoxicity of y irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01mediated abrogation of the G 2 checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. Methods: The effect of UCN-01 on cell cycle arrest induced by y irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone HI phosphorylation assay was employed to measure cyclin Bl/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of y irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells.
JNCI Journal of the National Cancer Institute, 1992
Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can ... more Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can inhibit tumor cell growth in vitro, and preliminary in vivo studies of the flavone L86-8275 have shown growth inhibition of LX529 and A549 lung carcinomas. L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] is a flavone of novel structure. The purpose of this study was to determine in vitro whether L86-8275 is a more potent inhibitor of growth in breast carcinoma and lung carcinoma cells than quercetin or genistein. We studied the effects of L86-8275 on cell growth in seven breast carcinoma cell lines and five lung carcinoma cell lines. MDA468 breast carcinoma was then selected for further study. Cell proliferation was measured by a colorimetric dye reduction assay; synthesis of DNA, RNA, and protein by incorporation of the radioactive metabolic precursors thymidine, uridine, or leucine, respectively; adenosine triphosphate (ATP) content by a luciferase-mediated bioluminescence reaction; and cell cycle progression by the use of cell-synchronizing drugs (aphidicolin and nocodazole) and flow cytometry. L86-8275 was not cytotoxic to stationary-phase cells but reversibly inhibited the growth of cells in exponential growth phase. At concentrations of 25-160 nM, L86-8275 inhibited growth of human breast and lung carcinoma cell lines by 50%. MDA468 breast carcinoma cells were 60-fold and 400-fold more sensitive to L86-8275 than to quercetin and genistein, respectively. By 24 hours after addition of L86-8275, DNA synthesis in MDA468 cells was inhibited by greater than 95%, protein synthesis by 80%, and RNA synthesis by 40%-60%, under conditions that preserved cellular ATP levels at approximately 80%-90% of control values. When MDA468 cells released from aphidicolin-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed the S phase but arrested in G2. When cells released from nocodazole-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed mitosis but arrested in G1. L86-8275 is a potent, yet reversible, growth-inhibitory flavone that can selectively block cell cycle progression in vitro at more than one point in the cell cycle. These findings suggest that L86-8275 is a candidate for further preclinical development, as well as a model for the synthesis of other flavonoids that might potently delay cell cycle progression to achieve inhibition of tumor growth. Future studies need to address optimal schedules for antiproliferative activity in vivo and inhibition of clonogenic activity.
Journal of Medicinal Chemistry, 2004
Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyri... more Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC(50)s in the low micromolar range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the 6-arylmethyl group resulted in approximately 100-fold increases in CDK4 affinity, giving ligands that were equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse xenograft model.
Gastroenterology, 1998
We have previously observed that breast cancer cell lines could ex hibit either epithelial or fib... more We have previously observed that breast cancer cell lines could ex hibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Som mers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49: 4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (, J. Cell. Physiol., 750: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibro blastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastineresistant ZR-75-B cell line have undergone such a transition. Adriamy cin-resistant MCF-7 cells express vimentin, have diminished keratin 19
Expert Review of Anticancer Therapy, 2003
The recent clinical and commercial success of anticancer antibodies, such as rituximab (Rituxan) ... more The recent clinical and commercial success of anticancer antibodies, such as rituximab (Rituxan) and trastuzumab (Herceptin) has created great interest in antibody-based therapeutics for hematopoietic malignancies and solid tumors. Given the likely lower toxicity for antibodies versus small molecules, the potential increase in efficacy by conjugation to radioisotopes and other cellular toxins and the ability to characterize the target with clinical laboratory diagnostics to improve the drug&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s clinical performance, it is anticipated that current and future antibody therapeutics will find substantial roles alone and in combination therapy strategies for the treatment of patients with cancer. It is also likely that conjugation strategies will add new radiolabeled and toxin-linked products to the market to complement the recent approvals of ibritumomab tiuxetan (Zevalin) and gemtuzumab ozogamycin (Mylotarg). However, although there are a large number of agents in both early and later stages of clinical development, only a handful will make it through regulatory approval and become successful products. This review considers the structure of anticancer therapeutic antibodies, the techniques used to reduce their antigenicity, factors that influence efficacy and toxicity, conjugation with isotopes and toxins and antibody target validation.
Critical Reviews in Biochemistry and Molecular Biology, 1990
Page 1. Biochemistry and Molecular Biology Structure and Function of Suppressor tRNAs in Higher E... more Page 1. Biochemistry and Molecular Biology Structure and Function of Suppressor tRNAs in Higher Eukaryotes Dolph L. Hatfield, David W. E. Smith, Byeong 3. Lee, Peter J. Worland, and Stephen Oroszlan 1. INTRODUCTION ...
