868 В. P. Czech, DH Desai, J. Koszuk, A. Czech, DA Babb, TW Robison and RA Bartsch Vol. 29 tho to... more 868 В. P. Czech, DH Desai, J. Koszuk, A. Czech, DA Babb, TW Robison and RA Bartsch Vol. 29 tho to the methyl group to form 4 in 76% yield. Reaction of 4 with triethylphosphite and freshly prepared, activated copper [16] provided a 72% yield of phosphonic acid di-ethyl ester 5 ...
Overexpression of casein kinase 2α (CK2α) is a common feature in lymphoid leukemias. Constitutive... more Overexpression of casein kinase 2α (CK2α) is a common feature in lymphoid leukemias. Constitutively active CK2α can disable transcriptional activity of lymphoid transcription factors like IKAROS that act as metabolic gatekeeper and limit the energy supply needed for oncogenic transformation of B cells. Our studies have shown that pharmacological inhibition of CK2α can restore the transcriptional activity of IKAROS and ablate leukemia. However, the role of CK2α in glucose metabolism has not been fully studied in B and T acute lymphoblastic leukemia (ALL). Therefore, in the present study we explored the metabolic alterations induced by the pharmacological inhibition of CK2α in B and T ALL. We studied the effect of CK2α inhibition using a specific inhibitor - CX4945 on cell proliferation, glucose utilization, lactate production, and intracellular ATP levels using established methods in human B-ALL (NALM6) cell line. Effect of CX4945 on glycolysis was studied using the Seahorse cell ana...
The Aurora kinases are serine/threonine kinases that are essential for mitosis. Aurora-B kinase i... more The Aurora kinases are serine/threonine kinases that are essential for mitosis. Aurora-B kinase is a component of the chromosomal passenger complex (CPC), chromosome condensation, the spindle-assembly checkpoint, and for cytokinesis. The activity of Aurora-B is essential for cellular division and proliferation. Inhibition of Aurora-B is cytotoxic, and inhibitor of Aurora-B - Barasertib (AZD1152) has been tested in clinical trials against different types of malignancies, including leukemia. However, regulation of Aurora-B expression is still largely unknown. Here, we present evidence that expression of Aurora-B in B-cell acute lymphoblastic leukemia (B-ALL) is regulated at the transcriptional level by Ikaros, a transcription factor and tumor suppressor protein. Analysis of global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) in several B-ALL primary human B-ALL cells and cell lines, showed a strong enrichment of Ikaros at the promoter of the Aurora-...
Ikzf1 encodes a zinc finger, DNA-binding protein that functions as a tumor suppressor in acute ly... more Ikzf1 encodes a zinc finger, DNA-binding protein that functions as a tumor suppressor in acute lymphoblastic leukemia (ALL). Deletion and/or loss of Ikaros function results in the development of high-risk leukemia. In the nucleus, Ikaros forms complexes with histone deacetylase complex, NuRD, and it participates in the formation of heterochromatin. The role of Ikaros-mediated formation of heterochromatin in tumor suppression in leukemia is unknown. We determined global genomic occupancy of Ikaros, global heterochromatin distribution, chromatin accessibility, DNA methylation landscape, and gene expression in primary human T-cell ALL (T-ALL), as well as in mouse T-ALL to analyze how Ikaros regulates heterochromatin landscape and gene expression in T-ALL. Results showed that Ikaros DNA occupancy is essential for the recruitment of histone deacetylase 1 (HDAC1), Polycomb repressive complex 2 (PRC2) and formation of facultative heterochromatin, as well as the formation of constitutive he...
Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be act... more Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be activated in leukemic stem cells (LSCs), especially in Pre-LSCs, and activation has been associated with poor prognosis in multiple cancers including AML. In AML, sorted CD34+/CD38- and aldehyde dehydrogenase-positive (ALDH+) cells have significantly phosphorylated STAT3 when compared to healthy controls. The Isatin analog, KS99 has shown selective anti-cancer activity against Multiple Myeloma (MM) which may, in part, be mediated by inhibition of BTK activation. Here we demonstrate that KS99 selectively targets AML CD34+/CD38- and ALDH+ cells and inhibits STAT3 phosphorylation while sparing normal hematopoietic stem and progenitor cells (HSPCs). Experimental Design: The pro-apoptotic activity of KS99 for primary human AML cells (n=21) and human AML cell lines (n=9) was evaluated using Annexin V and cell proliferation (MTS) assays. Colony forming assays were carried out to demonstrate the sp...
