p27 allosterically activates cyclin-dependent kinase 4 and antagonizes palbociclib inhibition.

The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors

The BET inhibitor/degrader ARV-825 prolongs the growth arrest response to Fulvestrant + Palbociclib and suppresses proliferative recovery in ER-positive breast cancer

Anti-estrogens or aromatase inhibitors in combination with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are the current standard of care for estrogen receptor-positive (ER+) Her-2 negative metastatic breast cancer. Although these combination therapies prolong progression-free survival compared to endocrine therapy alone, the growth-arrested state of residual tumor cells is clearly transient. Tumor cells that escape what might be considered a dormant or quiescent state and regain proliferative capacity often acquire resistance to further therapies. Our studies are based upon the observation that breast tumor cells arrested by Fulvestrant + Palbociclib enter into states of both autophagy and senescence from which a subpopulation ultimately escapes, potentially contributing to recurrent disease. Autophagy inhibition utilizing pharmacologic or genetic approaches only moderately enhanced the response to Fulvestrant + Palbociclib in ER+ MCF-7 breast tumor cells, slightly delaying proliferative recovery. In contrast, the BET inhibitor/degrader, ARV-825, prolonged the growth arrested state in both p53 wild type MCF-7 cells and p53 mutant T-47D cells and significantly delayed proliferative recovery. In addition, ARV-825 added after the Fulvestrant + Palbociclib combination promoted apoptosis and demonstrated efficacy in resistant RB deficient cell lines. These studies indicate that administration of BET inhibitors/degraders, which are currently being investigated in multiple clinical trials, may potentially improve standard of care therapy in metastatic ER+ breast cancer patients and may further prolong progression-free survival

Genotype-Phenotype Study of the Middle Gangetic Plain in India Shows Association of rs2470102 with Skin Pigmentation

Our understanding of the genetics of skin pigmentation has been largely skewed towards populations of European ancestry, imparting less attention to South Asian populations, who behold huge pigmentation diversity. Here, we investigate skin pigmentation variation in a cohort of 1,167 individuals in the Middle Gangetic Plain of the Indian subcontinent. Our data confirm the association of rs1426654 with skin pigmentation among South Asians, consistent with previous studies, and also show association for rs2470102 single nucleotide polymorphism. Our haplotype analyses further help us delineate the haplotype distribution across social categories and skin color. Taken together, our findings suggest that the social structure defined by the caste system in India has a profound influence on the skin pigmentation patterns of the subcontinent. In particular, social category and associated single nucleotide polymorphisms explain about 32% and 6.4%, respectively, of the total phenotypic variance. Phylogeography of the associated single nucleotide polymorphisms studied across 52 diverse populations of the Indian subcontinent shows wide presence of the derived alleles, although their frequencies vary across populations. Our results show that both polymorphisms (rs1426654 and rs2470102) play an important role in the skin pigmentation diversity of South Asians

Neuromuscular disease genetics in under-represented populations: increasing data diversity

Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses ‘solved’ or ‘possibly solved’ ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% ‘solved’ and ∼13% ‘possibly solved’ outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally

Neuromuscular disease genetics in underrepresented populations : increasing data diversity

DATA AVAILABILITY : At the end of the study, participants de-identified exome and genome data will be archived in the European Molecular Biology Laboratory European Bioinformatics Institute’s European Genome-Phenome Archive (EMBL EBI EGA), with community access to this and selected de-identified REDCap data managed via an ICGNMD Data Access Committee.Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses 'solved' or 'possibly solved' ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% 'solved' and ∼13% 'possibly solved' outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally.This work was supported by a Medical Research Council strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) MR/S005021/1. Additional ICGNMD support including travel and subsistence costs was received from the National Brain Appeal (UK Charity 290173) and University College London Global Engagement Funds. Fellowships for R.S.S.F. and K.N. were funded by the Guarantors of Brain (UK Charity 1197319). The authors acknowledge and are grateful for: conference bursaries from the World Muscle Society to R.S.S.F. S.R., K.N., O.Y.K., P.J.T., V.V.Y. S.V.D.M. and R.L. are members of the European Reference Network for Rare Neuromuscular Diseases (ERN EURO-MND). M.P.K.: National Institute of Neurological Disorders and Stroke (1K23NS112463), American Association of Neuromuscular & Electrodiagnostic Medicine Development Award and Allen Foundation. D.B.: National Institute of Neurological Disorders and Stroke (K23NS117310) and support from Biogen for the KCTN1 Natural History Study. G.M.R.: University College London and UCLH Biomedical Research Centre funding, Health Education England and University College London Hospitals NHS Foundation Trust Innovation Fund. R.M.F., R.W.T. and K.P.: Wellcome core support (203105/Z/16/Z). R.M.F. received additional support from the Lily Foundation and the Leigh Syndrome International Consortium. A.T.: EU Horizon 2020 research and innovation Solve-RD project, No. 779257. F.H.W., M.S., M.B. and A.V.: South African Medical Research Council award ‘The genetics of Neuromuscular Diseases in South African patient populations: the ICGNMD study’. K.T. is funded by a J. C. Bose Fellowship, Science and Engineering Research Board (SERB) Department of Science and Technology, India. P.G. is supported by the Centre for DNA Fingerprinting and Diagnostics (CDFD) Core Research Grant, Department of Biotechnology, Government of India. R.H.: Wellcome award 109915/Z/15/Z, UK Medical Research Council award MR/N025431/1, the Lily Foundation, Evelyn Trust Research Grant (Ref 19/14), Action for A-T and UK Research and Innovation Newton Fund (MR/NO27302/1). P.F.C.: Wellcome awards 212219/Z/18/Z and 224486/Z/21/Z, UK Medical Research Council awards MC_PC_21046, MR/S035699/1 and MR/ S01165X/1, LifeArc Philanthropic Fund, NIHR BioResource for Translational Research in Common and Rare Diseases, Alzheimer’s Society, NIHR BioResource for Genes and Cognition and Leverhulme Trust. R.D.S.P.: UK Medical Research Council MR/ S002065/1 and MC_PC_21046, and the Lily Foundation. H.H.: UK Medical Research Council, Wellcome, UCLH Biomedical Research Centre (NIHR-BRC), Rosetrees Trust, and SOLVE-RD. M.M.R.: Wellcome grant G104817, National Institute of Neurological Disorders and Stroke and Office of Rare Diseases grants U54NS065712 and 1UOINS109403-01 and Muscular Dystrophy Association grant.https://www.edusoft.ro/brain/index.php/brainam2024Paediatrics and Child HealthSDG-03:Good heatlh and well-bein