Cancer Research, 2004
MLN2704 is an antibody-chemotherapeutic conjugate designed to target prostate-specific membrane a... more MLN2704 is an antibody-chemotherapeutic conjugate designed to target prostate-specific membrane antigen (PSMA). PSMA is a transmembrane receptor whose expression is largely restricted to prostatic epithelium and prostate cancer cells with its expression level increasing during the progression of malignancy. MLN2704 consists of a de-immunized, monoclonal antibody that is specific for PSMA conjugated to drug maytansinoid 1 (DM1), a microtubule-depolymerizing compound. After antibody binding to PSMA and the subsequent cellular internalization of this complex, DM1 is released leading to cell death. MLN2704 has an approximate half-life of 39 hours in scid mice bearing CWR22 tumor tissue, and the antibody effectively penetrates xenograft tumor tissue. Optimization of dosage and schedule of MLN2704 administration defined interdependency between these conditions that maximized efficacy with no apparent toxicity. Tumor growth delays of ϳ100 days could be achieved on the optimized schedule of one dose of 60 mg/kg MLN2704 every 14 days for five doses (q14d؋5). The unconjugated antibody (MLN591) demonstrated essentially no antitumor activity and DM1 alone or a non-PSMA targeted antibody-DM1 conjugate was only weakly active. Furthermore, we show that MLN2704 is active in a novel model of osteoblastic prostate cancer metastasis.
Biochemical Pharmacology, 1993
The flavone L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)- piperidi... more The flavone L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] delayed the progression of aphidicolin-synchronized MDA-468 breast carcinoma cells through S phase and prevented progression through G2. L86-8275 prevented the G2-related increase in histone H1 kinase activity mediated by cyclin-dependent kinase-1 (p34cdc2 kinase). L86-8275 inhibited [32P]orthophosphate labeling of p34cdc2 threonine and tyrosine residues and decreased the phosphotyrosine content of p34cdc2. Diminution of p34cdc2 phosphotyrosine appeared selective, as a general depletion of cellular phosphotyrosine was not observed. The mass of p34cdc2 in L86-8275-exposed cells was not decreased during the period over which these effects occurred. [35S]Methionine labeling of p34cdc2 or other cellular proteins was not inhibited at concentrations that were effective for complete cellular growth inhibition. We hypothesize that L86-8275 interferes with the normal cell cycle-dependent phosphorylation of p34cdc2, resulting in decreased kinase activity and cell cycle arrest.
Biochemical and Biophysical Research Communications, 1994
L86-8275 [(-) cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1- methyl)-piperidinyl]-4H-benz... more L86-8275 [(-) cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1- methyl)-piperidinyl]-4H-benzopyran-4-one] directly inhibits immunoprecipitated Cdc2 kinase activity from G2/M synchronized MDA-MB-468 breast carcinoma cells and is at least 250-fold more potent than either quercetin or genistein. Purified sea-star Cdc2 kinase (IC50 = 0.5 microM) was inhibited with a similar potency to immunoprecipitated Cdc2 kinase from MDA-MB-468 cells (IC50 = 0.4 microM). This inhibition was competitive with respect to ATP (KiATP = 0.041 microM) and noncompetitive with respect to a synthetic peptide substrate, CDK1S1 (AAKAKKTPKKAKK-CONH2, KiCDK1S1 = 0.14 microM). These data suggest L86-8275 as a lead structure for the development of inhibitors of the cyclin-dependent kinases.
Radiation Oncology Investigations, 1993
We have previously reported the radiation-induced malignant transformation of immortalized human ... more We have previously reported the radiation-induced malignant transformation of immortalized human epidermal keratinocytes. Two sets of tumorigenic transformants (treated with either 2 Gy or 4 Gy twice) and their respective clonal derivatives were characterized [Thraves et al.: Proc Natl Acad Sci USA 87:117&1177, 19901. We have tested the hypothesis that the multiple changes which take place during cellular transformation are reflected at the level of protein expression. Toward this end, we have compared protein expression in the non-tumorigenic, parental keratinocytes and their radiationtransformed derivatives during the various stages of progression to tumorigenicity. We employed two-dimensional gel electrophoresis (2D-PAGE) coupled with silver staining and computer-assisted data analyses. Eighteen differentially expressed proteins were identified in the various transformed cultures. Changes in 8 of these proteins exhibited a similar pattern in both the 2 and 4 Gy transformants, while the expression of the other 10 varied. Two of the eight proteins that showed identical changes in both sets of transformed keratinocytes have been identified as tropomyosin and proliferating cell nuclear antigen (PCNA) by their characteristic 2D-map locations and reactivity with specific antisera. The identification of these differentially expressed polypeptides in the tumorigenic keratinocytes constitutes a preliminary step in the study of the complex process of radiationinduced transformation and its regulation by specific gene products.