D,L-Sulforaphane (SFN) is a synthetic racemic analogue of broccoli constituent L-sulforaphane wit... more D,L-Sulforaphane (SFN) is a synthetic racemic analogue of broccoli constituent L-sulforaphane with in vivo evidence of cancer chemopreventive effect in rodent models. For example, we showed previously that oral administration of 6 μmol (about 1 mg/mouse) SFN (3x/week) modestly inhibited incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated cancer, but not poorly-differentiated (advanced) prostate cancer, in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice in association with apoptosis induction. Previous studies from our laboratory have also revealed that SFN treatment causes autophagy in cultured human prostate cancer cells, which serves to inhibit apoptotic cell death by delaying release of cytochrome c due to sequestration of mitochondria in autophagosomes. However, the in vivo significance of these cellular observations was unclear. The present study demonstrates, for the first time, in vivo augmentation of SFN-mediated prostate cancer chemoprevention by pharmacologic suppression of autophagy using chloroquine (CQ). A total of 128 four-week old TRAMP mice were randomized into one of the following four groups: (1) control (n=32), (2) SFN (n=32), (3) CQ (n=35), and (4) SFN + CQ (n=29). Termination of the experiment was planned after 18 weeks of treatment, but early sacrifice was necessary for some mice in each group (mostly from groups 1-3) due to a variety of reasons but not unique to any treatment group, including weight loss, premature death, morbidity, or early tumor onset. Nevertheless, % mice with prostate weight of >1g, an indicator of advanced (poorly-differentiated) prostate cancer, was significantly lower in the SFN + CQ group compared with control (P=0.006 by Fisher's exact test), SFN alone (P=0.017), and CQ alone group (P=0.072). Likewise, the incidence and burden of microscopic poorly-differentiated prostate cancer was significantly lower in the mice of the SFN + CQ group compared with control. Transmission electron microscopy confirmed in vivo autophagy induction by SFN administration in the dorsolateral prostate. Augmentation of SFN-mediated prostate cancer chemoprevention by CQ was associated with reduced cell proliferation. Plasma proteomics indicated protein level changes unique to the SFN + CQ combination compared with other groups (control or SFN), including hemopexin, serpina1c protein, and fructose-bisphosphate aldolase A isoform precursor. Cluster analysis of proteomics data revealed significant enrichment for gene ontology (GO) terms proteasome and threonine protease (2.8), protease inhibitor (2.7), and protein-lipid complex (2.6). These results merit determination of the efficacy of SFN + CQ combination for prevention of prostate cancer in a clinical setting. This investigation was supported by the NCI grant CA115498-07. Citation Format: Avani R. Vyas, Eun-Ryeong Hahm, Julie A. Arlotti, Dhimant Desai, Shantu Amin, Shivendra V. Singh. Augmentation of D,L-sulforaphane-mediated prostate cancer chemoprevention by pharmacologic inhibition of autophagy using chloroquine in a transgenic mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2013-3695
Despite public awareness and availability of sunscreen, incidence and mortality rates for maligna... more Despite public awareness and availability of sunscreen, incidence and mortality rates for malignant melanoma continue to increase every year. Novel strategies are required to prevent melanoma development as the causes for ∼60% of cases remaining unknown. While sunscreen is effective at protecting skin from UV radiation induced damage, inhibiting melanocytic lesion development using body creams or sunscreens containing chemopreventive agents could be a novel approach to fight this disease. Hence, the chemopreventive efficacy of PBISe, a selenium containing isosteric analogue of PBIT, has been examined in laboratory generated skin reconstructs and melanoma tumor xenograft models. The data demonstrate that PBISe treated skin reconstructs containing melanocytic lesion cells had 60–70% smaller tumor lesions compared to control treated skins. Furthermore, mice topically treated with PBISe had ∼50% fewer tumors compared to PBIT or acetone vehicle treated control groups. Mechanistically, PBISe elevated MAP kinase pathway activity as indicated by increased pErk1/2 levels to amounts that induced cellular senescence. Furthermore, PBISe treatment down-regulated cyclin-D1, upregulated the expression of cell cycle inhibitors p21 and p27 as well as apoptotic markers cleaved caspase-3 and PARP to inhibit cell proliferation and induce apoptosis. In conclusion, the results demonstrate the potential utility of topical PBISe application for preventing melanoma leision development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A96.