Targeting the Transcription of RET Oncogene in Medullary Thyroid Carcinoma Using Drug-Like Small Molecules

Dominant-activating mutations in the RET (Rearranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, was identified as the major cause for the development of medullary thyroid carcinoma (MTC). Hence, the RET protein has been explored as an excellent molecular target for therapeutic intervention to treat progressive and advanced MTC. The main objective of this dissertation is to highlight a clinically relevant therapeutic strategy for the treatment of MTC by selectively suppressing the transcription of mutant RET oncogene. The basal transcriptional activity of the RET gene is mainly regulated by the polypurine/polypyrimidine (pPu/pPy) tract, present within the proximal promoter region of this gene (-51 to -33 relative to transcription start site). The pPu/pPy tract is rich in guanine-repeat sequence containing five runs of consecutive guanine residues that serve as the binding site for transcriptional factors. This stretch of nucleotides in the promoter region is highly dynamic in nature and tend to adopt non-B DNA secondary structures called G-quadruplexes. As we have recently shown, the formation of G-quadruplex structures on the RET promoter region negatively regulates the transcription of this gene through sequestration of the transcription factor binding sites. Hence, we hypothesized that the stabilization of these structures using small molecules could exert transcriptional inhibitory effect on the RET gene. In our study, we validated that the RET promoter G-quadruplex structure is an amenable drug target to silence RET gene expression by demonstrating the anti-RET activity of a well-characterized G-quadruplex stabilizing agent, berberine. This compound inhibits the RET gene transcription in an MTC derived TT cell line by specifically targeting its promoter G-quadruplex structure. Interestingly, the inhibitory effect of berberine on the RET expression is lost in papillary thyroid carcinoma cell line, TPC1, which lacks the G-quadruplex forming sequence on the RET promoter region due to chromosomal rearrangement. Furthermore, the RET downregulation by berberine results in the inhibition of TT-cell proliferation through cell-cycle arrest and activation of apoptosis. Overall, these data strongly suggest that the formation of G-quadruplex structure and its stabilization by small molecules is an ideal approach for the transcriptional repression of the RET oncogene. In continuation of our study, we next explored for more drug-like small molecules as potential RET G-quadruplex stabilizers that could be used for MTC patients in a clinical setting. We identified the anti-cancer drug, ellipticine, and its derivatives as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that one of the ellipticine analogs, NSC311153 that incorporates a ethylpiperidine group, showed improved binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound possessed selective inhibitory effect on RET transcription in TT cell line over the TPC1 cells. The RET downregulation by NSC311153 selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In this study, we also showed that the systemic administration of a structural analog of NSC311153 in mice, bearing MTC xenografts, results in the inhibition of tumor growth through RET downregulation. Overall, our study supports the rationale for the development of RET G-quadruplex stabilizing-based therapeutic approaches for the treatment of progressive MTC.Release after 01-Oct-201

Demonstration of a potent RET transcriptional inhibitor for the treatment of medullary thyroid carcinoma based on an ellipticine derivative

