American Journal of Physiology-Endocrinology and Metabolism, 2000
The catabolic properties of glucocorticoid hormones are largely attributable to dual regulation o... more The catabolic properties of glucocorticoid hormones are largely attributable to dual regulation of protein degradation and synthesis. With regard to the latter, glucocorticoids modulate the translational machinery, namely that component functional in translation initiation. This investigation revealed that in L6 myoblasts, dexamethasone, a synthetic glucocorticoid, deactivated the ribosomal protein S6 kinase (p70S6k) within 4 h, as evidenced by diminished phosphorylation of its physiological substrate, the 40S ribosomal protein S6. This deactivation correlated with dephosphorylation of p70S6kat Thr389, whereas phosphorylation of Ser411was unaffected. Furthermore, glucocorticoid administration induced dephosphorylation of the cap-dependent translational repressor, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), thereby facilitating conjunction of the inhibitor and eIF4E. The mechanism of action is reminiscent of classical transcriptional regulation by steroid horm...
American Journal of Physiology-Endocrinology and Metabolism, 2000
Glucocorticoids are diabetogenic factors that not only antagonize the action of insulin in target... more Glucocorticoids are diabetogenic factors that not only antagonize the action of insulin in target tissues but also render these tissues catabolic. Therefore, in rats, we endeavored to characterize the effects in skeletal muscle of glucocorticoids on translation initiation, a regulated process that, in part, governs overall protein synthesis through the modulated activities of eukaryotic initiation factors (eIFs). Four hours after intraperitoneal administration of dexamethasone (100 μg/100 g body wt), protein synthesis in skeletal muscle was reduced to 59% of the value recorded in untreated control animals. Furthermore, translation initiation factor eIF4E preferred association with its endogenous inhibitor 4E-BP1 rather than eIF4G. Dexamethasone treatment resulted in dephosphorylation of both 4E-BP1 and the 40S ribosomal protein S6 kinase concomitant with enhanced phosphorylation of eIF4E. Moreover, the guanine nucleotide exchange activity of eIF2B was unaffected as was phosphorylati...
American Journal of Physiology-Endocrinology and Metabolism, 2000
Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesi... more Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses ...
American Journal of Physiology-Endocrinology and Metabolism, 2000
Glucocorticoids comprise an important class of hormonal mediators of fuel and protein homeostasis... more Glucocorticoids comprise an important class of hormonal mediators of fuel and protein homeostasis in normal and pathological scenarios. In skeletal muscle, exposure to glucocorticoids is characterized by a reduction in protein synthetic rate coincident with hampered translation initiation. However, it is unclear whether this involves attenuation of anabolic stimuli or is simply due to inhibition of the basally activated translational apparatus. Therefore, this inquiry was designed to determine whether leucine, administered orally, could rescue the translational inhibition induced by glucocorticoids. Dexamethasone, injected intraperitoneally, acutely diminished protein synthetic rates to 80% of control values in skeletal muscle from rat hindlimb. The eukaryotic initiation factor (eIF)4 regulatory element was simultaneously and negatively impacted via sequestration of eIF4E by the hypophosphorylated form of the translational suppressor, eIF4E binding protein 1 (4E-BP1). The 70-kDa rib...
There has been renewed interest in the field of cancer epigenetics from the pharmaceutical and bi... more There has been renewed interest in the field of cancer epigenetics from the pharmaceutical and biotech sectors owing to the discovery of druggable epigenetic modulators that undergo oncogenic mutation and thus represent attractive cancer drug targets. A challenge in this field has been that inhibition of some epigenetic targets results in a significantly delayed drug response that is not revealed by conventional, short-term cell growth assays. We therefore set out to define the two- and three-dimensional assay conditions necessary to yield robust cell line profiling data for therapeutics requiring an extended assay duration. The Eurofins Panlabs’ OncoPanel is a collection of 240 genomically characterized cell lines for which doubling times have been meticulously recorded and compiled over the last several years. Using historical doubling times to compute seeding densities, we demonstrate here that subconfluent, exponential growth can be achieved in the majority of OncoPanel cell lin...
