9 research outputs found
Mechanisms of IKBKE Activation in Cancer
Cancer is the second leading cause of death in the USA and it is expected to surpass heart diseases making it important to understand the underlying mechanisms of cancer. The efforts to target single signaling molecule showed little success in increasing the patient survival and it can be due to increased compensation for cell survival by alternative pathway activations. Hence comprehensive understanding of the alternative signaling pathways may help us treat cancer better. Chronic inflammation is attributed to increased risk of cancer and emerging studies show the growing importance of both canonical and non-canonical IκB kinases such as IKKα, IKKβ, IKBKE and TBK1 in human cancer pathogenesis. Initially identified as activator of NFκB pathway, IKBKE was shown to play an important oncogenic role by regulating multiple pathways downstream. Although IKBKE is implicated in tumorigenesis for over a decade, therapeutic targeting of this pathway has been a challenge. Recently, amlexanox and CYT387, which are in clinical trials for Type II diabetes and myeloproliferative disorders respectively, were identified as potential IKBKE inhibitors. In this study, we uncovered specific novel mechanisms of activation of IKBKE in tumor cells and the outcomes of targeting IKBKE pathway.
Oncogenic mutations are a cause of several human malignancies. Mutations in EGFR are observed in 15% of non-small cell lung cancer patients. While cells expressing these mutations respond better to the first generation TKIs, patients become resistant to these inhibitors due to secondary mutations in EGFR. These mutations were shown to make EGFR constitutively active even in the absence of ligands. Direct targeting of EGFR with secondary mutations has been challenging as EGFR acquires novel mutations upon inhibitor treatment, which confer resistance to the EGFR-TKIs. Hence, it is important to improve our knowledge of the downstream signaling pathways of EGFR. Although PI3K, MEK signaling are well established, mutant EGFR was shown to activate several novel signaling pathways such as miRNA processing and autophagy that are implicated in resistance to EGFR-TKIs.
Here, we show that IKBKE acts downstream of mutant EGFR to activate the NFκB and AKT pathways. In addition, we show that mutant EGFR but not wildtype EGFR can directly phosphorylate IKBKE at Tyrosine 153 and Tyrosine 179 residues that are important for activation of IKBKE kinase. We also found that the IKBKE/TBK1 inhibitor Amlexanox exhibits increased efficacy in inhibiting cell viability in NSCLC cells with activating EGFR mutations. Furthermore, we also found that IKBKE inhibitors activate the MAPK pathway, and EGFR-TKI resistant NSCLCs exhibit enhanced response to co-treatment with IKBKE inhibitors and MEK inhibitors.
Similar to lung cancer, pancreatic cancer is a challenging disease due to lack of direct inhibitors of the KRas mutations that are observed in more than 95% of pancreatic cancer patients. IKBKE/TBK1 pathway is important for KRas signaling, but the efficacy of IKBKE inhibitors in pancreatic cancers is not well studied. Here, we show that IKBKE is an important target in pancreatic cancers that regulates pancreatic cell viability, cell migration and cancer stem cells. Importantly, we provide mechanistic insights into the effects of IKBKE inhibitors on specific signaling pathways. We found that IKBKE inhibition results in significantly increased expression of RTKs, such as ErbB3 and IGF1-R, which increases ERK1/2 activation. Our findings provide support for novel combination strategies for pancreatic cancer.
Metastasis is a poor prognostic factor for ovarian cancer. Although patients with early stage ovarian cancer with no distal metastasis exhibit a 70% 5-year survival rate, Stage IV patients with distal metastasis exhibit only 20% 5-year survival rate. Hence, ongoing efforts are aimed at targeting the pathways that regulate metastasis in ovarian cancers. IKBKE is upregulated in ovarian cancer patients, and IKBKE expression is known to regulate the expression of several genes important for cell motility in ovarian cancers. IKBKE is also implicated in chemo-resistance in ovarian cancer, and siRNA knockdown of IKBKE increases sensitivity towards chemotherapy. However, the mechanistic role of IKBKE in chemo-resistance in ovarian cancer is not known. EphA2 is another well studied oncogene in ovarian cancer as 70% of ovarian cancer patients exhibit elevated levels of EphA2. By activating Focal Adhesion Kinases (FAK), EphA2 can induce metastasis in ovarian cancers. In this study, we show that the clinical PARP inhibitor Olaparib (AZD2281) activates IKBKE by EphA2-mediated tyrosine phosphorylation. We also found that phosphorylation of EphA2 or IKBKE expression can be used as a biomarker for olaparib resistance.