Journal of Medicinal Chemistry, 2015
We report here the synthesis and structure-activity relationship (SAR) of a novel series of triaz... more We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC 115 for clinical development.
Journal of Medicinal Chemistry, 2015
We report here the synthesis and structure-activity relationship (SAR) of a novel series of mamma... more We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development.
Molecular cancer therapeutics, Jan 8, 2015
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth,... more The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, proliferation and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K/AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. While rapamycin analogs, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity; it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 (pAKT(S473)) in cellular systems. Growth inhibitory activity was demonstrated in hematological and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of th...
Cancer research, Jan 15, 1999
Flavopiridol is a novel flavonoid that induces cell cycle arrest at different stages of the cell ... more Flavopiridol is a novel flavonoid that induces cell cycle arrest at different stages of the cell cycle because of the inhibition of cyclin-dependent kinases (cdks). In previous studies from our laboratory, (B. A. Carlson et al., Cancer Res., 56: 2973-2978, 1996), we observed that exposure of the MCF-7 breast carcinoma cell line to flavopiridol resulted in G1-S arrest, which was associated with the loss of cdk4 and cdk2 activity by 24 h of exposure. Along with this inhibition, flavopiridol decreased total cyclin-D protein levels in this cell line. In this work, we demonstrate that using isoform-specific antibodies, flavopiridol induces an early (by 6 h) decrease in cyclin D1 protein levels. This decline is followed by a decline in cyclin D3 with no effect on cyclin D2 or cyclin E levels by 10 h. Furthermore, at early time points (up to 8 h), the activity of cdk4 and the expression of endogenous phosphorylated retinoblastoma species from intact cells exposed to flavopiridol are unchan...
Cancer research, 1997
Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result o... more Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result of screening. Because traditional prognostic parameters are either lacking (tumor size) or rare (nodal metastases), a marker(s) is needed to identify the subset of patients who could benefit from adjuvant therapy. A retrospective series of 202 patients with stage T1a,b invasive breast carcinomas was evaluated. The clinicopathological features (age, histological grade, extensive in situ carcinoma, hormone receptor status, and nodal metastasis) as well as microvessel density and the expression of c-erb-B2, p53, MIB-1/Ki-67, and cdc25B were assessed. In addition, expression of the cell cycle inhibitor p27 was evaluated. Nineteen patients (18% of patients who had axillary dissection) had locoregional lymph node metastases. Forty-two % of them died of disease (median survival, 112 months), whereas mortality was 11% in node-negative patients (median survival, 168 months; P = 0.0055). Patients w...
Cancer research, 1989
To characterize differences in gene expression between hormone-dependent and hormone-independent ... more To characterize differences in gene expression between hormone-dependent and hormone-independent mammary carcinoma, we cloned complementary DNAs of genes expressed in a hormone-independent breast carcinoma cell line that were not expressed in a hormone-dependent line. One clone, which was isolated in many copies, coded for the intermediate filament protein vimentin. A complementary DNA clone 1.8 kilobases long included the entire protein-coding region for vimentin. Vimentin was expressed by more than one-half of the hormone-independent breast carcinoma cell lines tested but not by the hormone-dependent cell lines. The cell lines which expressed vimentin expressed only low levels of cytokeratins. The correlation between vimentin expression and more advanced stages of mammary cell transformation was tested in a model system in which immortal, nontumorigenic human mammary epithelial cells or derivative lines transformed with v-ras-H or SV40 T-antigen were found not to express vimentin,...
Flavopiridol (L86-8275), a JV-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle pro... more Flavopiridol (L86-8275), a JV-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G, or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cycIin Dl and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack
The polypeptide patterns of MCF-7 human breast cancer cells (MCF- 7gpt) and a stably v-H-roi-tran... more The polypeptide patterns of MCF-7 human breast cancer cells (MCF- 7gpt) and a stably v-H-roi-transfected subclone (MCF-7ras) have been analyzed following estradili! treatment. Since both estradili! and \-H-ras transfection increase tumorigenicity of MCF-7 cells, this study was designed to ascertain if specific changes in polypeptides were common in both treatments. Separation of cellular and secreted polypeptides was accomplished by 2-dimensional
Somatic Cell and Molecular Genetics, 1997
O, of l)NA monoadducts normalO:. Our results are compatible with repair o[" DNA crosslinks being ... more O, of l)NA monoadducts normalO:. Our results are compatible with repair o[" DNA crosslinks being slower in leA than in nornud cells and FA cells having normal cell cycle ('heckl.~oints.