Nonalcoholic steatohepatitis (NASH) is a rapidly developing pathology around the world, with limi... more Nonalcoholic steatohepatitis (NASH) is a rapidly developing pathology around the world, with limited treatment options available. Some farnesoid X receptor (FXR) agonists have been applied in clinical trials for NASH, but side effects such as pruritus and low‐density lipoprotein elevation have been reported. Intestinal FXR is recognized as a promising therapeutic target for metabolic diseases. Glycine‐β‐muricholic acid (Gly‐MCA) is an intestine‐specific FXR antagonist previously shown to have favorable metabolic effects on obesity and insulin resistance. Herein, we identify a role for Gly‐MCA in the pathogenesis of NASH, and explore the underlying molecular mechanism. Gly‐MCA improved lipid accumulation, inflammatory response, and collagen deposition in two different NASH models. Mechanistically, Gly‐MCA decreased intestine‐derived ceramides by suppressing ceramide synthesis–related genes via decreasing intestinal FXR signaling, leading to lower liver endoplasmic reticulum (ER) stress and proinflammatory cytokine production. The role of bile acid metabolism and adiposity was excluded in the suppression of NASH by Gly‐MCA, and a correlation was found between intestine‐derived ceramides and NASH severity. This study revealed that Gly‐MCA, an intestine‐specific FXR antagonist, has beneficial effects on NASH by reducing ceramide levels circulating to liver via lowering intestinal FXR signaling, and ceramide production, followed by decreased liver ER stress and NASH progression. Intestinal FXR is a promising drug target and Gly‐MCA a novel agent for the prevention and treatment of NASH.
Colorectal cancer (CRC) is a multistep disorder resulting from genetic and epigenetic genome chan... more Colorectal cancer (CRC) is a multistep disorder resulting from genetic and epigenetic genome changes. It is the third most common malignancy in developed nations accounting for roughly 600,000 deaths annually. Persistent gut inflammation, as observed in inflammatory bowel disease (IBD), is a key risk factor for CRC development. From an epigenetic viewpoint, the pharmacological inhibition of HDACs using HDAC inhibitors such as SAHA has emerged as a suitable anticancer strategy in the recent past. However, the clinical success of these strategies is limited and has risk factors associated with their uses. Thus, considering the critical involvement of epigenetic regulation of key molecular mechanisms in carcinogenesis as well as HDAC inhibitory and anti-tumorigenic properties of Selenium (Se), we aimed to explore the potentially safer and enhanced chemotherapeutic potential of a Se derivative of SAHA namely SelSA-1, in an experimental model of colitis-associated experimental cancer (CA...
868 В. P. Czech, DH Desai, J. Koszuk, A. Czech, DA Babb, TW Robison and RA Bartsch Vol. 29 tho to... more 868 В. P. Czech, DH Desai, J. Koszuk, A. Czech, DA Babb, TW Robison and RA Bartsch Vol. 29 tho to the methyl group to form 4 in 76% yield. Reaction of 4 with triethylphosphite and freshly prepared, activated copper [16] provided a 72% yield of phosphonic acid di-ethyl ester 5 ...
Overexpression of casein kinase 2α (CK2α) is a common feature in lymphoid leukemias. Constitutive... more Overexpression of casein kinase 2α (CK2α) is a common feature in lymphoid leukemias. Constitutively active CK2α can disable transcriptional activity of lymphoid transcription factors like IKAROS that act as metabolic gatekeeper and limit the energy supply needed for oncogenic transformation of B cells. Our studies have shown that pharmacological inhibition of CK2α can restore the transcriptional activity of IKAROS and ablate leukemia. However, the role of CK2α in glucose metabolism has not been fully studied in B and T acute lymphoblastic leukemia (ALL). Therefore, in the present study we explored the metabolic alterations induced by the pharmacological inhibition of CK2α in B and T ALL. We studied the effect of CK2α inhibition using a specific inhibitor - CX4945 on cell proliferation, glucose utilization, lactate production, and intracellular ATP levels using established methods in human B-ALL (NALM6) cell line. Effect of CX4945 on glycolysis was studied using the Seahorse cell ana...
The Aurora kinases are serine/threonine kinases that are essential for mitosis. Aurora-B kinase i... more The Aurora kinases are serine/threonine kinases that are essential for mitosis. Aurora-B kinase is a component of the chromosomal passenger complex (CPC), chromosome condensation, the spindle-assembly checkpoint, and for cytokinesis. The activity of Aurora-B is essential for cellular division and proliferation. Inhibition of Aurora-B is cytotoxic, and inhibitor of Aurora-B - Barasertib (AZD1152) has been tested in clinical trials against different types of malignancies, including leukemia. However, regulation of Aurora-B expression is still largely unknown. Here, we present evidence that expression of Aurora-B in B-cell acute lymphoblastic leukemia (B-ALL) is regulated at the transcriptional level by Ikaros, a transcription factor and tumor suppressor protein. Analysis of global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) in several B-ALL primary human B-ALL cells and cell lines, showed a strong enrichment of Ikaros at the promoter of the Aurora-...