Although current therapies that target driver mutations often show dramatic short-term benefit, r... more Although current therapies that target driver mutations often show dramatic short-term benefit, resistance to therapy is common as cancer cells respond by shifting to a reliance on other driver mutations not affected by treatment. Antibody-drug conjugates (ADCs) represent an alternative therapeutic modality that delivers a general toxin selectively to tumors by virtue of linkage to a monoclonal antibody that binds specifically to antigens expressed on the surface of cancer cells. ADCs are now being widely developed, with greater than 20 currently under evaluation in clinical trials. The mechanism of action of ADCs involves a specific sequence of events, all of which are requisite for efficient payload delivery: (1) the ADC must bind native antigen expressed on the cancer cell surface; (2) the resulting antigen-ADC complex must be internalized upon binding; (3) the internalized ADC must be trafficked to the lysosome; and (4) the payload must be successfully released to engage its bio...
Objective: To evaluate the diagnostic accuracy of Alvarado score for the prediction of acute appe... more Objective: To evaluate the diagnostic accuracy of Alvarado score for the prediction of acute appendicitis. Study Design: Analytical study. Place and Duration of Study: This study was carried out in the Department of Surgery, Pakistan Institute of Medical Sciences (PIMS), Islamabad, during the period from January 15, 2009 to July 15, 2010. Methodology: The study included all adult patients of either gender who presented with clinical findings suggestive of acute appendicitis, who were assigned Alvarado score of < 4 pre-operatively and subsequently underwent emergency appendicectomy with histological examination of the resected specimens. Based on the Alvarado score, the patients were stratified into two groups. i.e. Group I (with a score of > 7) and Group II (with a score of 5-7). Alvarado score was compared with the histopathology. The data was subjected to statistical analysis to measure the objective. Results: The overall sensitivity, specificity, positive predictive value a...
While drug response profiling of cancer cells in two-dimensional culture has been a mainstay of p... more While drug response profiling of cancer cells in two-dimensional culture has been a mainstay of predictive biomarker discovery and anti-cancer drug development, there are aspects of tumor biology that are not replicated in a two-dimensional cell culture environment. This is particularly true for solid tumor models where the three-dimensional organization of the tumor creates distinct, regional microenvironments that influence drug response. For example, as the radius of solid tumors approaches the diffusion limit of oxygen and other nutrients, the tumor core becomes hypoxic and accumulates metabolic waste products, resulting in cell death through mechanisms involving apoptosis and necrosis. Moreover, without proper vascularization, the tumor interior also exhibits increased interstitial pressure, which limits drug penetration into the tumor. We have characterized the three-dimensional growth of 240 cancer cell lines from various tumor origins by high-content imaging. This characteri...
The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell... more The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in H...
Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcriptio... more Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in JAK2V617F-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by ac...
OncoPanelTM is a collection of 240 genomically and histologically diverse cancer cell lines that ... more OncoPanelTM is a collection of 240 genomically and histologically diverse cancer cell lines that comprise an unparalleled platform for multiplexed high-content imaging analysis of potential therapeutic targets in oncology. High-content imaging is ideally suited to detect binding, localization, and trafficking of therapeutic antibodies, adding an important level of resolution when interpreting drug response data. Moreover, understanding relative antigen expression is important both to define the patient population most likely to benefit from therapy as well as to define the antigen expression threshold needed to elicit biologic response. Here we describe the high-throughput parallelization of quantitative, comparative therapeutic antibody binding measurements and drug response profiling across the OncoPanel using high-content imaging. High-quality binding data for monoclonal antibodies was achieved by analysis of referenced, background-subtracted fluorescence intensities on a per cel...
ABT-348 is a novel, potent and orally bioavailable inhibitor of the Aurora kinases as well as the... more ABT-348 is a novel, potent and orally bioavailable inhibitor of the Aurora kinases as well as the VEGF and PDGF families of receptor tyrosine kinases. ABT-348 is broadly efficacious preclinically as a single agent against a wide range of tumor types and is currently in Phase I/II clinical trials. Several published studies have supported the potential utility of combining targeted anti-proliferative agents with cytotoxic chemotherapies. Herein, the efficacy of combining the neocytotoxic agent, ABT-348, with CDK4/6 inhibition in the treatment of solid tumor cells has been evaluated. Inhibition of CDK4/6 via siRNA elicits a potent cytostatic response in cells that harbor a wild-type, functional RB. We demonstrate that features of cellular senescence are induced in human tumor cells with siRNAs targeting both CDK4 and CDK6. Dual inhibition of CDK4 and CDK6 is required for antiproliferative activity in most cancer cell lines and this is not accompanied by induction of apoptosis. In RB wi...