Together, these studies have shed light on novel mechanisms of regulation of IKBKE and their importance in therapy resistance. These observations form a strong pre-clinical proof-of-concept to study the inhibitors further in the clinic
Ack1 Mediated AKT/PKB Tyrosine 176 Phosphorylation Regulates Its Activation
The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery
Mechanisms of IKBKE Activation in Cancer
Cancer is the second leading cause of death in the USA and it is expected to surpass heart diseases making it important to understand the underlying mechanisms of cancer. The efforts to target single signaling molecule showed little success in increasing the patient survival and it can be due to increased compensation for cell survival by alternative pathway activations. Hence comprehensive understanding of the alternative signaling pathways may help us treat cancer better. Chronic inflammation is attributed to increased risk of cancer and emerging studies show the growing importance of both canonical and non-canonical IκB kinases such as IKKα, IKKβ, IKBKE and TBK1 in human cancer pathogenesis. Initially identified as activator of NFκB pathway, IKBKE was shown to play an important oncogenic role by regulating multiple pathways downstream. Although IKBKE is implicated in tumorigenesis for over a decade, therapeutic targeting of this pathway has been a challenge. Recently, amlexanox and CYT387, which are in clinical trials for Type II diabetes and myeloproliferative disorders respectively, were identified as potential IKBKE inhibitors. In this study, we uncovered specific novel mechanisms of activation of IKBKE in tumor cells and the outcomes of targeting IKBKE pathway.
Oncogenic mutations are a cause of several human malignancies. Mutations in EGFR are observed in 15% of non-small cell lung cancer patients. While cells expressing these mutations respond better to the first generation TKIs, patients become resistant to these inhibitors due to secondary mutations in EGFR. These mutations were shown to make EGFR constitutively active even in the absence of ligands. Direct targeting of EGFR with secondary mutations has been challenging as EGFR acquires novel mutations upon inhibitor treatment, which confer resistance to the EGFR-TKIs. Hence, it is important to improve our knowledge of the downstream signaling pathways of EGFR. Although PI3K, MEK signaling are well established, mutant EGFR was shown to activate several novel signaling pathways such as miRNA processing and autophagy that are implicated in resistance to EGFR-TKIs.
Here, we show that IKBKE acts downstream of mutant EGFR to activate the NFκB and AKT pathways. In addition, we show that mutant EGFR but not wildtype EGFR can directly phosphorylate IKBKE at Tyrosine 153 and Tyrosine 179 residues that are important for activation of IKBKE kinase. We also found that the IKBKE/TBK1 inhibitor Amlexanox exhibits increased efficacy in inhibiting cell viability in NSCLC cells with activating EGFR mutations. Furthermore, we also found that IKBKE inhibitors activate the MAPK pathway, and EGFR-TKI resistant NSCLCs exhibit enhanced response to co-treatment with IKBKE inhibitors and MEK inhibitors.
Similar to lung cancer, pancreatic cancer is a challenging disease due to lack of direct inhibitors of the KRas mutations that are observed in more than 95% of pancreatic cancer patients. IKBKE/TBK1 pathway is important for KRas signaling, but the efficacy of IKBKE inhibitors in pancreatic cancers is not well studied. Here, we show that IKBKE is an important target in pancreatic cancers that regulates pancreatic cell viability, cell migration and cancer stem cells. Importantly, we provide mechanistic insights into the effects of IKBKE inhibitors on specific signaling pathways. We found that IKBKE inhibition results in significantly increased expression of RTKs, such as ErbB3 and IGF1-R, which increases ERK1/2 activation. Our findings provide support for novel combination strategies for pancreatic cancer.
Metastasis is a poor prognostic factor for ovarian cancer. Although patients with early stage ovarian cancer with no distal metastasis exhibit a 70% 5-year survival rate, Stage IV patients with distal metastasis exhibit only 20% 5-year survival rate. Hence, ongoing efforts are aimed at targeting the pathways that regulate metastasis in ovarian cancers. IKBKE is upregulated in ovarian cancer patients, and IKBKE expression is known to regulate the expression of several genes important for cell motility in ovarian cancers. IKBKE is also implicated in chemo-resistance in ovarian cancer, and siRNA knockdown of IKBKE increases sensitivity towards chemotherapy. However, the mechanistic role of IKBKE in chemo-resistance in ovarian cancer is not known. EphA2 is another well studied oncogene in ovarian cancer as 70% of ovarian cancer patients exhibit elevated levels of EphA2. By activating Focal Adhesion Kinases (FAK), EphA2 can induce metastasis in ovarian cancers. In this study, we show that the clinical PARP inhibitor Olaparib (AZD2281) activates IKBKE by EphA2-mediated tyrosine phosphorylation. We also found that phosphorylation of EphA2 or IKBKE expression can be used as a biomarker for olaparib resistance.