Ikzf1 encodes a zinc finger, DNA-binding protein that functions as a tumor suppressor in acute ly... more Ikzf1 encodes a zinc finger, DNA-binding protein that functions as a tumor suppressor in acute lymphoblastic leukemia (ALL). Deletion and/or loss of Ikaros function results in the development of high-risk leukemia. In the nucleus, Ikaros forms complexes with histone deacetylase complex, NuRD, and it participates in the formation of heterochromatin. The role of Ikaros-mediated formation of heterochromatin in tumor suppression in leukemia is unknown. We determined global genomic occupancy of Ikaros, global heterochromatin distribution, chromatin accessibility, DNA methylation landscape, and gene expression in primary human T-cell ALL (T-ALL), as well as in mouse T-ALL to analyze how Ikaros regulates heterochromatin landscape and gene expression in T-ALL. Results showed that Ikaros DNA occupancy is essential for the recruitment of histone deacetylase 1 (HDAC1), Polycomb repressive complex 2 (PRC2) and formation of facultative heterochromatin, as well as the formation of constitutive he...
Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be act... more Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be activated in leukemic stem cells (LSCs), especially in Pre-LSCs, and activation has been associated with poor prognosis in multiple cancers including AML. In AML, sorted CD34+/CD38- and aldehyde dehydrogenase-positive (ALDH+) cells have significantly phosphorylated STAT3 when compared to healthy controls. The Isatin analog, KS99 has shown selective anti-cancer activity against Multiple Myeloma (MM) which may, in part, be mediated by inhibition of BTK activation. Here we demonstrate that KS99 selectively targets AML CD34+/CD38- and ALDH+ cells and inhibits STAT3 phosphorylation while sparing normal hematopoietic stem and progenitor cells (HSPCs). Experimental Design: The pro-apoptotic activity of KS99 for primary human AML cells (n=21) and human AML cell lines (n=9) was evaluated using Annexin V and cell proliferation (MTS) assays. Colony forming assays were carried out to demonstrate the sp...
D,L-Sulforaphane (SFN) is a synthetic racemic analogue of broccoli constituent L-sulforaphane wit... more D,L-Sulforaphane (SFN) is a synthetic racemic analogue of broccoli constituent L-sulforaphane with in vivo evidence of cancer chemopreventive effect in rodent models. For example, we showed previously that oral administration of 6 μmol (about 1 mg/mouse) SFN (3x/week) modestly inhibited incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated cancer, but not poorly-differentiated (advanced) prostate cancer, in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice in association with apoptosis induction. Previous studies from our laboratory have also revealed that SFN treatment causes autophagy in cultured human prostate cancer cells, which serves to inhibit apoptotic cell death by delaying release of cytochrome c due to sequestration of mitochondria in autophagosomes. However, the in vivo significance of these cellular observations was unclear. The present study demonstrates, for the first time, in vivo augmentation of SFN-mediated prostate cancer chemoprevention by pharmacologic suppression of autophagy using chloroquine (CQ). A total of 128 four-week old TRAMP mice were randomized into one of the following four groups: (1) control (n=32), (2) SFN (n=32), (3) CQ (n=35), and (4) SFN + CQ (n=29). Termination of the experiment was planned after 18 weeks of treatment, but early sacrifice was necessary for some mice in each group (mostly from groups 1-3) due to a variety of reasons but not unique to any treatment group, including weight loss, premature death, morbidity, or early tumor onset. Nevertheless, % mice with prostate weight of >1g, an indicator of advanced (poorly-differentiated) prostate cancer, was significantly lower in the SFN + CQ group compared with control (P=0.006 by Fisher's exact test), SFN alone (P=0.017), and CQ alone group (P=0.072). Likewise, the incidence and burden of microscopic poorly-differentiated prostate cancer was significantly lower in the mice of the SFN + CQ group compared with control. Transmission electron microscopy confirmed in vivo autophagy induction by SFN administration in the dorsolateral prostate. Augmentation of SFN-mediated prostate cancer chemoprevention by CQ was associated with reduced cell proliferation. Plasma proteomics indicated protein level changes unique to the SFN + CQ combination compared with other groups (control or SFN), including hemopexin, serpina1c protein, and fructose-bisphosphate aldolase A isoform precursor. Cluster analysis of proteomics data revealed significant enrichment for gene ontology (GO) terms proteasome and threonine protease (2.8), protease inhibitor (2.7), and protein-lipid complex (2.6). These results merit determination of the efficacy of SFN + CQ combination for prevention of prostate cancer in a clinical setting. This investigation was supported by the NCI grant CA115498-07. Citation Format: Avani R. Vyas, Eun-Ryeong Hahm, Julie A. Arlotti, Dhimant Desai, Shantu Amin, Shivendra V. Singh. Augmentation of D,L-sulforaphane-mediated prostate cancer chemoprevention by pharmacologic inhibition of autophagy using chloroquine in a transgenic mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3695. doi:10.1158/1538-7445.AM2013-3695