Proceedings of the National Academy of Sciences, 2010
Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinic... more Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This “addiction” can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.
Three p160 family members, p/CIP, SRC1, and TIF2, have been identified as transcriptional coactiv... more Three p160 family members, p/CIP, SRC1, and TIF2, have been identified as transcriptional coactivators for nuclear hormone receptors and other transcription factors in vitro. In a previous study, we reported initial characterization of the obesityresistant phenotypes of p/CIP and SRC-1 double knockout (DKO) mice, which exhibit increased energy expenditure, and suggested that nuclear hormone receptor target genes were involved in these phenotypes. In this study, we demonstrate that p/CIP and SRC1 control insulin signaling in a cell-autonomous manner both in vitro and in vivo. Genetic deletion of p/ CIP and SRC-1 increases glucose uptake and enhances insulin sensitivity in both regular chow-and high fat diet-fed DKO mice despite increased food intake. Interestingly, we discover that loss of p/CIP and SRC-1 results in resistance to agerelated obesity and glucose intolerance. We show that expression levels of a key insulin signaling component, insulin receptor substrate 1 (IRS1), are significantly increased in two cell lines representing fat and muscle lineages with p/CIP and SRC-1 deletions and in white adipose tissue and skeletal muscle of DKO mice; this may account for increased glucose metabolism and insulin sensitivity. This is the first evidence that the p160 coactivators control insulin signaling and glucose metabolism through IRS1. Therefore, our studies indicate that p/CIP and SRC-1 are potential therapeutic targets not only for obesity but also for diabetes.
Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that... more Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated.
American Journal of Physiology-Endocrinology and Metabolism, 2000
The catabolic properties of glucocorticoid hormones are largely attributable to dual regulation o... more The catabolic properties of glucocorticoid hormones are largely attributable to dual regulation of protein degradation and synthesis. With regard to the latter, glucocorticoids modulate the translational machinery, namely that component functional in translation initiation. This investigation revealed that in L6 myoblasts, dexamethasone, a synthetic glucocorticoid, deactivated the ribosomal protein S6 kinase (p70S6k) within 4 h, as evidenced by diminished phosphorylation of its physiological substrate, the 40S ribosomal protein S6. This deactivation correlated with dephosphorylation of p70S6kat Thr389, whereas phosphorylation of Ser411was unaffected. Furthermore, glucocorticoid administration induced dephosphorylation of the cap-dependent translational repressor, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), thereby facilitating conjunction of the inhibitor and eIF4E. The mechanism of action is reminiscent of classical transcriptional regulation by steroid horm...
American Journal of Physiology-Endocrinology and Metabolism, 2000
Glucocorticoids are diabetogenic factors that not only antagonize the action of insulin in target... more Glucocorticoids are diabetogenic factors that not only antagonize the action of insulin in target tissues but also render these tissues catabolic. Therefore, in rats, we endeavored to characterize the effects in skeletal muscle of glucocorticoids on translation initiation, a regulated process that, in part, governs overall protein synthesis through the modulated activities of eukaryotic initiation factors (eIFs). Four hours after intraperitoneal administration of dexamethasone (100 μg/100 g body wt), protein synthesis in skeletal muscle was reduced to 59% of the value recorded in untreated control animals. Furthermore, translation initiation factor eIF4E preferred association with its endogenous inhibitor 4E-BP1 rather than eIF4G. Dexamethasone treatment resulted in dephosphorylation of both 4E-BP1 and the 40S ribosomal protein S6 kinase concomitant with enhanced phosphorylation of eIF4E. Moreover, the guanine nucleotide exchange activity of eIF2B was unaffected as was phosphorylati...