Together, these studies have shed light on novel mechanisms of regulation of IKBKE and their importance in therapy resistance. These observations form a strong pre-clinical proof-of-concept to study the inhibitors further in the clinic
MARTs and MARylation in the Cytosol: Biological Functions, Mechanisms of Action, and Therapeutic Potential
Mono(ADP-ribosyl)ation (MARylation) is a regulatory post-translational modification of proteins that controls their functions through a variety of mechanisms. MARylation is catalyzed by mono(ADP-ribosyl) transferase (MART) enzymes, a subclass of the poly(ADP-ribosyl) polymerase (PARP) family of enzymes. Although the role of PARPs and poly(ADP-ribosyl)ation (PARylation) in cellular pathways, such as DNA repair and transcription, is well studied, the role of MARylation and MARTs (i.e., the PARP ‘monoenzymes’) are not well understood. Moreover, compared to PARPs, the development of MART-targeted therapeutics is in its infancy. Recent studies are beginning to shed light on the structural features, catalytic targets, and biological functions of MARTs. The development of new technologies to study MARTs have uncovered essential roles for these enzymes in the regulation of cellular processes, such as RNA metabolism, cellular transport, focal adhesion, and stress responses. These insights have increased our understanding of the biological functions of MARTs in cancers, neuronal development, and immune responses. Furthermore, several novel inhibitors of MARTs have been developed and are nearing clinical utility. In this review, we summarize the biological functions and molecular mechanisms of MARTs and MARylation, as well as recent advances in technology that have enabled detection and inhibition of their activity. We emphasize PARP-7, which is at the forefront of the MART subfamily with respect to understanding its biological roles and the development of therapeutically useful inhibitors. Collectively, the available studies reveal a growing understanding of the biochemistry, chemical biology, physiology, and pathology of MARTs
Ionic liquid-based vortex-assisted DLLME followed by RP-LC-PDA method for bioassay of daclatasvir in rat serum: application to pharmacokinetics
Abstract Background Daclatasvir is a direct-acting antiviral agent against hepatitis C virus (HCV) used for the treatment of chronic HCV genotype infections of 1 and 3. Hepatitis C is an infectious liver disease caused by infection with hepatitis C virus (HCV). There are no reports found to be daclatasvir in ionic liquid-based extraction. Methods A simple vortex assisted an environmental eco-friendly ionic liquid dispersive liquid–liquid microextraction method for determination of daclatasvir form rat serum. For the sample extraction, various green solvents, like ionic liquids, were used. The repercussion of various dispersive solvents, extractant, and disperser ratios was evaluated; non-identical ionic liquids assess the salt concentration on sample recoveries and enrichment factors were examined. Amid all the ionic liquids that were scrutinized, 1-butyl-3-methylimidazolium hexafluorophosphate was selected as the most effective ionic liquid. Results The present bioassay recoveries were found to be more than 99.4% at an extractant and disperser ratio of 0.43 with an addition of 5.0% NaCl (sodium chloride) that was selected as an effective salt concentration for present extraction. Compared to protein precipitation, the enhanced detection and quantification limits attained were 0.015 μg/mL and 0.045 μg/mL, respectively. A linear relationship in the range of 0.05–10.0 μg/mL respectively with a correlation coefficient of (r 2) 0.9996 was observed. Conclusion The developed method was successfully applied to study the pharmacokinetics of daclatasvir in rat serum according to the bioanalytical method validation guidelines
Identification of PARP-7 substrates reveals a role for MARylation in microtubule control in ovarian cancer cells
PARP-7 (TiPARP) is a mono(ADP-ribosyl) transferase whose protein substrates and biological activities are poorly understood. We observed that PARP7 mRNA levels are lower in ovarian cancer patient samples compared to non-cancerous tissue, but PARP-7 protein nonetheless contributes to several cancer-related biological endpoints in ovarian cancer cells (e.g. growth, migration). Global gene expression analyses in ovarian cancer cells subjected to PARP-7 depletion indicate biological roles for PARP-7 in cell-cell adhesion and gene regulation. To identify the MARylated substrates of PARP-7 in ovarian cancer cells, we developed an NAD+ analog-sensitive approach, which we coupled with mass spectrometry to identify the PARP-7 ADP-ribosylated proteome in ovarian cancer cells, including cell-cell adhesion and cytoskeletal proteins. Specifically, we found that PARP-7 MARylates α-tubulin to promote microtubule instability, which may regulate ovarian cancer cell growth and motility. In sum, we identified an extensive PARP-7 ADP-ribosylated proteome with important roles in cancer-related cellular phenotypes
One-Bead–Two-Compound Thioether Bridged Macrocyclic γ‑AApeptide Screening Library against EphA2
Identification of molecular ligands
that recognize peptides or
proteins is significant but poses a fundamental challenge in chemical
biology and biomedical sciences. Development of cyclic peptidomimetic
library is scarce, and thus discovery of cyclic peptidomimetic ligands
for protein targets is rare. Herein we report the unprecedented one-bead–two-compound
(OBTC) combinatorial library based on a novel class of the macrocyclic
peptidomimetics γ-AApeptides. In the library, we utilized the
coding peptide tags synthesized with Dde-protected α-amino acids,
which were orthogonal to solid phase synthesis of γ-AApeptides.
Employing the thioether linkage, the desired macrocyclic γ-AApeptides
were found to be effective for ligand identification. Screening the
library against the receptor tyrosine kinase EphA2 led to the discovery
of one lead compound that tightly bound to EphA2 (<i>K</i><sub>d</sub> = 81 nM) and potently antagonized EphA2-mediated signaling.
This new approach of macrocyclic peptidomimetic library may lead to
a novel platform for biomacromolecular surface recognition and function
modulation