Dominant-activating mutations in the RET (rear-ranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, is often associated with the development of medullary thyroid carcinoma (MTC). The proximal promoter region of the RET gene consists of a guanine-rich sequence containing five runs of three consecutive guanine residues that serve as the binding site for transcriptional factors. As we have recently shown, this stretch of nucleotides in the promoter region is highly dynamic in nature and tend to form non-B DNA secondary structures called G-quadruplexes, which suppress the transcription of the RET gene. In the present study, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that the incorporation of a piperidine ring in an ellipticine derivative, NSC311153 improves its binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound also interfered with the transcriptional mechanism of the RET gene in an MTC derived cell line, TT cells and significantly decreased the endogenous RET protein expression. We demonstrated the specificity of NSC311153 by using papillary thyroid carcinoma (PTC) cells, the TPC1 cell line which lacks the G-quadruplex forming sequence in the promoter region due to chromosomal rearrangement. The RET downregulation selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In the present study, we also showed that the systemic administration of a water soluble NSC311153 analog in a mouse MTC xenograft model inhibited the tumor growth through RET downregulation.6 month embargo; Published online: May 11, 2017This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Power quality analysis in electrical drives and a case study of artificial intelligence prediction algorithm for fault deterrent electrical drives

Since electrical drives have become an integral element of any industrial sector, power quality difficulties have been well expected, and delivering genuine quality of the same has proven to be a difficult challenge. Since power quality relies on load side non-linearity and high semiconductor technology consumption, it is a serious concern. The efficiency of the drive segment employed in the sector is increasingly becoming a topic of discussion in today’s market. Numerous reviews of available literature have found problems with the load side as well as with electrical drive proficiency, as a result of the issues listed above. A high level of power quality vulnerability is simply too much. Even the most advanced technology has its limits when it comes to drive operation. Research on the grid-side quality issues of electrical drives is the focus of this article. After field testing of grid power quality, each parametric analysis is performed to identify crucial parameters that can cause industrial drives to fail. Based on this discovery, a machine learning strategy was developed and an artificial intelligence technique was proposed to administer the fault deterrent prediction algorithm. An accurate forecast of anomalous behavior on the grid side ensures safe and dependable grid operation such that shutdown or failure probability is minimized to a greater extent by the results. Additional information gleaned from historical data will prove useful to equipment manufacturers in the future, providing a solution to this problem

Salinomycin and its derivatives as potent RET transcriptional inhibitors for the treatment of medullary thyroid carcinoma

Rearranged during transfection kinase (RET) is a validated molecular target in medullary thyroid cancer (MTC), as activating mutations in RET are often associated with the development of MTC. The present study reports the first preclinical characterization of salinomycin and selected analogs as potent RET transcriptional inhibitors. Reverse transcription‑PCR and immunoblotting revealed that salinomycin profoundly decreased RET expression in the TT human MTC cell line by inhibiting RET transcription. Moreover, salinomycin resulted in remarkable anti‑proliferative activity against MTC that is driven by RET (gain of function mutation) by selectively inhibiting the intracellular PI3K/Akt/mTOR signaling pathway. Also, flow cytometry and fluorescence‑activated cell sorting showed that salinomycin induces G1 phase arrest and apoptosis by reducing the expression of retinoblastoma protein, E2F1, cyclin D and CDK4. The structure‑activity relationship of salinomycin was investigated in this study. Some of the salinomycin derivatives showed the ability to reduce RET expression where others fail to alter RET expression. These results suggest that the RET‑suppressing effect of salinomycin may be largely attributed to disruption of the Wnt pathway, presumably through interference with the ternary LRP6‑Frizzled‑Wnt complex. Furthermore, these findings support the further preclinical evaluation of salinomycin and its analogs as a promising new class of therapeutic agents for the improved treatment of MTC.American Cancer Society [RSGM-12-046-01-CDD]6 month embargo; published online: 20 November 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine

BACKGROUND: The gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs). Thus, the RET protein has been explored as an excellent target for progressive and advanced MTC. In this study we have demonstrated a therapeutic strategy for MTC by suppressing the transcription of RET proto-oncogene though the stabilization of G-quadruplex structure formed on the promoter region of this gene using a natural product berberine. METHODS: Medullary thyroid carcinoma (MTC) TT cell line has been used to evaluate the effects of berberine on RET expression and its downstream signaling pathways. The specificity of berberine was demonstrated by using the papillary thyroid carcinoma TPC1 cell line, which lacks the G-quadruplex forming sequence on the RET promoter region due to chromosomal rearrangement. RESULTS: Berberine suppressed the RET expression by more than 90 % in MTC TT cells at a concentration of 2.5 μg/ml with minimal effect on the TPC1 cells. Canadine, which is a structural analogue of berberine, showed little interaction with RET G-quadruplex and also had no effect on RET expression in MTC TT cells. The down-regulation of RET with berberine further inhibited the cell proliferation through cell cycle arrest and activation of apoptosis in TT cells, which was confirmed by a 2-fold increase in the caspase-3 activity and the down-regulation of cell-cycle regulatory proteins. CONCLUSION: Our data strongly suggest that the G-quadruplex forming region and the stabilization of this structure play a critical role in mediating the repressive effect of berberine on RET transcription.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]