Despite public awareness and availability of sunscreen, incidence and mortality rates for maligna... more Despite public awareness and availability of sunscreen, incidence and mortality rates for malignant melanoma continue to increase every year. Novel strategies are required to prevent melanoma development as the causes for ∼60% of cases remaining unknown. While sunscreen is effective at protecting skin from UV radiation induced damage, inhibiting melanocytic lesion development using body creams or sunscreens containing chemopreventive agents could be a novel approach to fight this disease. Hence, the chemopreventive efficacy of PBISe, a selenium containing isosteric analogue of PBIT, has been examined in laboratory generated skin reconstructs and melanoma tumor xenograft models. The data demonstrate that PBISe treated skin reconstructs containing melanocytic lesion cells had 60–70% smaller tumor lesions compared to control treated skins. Furthermore, mice topically treated with PBISe had ∼50% fewer tumors compared to PBIT or acetone vehicle treated control groups. Mechanistically, PBISe elevated MAP kinase pathway activity as indicated by increased pErk1/2 levels to amounts that induced cellular senescence. Furthermore, PBISe treatment down-regulated cyclin-D1, upregulated the expression of cell cycle inhibitors p21 and p27 as well as apoptotic markers cleaved caspase-3 and PARP to inhibit cell proliferation and induce apoptosis. In conclusion, the results demonstrate the potential utility of topical PBISe application for preventing melanoma leision development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A96.
Nonalcoholic steatohepatitis (NASH) is a rapidly developing pathology around the world, with limi... more Nonalcoholic steatohepatitis (NASH) is a rapidly developing pathology around the world, with limited treatment options available. Some farnesoid X receptor (FXR) agonists have been applied in clinical trials for NASH, but side effects such as pruritus and low‐density lipoprotein elevation have been reported. Intestinal FXR is recognized as a promising therapeutic target for metabolic diseases. Glycine‐β‐muricholic acid (Gly‐MCA) is an intestine‐specific FXR antagonist previously shown to have favorable metabolic effects on obesity and insulin resistance. Herein, we identify a role for Gly‐MCA in the pathogenesis of NASH, and explore the underlying molecular mechanism. Gly‐MCA improved lipid accumulation, inflammatory response, and collagen deposition in two different NASH models. Mechanistically, Gly‐MCA decreased intestine‐derived ceramides by suppressing ceramide synthesis–related genes via decreasing intestinal FXR signaling, leading to lower liver endoplasmic reticulum (ER) stress and proinflammatory cytokine production. The role of bile acid metabolism and adiposity was excluded in the suppression of NASH by Gly‐MCA, and a correlation was found between intestine‐derived ceramides and NASH severity. This study revealed that Gly‐MCA, an intestine‐specific FXR antagonist, has beneficial effects on NASH by reducing ceramide levels circulating to liver via lowering intestinal FXR signaling, and ceramide production, followed by decreased liver ER stress and NASH progression. Intestinal FXR is a promising drug target and Gly‐MCA a novel agent for the prevention and treatment of NASH.
Colorectal cancer (CRC) is a multistep disorder resulting from genetic and epigenetic genome chan... more Colorectal cancer (CRC) is a multistep disorder resulting from genetic and epigenetic genome changes. It is the third most common malignancy in developed nations accounting for roughly 600,000 deaths annually. Persistent gut inflammation, as observed in inflammatory bowel disease (IBD), is a key risk factor for CRC development. From an epigenetic viewpoint, the pharmacological inhibition of HDACs using HDAC inhibitors such as SAHA has emerged as a suitable anticancer strategy in the recent past. However, the clinical success of these strategies is limited and has risk factors associated with their uses. Thus, considering the critical involvement of epigenetic regulation of key molecular mechanisms in carcinogenesis as well as HDAC inhibitory and anti-tumorigenic properties of Selenium (Se), we aimed to explore the potentially safer and enhanced chemotherapeutic potential of a Se derivative of SAHA namely SelSA-1, in an experimental model of colitis-associated experimental cancer (CA...
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