American Journal of Physiology-Endocrinology and Metabolism, 2000
Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesi... more Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses ...
American Journal of Physiology-Endocrinology and Metabolism, 2000
Glucocorticoids comprise an important class of hormonal mediators of fuel and protein homeostasis... more Glucocorticoids comprise an important class of hormonal mediators of fuel and protein homeostasis in normal and pathological scenarios. In skeletal muscle, exposure to glucocorticoids is characterized by a reduction in protein synthetic rate coincident with hampered translation initiation. However, it is unclear whether this involves attenuation of anabolic stimuli or is simply due to inhibition of the basally activated translational apparatus. Therefore, this inquiry was designed to determine whether leucine, administered orally, could rescue the translational inhibition induced by glucocorticoids. Dexamethasone, injected intraperitoneally, acutely diminished protein synthetic rates to 80% of control values in skeletal muscle from rat hindlimb. The eukaryotic initiation factor (eIF)4 regulatory element was simultaneously and negatively impacted via sequestration of eIF4E by the hypophosphorylated form of the translational suppressor, eIF4E binding protein 1 (4E-BP1). The 70-kDa rib...
There has been renewed interest in the field of cancer epigenetics from the pharmaceutical and bi... more There has been renewed interest in the field of cancer epigenetics from the pharmaceutical and biotech sectors owing to the discovery of druggable epigenetic modulators that undergo oncogenic mutation and thus represent attractive cancer drug targets. A challenge in this field has been that inhibition of some epigenetic targets results in a significantly delayed drug response that is not revealed by conventional, short-term cell growth assays. We therefore set out to define the two- and three-dimensional assay conditions necessary to yield robust cell line profiling data for therapeutics requiring an extended assay duration. The Eurofins Panlabs’ OncoPanel is a collection of 240 genomically characterized cell lines for which doubling times have been meticulously recorded and compiled over the last several years. Using historical doubling times to compute seeding densities, we demonstrate here that subconfluent, exponential growth can be achieved in the majority of OncoPanel cell lin...
Although current therapies that target driver mutations often show dramatic short-term benefit, r... more Although current therapies that target driver mutations often show dramatic short-term benefit, resistance to therapy is common as cancer cells respond by shifting to a reliance on other driver mutations not affected by treatment. Antibody-drug conjugates (ADCs) represent an alternative therapeutic modality that delivers a general toxin selectively to tumors by virtue of linkage to a monoclonal antibody that binds specifically to antigens expressed on the surface of cancer cells. ADCs are now being widely developed, with greater than 20 currently under evaluation in clinical trials. The mechanism of action of ADCs involves a specific sequence of events, all of which are requisite for efficient payload delivery: (1) the ADC must bind native antigen expressed on the cancer cell surface; (2) the resulting antigen-ADC complex must be internalized upon binding; (3) the internalized ADC must be trafficked to the lysosome; and (4) the payload must be successfully released to engage its bio...
Objective: To evaluate the diagnostic accuracy of Alvarado score for the prediction of acute appe... more Objective: To evaluate the diagnostic accuracy of Alvarado score for the prediction of acute appendicitis. Study Design: Analytical study. Place and Duration of Study: This study was carried out in the Department of Surgery, Pakistan Institute of Medical Sciences (PIMS), Islamabad, during the period from January 15, 2009 to July 15, 2010. Methodology: The study included all adult patients of either gender who presented with clinical findings suggestive of acute appendicitis, who were assigned Alvarado score of < 4 pre-operatively and subsequently underwent emergency appendicectomy with histological examination of the resected specimens. Based on the Alvarado score, the patients were stratified into two groups. i.e. Group I (with a score of > 7) and Group II (with a score of 5-7). Alvarado score was compared with the histopathology. The data was subjected to statistical analysis to measure the objective. Results: The overall sensitivity, specificity, positive predictive value a...