Oncogene, 2000
Replicative senescence may be an important tumor suppressive mechanism for human cells. We invest... more Replicative senescence may be an important tumor suppressive mechanism for human cells. We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of`self-selection', and as a consequence exhibit diminished p16 INK4A levels. As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age. In contrast to observations in ®broblast, p21 Cip1 was not increased at senescence in HMECs. There was no increase in p27 Kip1 levels nor in KIP association with targets cdks. While p15 INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15 INK4B . The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A. Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence. Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase. Oncogene 19, 5314 ± 5323.
JNCI Journal of the National Cancer Institute, 1996
Background: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viabilit... more Background: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G 2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G 2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. Purpose: We studied the effect of UCN-01 on G 2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 response to DNA damage have been characterized. We also assessed the ability of UCN-01 to enhance the cytotoxicity of y irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01mediated abrogation of the G 2 checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. Methods: The effect of UCN-01 on cell cycle arrest induced by y irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone HI phosphorylation assay was employed to measure cyclin Bl/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of y irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells.
JNCI Journal of the National Cancer Institute, 1992
Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can ... more Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can inhibit tumor cell growth in vitro, and preliminary in vivo studies of the flavone L86-8275 have shown growth inhibition of LX529 and A549 lung carcinomas. L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] is a flavone of novel structure. The purpose of this study was to determine in vitro whether L86-8275 is a more potent inhibitor of growth in breast carcinoma and lung carcinoma cells than quercetin or genistein. We studied the effects of L86-8275 on cell growth in seven breast carcinoma cell lines and five lung carcinoma cell lines. MDA468 breast carcinoma was then selected for further study. Cell proliferation was measured by a colorimetric dye reduction assay; synthesis of DNA, RNA, and protein by incorporation of the radioactive metabolic precursors thymidine, uridine, or leucine, respectively; adenosine triphosphate (ATP) content by a luciferase-mediated bioluminescence reaction; and cell cycle progression by the use of cell-synchronizing drugs (aphidicolin and nocodazole) and flow cytometry. L86-8275 was not cytotoxic to stationary-phase cells but reversibly inhibited the growth of cells in exponential growth phase. At concentrations of 25-160 nM, L86-8275 inhibited growth of human breast and lung carcinoma cell lines by 50%. MDA468 breast carcinoma cells were 60-fold and 400-fold more sensitive to L86-8275 than to quercetin and genistein, respectively. By 24 hours after addition of L86-8275, DNA synthesis in MDA468 cells was inhibited by greater than 95%, protein synthesis by 80%, and RNA synthesis by 40%-60%, under conditions that preserved cellular ATP levels at approximately 80%-90% of control values. When MDA468 cells released from aphidicolin-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed the S phase but arrested in G2. When cells released from nocodazole-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed mitosis but arrested in G1. L86-8275 is a potent, yet reversible, growth-inhibitory flavone that can selectively block cell cycle progression in vitro at more than one point in the cell cycle. These findings suggest that L86-8275 is a candidate for further preclinical development, as well as a model for the synthesis of other flavonoids that might potently delay cell cycle progression to achieve inhibition of tumor growth. Future studies need to address optimal schedules for antiproliferative activity in vivo and inhibition of clonogenic activity.
Journal of Medicinal Chemistry, 2004
Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyri... more Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC(50)s in the low micromolar range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the 6-arylmethyl group resulted in approximately 100-fold increases in CDK4 affinity, giving ligands that were equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse xenograft model.
Gastroenterology, 1998
We have previously observed that breast cancer cell lines could ex hibit either epithelial or fib... more We have previously observed that breast cancer cell lines could ex hibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Som mers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49: 4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (, J. Cell. Physiol., 750: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibro blastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastineresistant ZR-75-B cell line have undergone such a transition. Adriamy cin-resistant MCF-7 cells express vimentin, have diminished keratin 19
Expert Review of Anticancer Therapy, 2003
The recent clinical and commercial success of anticancer antibodies, such as rituximab (Rituxan) ... more The recent clinical and commercial success of anticancer antibodies, such as rituximab (Rituxan) and trastuzumab (Herceptin) has created great interest in antibody-based therapeutics for hematopoietic malignancies and solid tumors. Given the likely lower toxicity for antibodies versus small molecules, the potential increase in efficacy by conjugation to radioisotopes and other cellular toxins and the ability to characterize the target with clinical laboratory diagnostics to improve the drug&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s clinical performance, it is anticipated that current and future antibody therapeutics will find substantial roles alone and in combination therapy strategies for the treatment of patients with cancer. It is also likely that conjugation strategies will add new radiolabeled and toxin-linked products to the market to complement the recent approvals of ibritumomab tiuxetan (Zevalin) and gemtuzumab ozogamycin (Mylotarg). However, although there are a large number of agents in both early and later stages of clinical development, only a handful will make it through regulatory approval and become successful products. This review considers the structure of anticancer therapeutic antibodies, the techniques used to reduce their antigenicity, factors that influence efficacy and toxicity, conjugation with isotopes and toxins and antibody target validation.
Critical Reviews in Biochemistry and Molecular Biology, 1990
Page 1. Biochemistry and Molecular Biology Structure and Function of Suppressor tRNAs in Higher E... more Page 1. Biochemistry and Molecular Biology Structure and Function of Suppressor tRNAs in Higher Eukaryotes Dolph L. Hatfield, David W. E. Smith, Byeong 3. Lee, Peter J. Worland, and Stephen Oroszlan 1. INTRODUCTION ...
Cancer Research, 2004
MLN2704 is an antibody-chemotherapeutic conjugate designed to target prostate-specific membrane a... more MLN2704 is an antibody-chemotherapeutic conjugate designed to target prostate-specific membrane antigen (PSMA). PSMA is a transmembrane receptor whose expression is largely restricted to prostatic epithelium and prostate cancer cells with its expression level increasing during the progression of malignancy. MLN2704 consists of a de-immunized, monoclonal antibody that is specific for PSMA conjugated to drug maytansinoid 1 (DM1), a microtubule-depolymerizing compound. After antibody binding to PSMA and the subsequent cellular internalization of this complex, DM1 is released leading to cell death. MLN2704 has an approximate half-life of 39 hours in scid mice bearing CWR22 tumor tissue, and the antibody effectively penetrates xenograft tumor tissue. Optimization of dosage and schedule of MLN2704 administration defined interdependency between these conditions that maximized efficacy with no apparent toxicity. Tumor growth delays of ϳ100 days could be achieved on the optimized schedule of one dose of 60 mg/kg MLN2704 every 14 days for five doses (q14d؋5). The unconjugated antibody (MLN591) demonstrated essentially no antitumor activity and DM1 alone or a non-PSMA targeted antibody-DM1 conjugate was only weakly active. Furthermore, we show that MLN2704 is active in a novel model of osteoblastic prostate cancer metastasis.
Biochemical Pharmacology, 1993
The flavone L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)- piperidi... more The flavone L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] delayed the progression of aphidicolin-synchronized MDA-468 breast carcinoma cells through S phase and prevented progression through G2. L86-8275 prevented the G2-related increase in histone H1 kinase activity mediated by cyclin-dependent kinase-1 (p34cdc2 kinase). L86-8275 inhibited [32P]orthophosphate labeling of p34cdc2 threonine and tyrosine residues and decreased the phosphotyrosine content of p34cdc2. Diminution of p34cdc2 phosphotyrosine appeared selective, as a general depletion of cellular phosphotyrosine was not observed. The mass of p34cdc2 in L86-8275-exposed cells was not decreased during the period over which these effects occurred. [35S]Methionine labeling of p34cdc2 or other cellular proteins was not inhibited at concentrations that were effective for complete cellular growth inhibition. We hypothesize that L86-8275 interferes with the normal cell cycle-dependent phosphorylation of p34cdc2, resulting in decreased kinase activity and cell cycle arrest.
Biochemical and Biophysical Research Communications, 1994
L86-8275 [(-) cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1- methyl)-piperidinyl]-4H-benz... more L86-8275 [(-) cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1- methyl)-piperidinyl]-4H-benzopyran-4-one] directly inhibits immunoprecipitated Cdc2 kinase activity from G2/M synchronized MDA-MB-468 breast carcinoma cells and is at least 250-fold more potent than either quercetin or genistein. Purified sea-star Cdc2 kinase (IC50 = 0.5 microM) was inhibited with a similar potency to immunoprecipitated Cdc2 kinase from MDA-MB-468 cells (IC50 = 0.4 microM). This inhibition was competitive with respect to ATP (KiATP = 0.041 microM) and noncompetitive with respect to a synthetic peptide substrate, CDK1S1 (AAKAKKTPKKAKK-CONH2, KiCDK1S1 = 0.14 microM). These data suggest L86-8275 as a lead structure for the development of inhibitors of the cyclin-dependent kinases.