While drug response profiling of cancer cells in two-dimensional culture has been a mainstay of p... more While drug response profiling of cancer cells in two-dimensional culture has been a mainstay of predictive biomarker discovery and anti-cancer drug development, there are aspects of tumor biology that are not replicated in a two-dimensional cell culture environment. This is particularly true for solid tumor models where the three-dimensional organization of the tumor creates distinct, regional microenvironments that influence drug response. For example, as the radius of solid tumors approaches the diffusion limit of oxygen and other nutrients, the tumor core becomes hypoxic and accumulates metabolic waste products, resulting in cell death through mechanisms involving apoptosis and necrosis. Moreover, without proper vascularization, the tumor interior also exhibits increased interstitial pressure, which limits drug penetration into the tumor. We have characterized the three-dimensional growth of 240 cancer cell lines from various tumor origins by high-content imaging. This characteri...
The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell... more The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in H...
Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcriptio... more Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in JAK2V617F-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by ac...
OncoPanelTM is a collection of 240 genomically and histologically diverse cancer cell lines that ... more OncoPanelTM is a collection of 240 genomically and histologically diverse cancer cell lines that comprise an unparalleled platform for multiplexed high-content imaging analysis of potential therapeutic targets in oncology. High-content imaging is ideally suited to detect binding, localization, and trafficking of therapeutic antibodies, adding an important level of resolution when interpreting drug response data. Moreover, understanding relative antigen expression is important both to define the patient population most likely to benefit from therapy as well as to define the antigen expression threshold needed to elicit biologic response. Here we describe the high-throughput parallelization of quantitative, comparative therapeutic antibody binding measurements and drug response profiling across the OncoPanel using high-content imaging. High-quality binding data for monoclonal antibodies was achieved by analysis of referenced, background-subtracted fluorescence intensities on a per cel...
ABT-348 is a novel, potent and orally bioavailable inhibitor of the Aurora kinases as well as the... more ABT-348 is a novel, potent and orally bioavailable inhibitor of the Aurora kinases as well as the VEGF and PDGF families of receptor tyrosine kinases. ABT-348 is broadly efficacious preclinically as a single agent against a wide range of tumor types and is currently in Phase I/II clinical trials. Several published studies have supported the potential utility of combining targeted anti-proliferative agents with cytotoxic chemotherapies. Herein, the efficacy of combining the neocytotoxic agent, ABT-348, with CDK4/6 inhibition in the treatment of solid tumor cells has been evaluated. Inhibition of CDK4/6 via siRNA elicits a potent cytostatic response in cells that harbor a wild-type, functional RB. We demonstrate that features of cellular senescence are induced in human tumor cells with siRNAs targeting both CDK4 and CDK6. Dual inhibition of CDK4 and CDK6 is required for antiproliferative activity in most cancer cell lines and this is not accompanied by induction of apoptosis. In RB wi...
Proceedings of the National Academy of Sciences, 2010
Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinic... more Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This “addiction” can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.
Three p160 family members, p/CIP, SRC1, and TIF2, have been identified as transcriptional coactiv... more Three p160 family members, p/CIP, SRC1, and TIF2, have been identified as transcriptional coactivators for nuclear hormone receptors and other transcription factors in vitro. In a previous study, we reported initial characterization of the obesityresistant phenotypes of p/CIP and SRC-1 double knockout (DKO) mice, which exhibit increased energy expenditure, and suggested that nuclear hormone receptor target genes were involved in these phenotypes. In this study, we demonstrate that p/CIP and SRC1 control insulin signaling in a cell-autonomous manner both in vitro and in vivo. Genetic deletion of p/ CIP and SRC-1 increases glucose uptake and enhances insulin sensitivity in both regular chow-and high fat diet-fed DKO mice despite increased food intake. Interestingly, we discover that loss of p/CIP and SRC-1 results in resistance to agerelated obesity and glucose intolerance. We show that expression levels of a key insulin signaling component, insulin receptor substrate 1 (IRS1), are significantly increased in two cell lines representing fat and muscle lineages with p/CIP and SRC-1 deletions and in white adipose tissue and skeletal muscle of DKO mice; this may account for increased glucose metabolism and insulin sensitivity. This is the first evidence that the p160 coactivators control insulin signaling and glucose metabolism through IRS1. Therefore, our studies indicate that p/CIP and SRC-1 are potential therapeutic targets not only for obesity but also for diabetes.
Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that... more Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated.
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Papers by Jameel Shah