Publications by authors named "Kiran Kodali"

10 Publications

  • Page 1 of 1

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

J Med Chem 2021 06 27;64(11):7296-7311. Epub 2021 May 27.

Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 and , and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201443PMC
June 2021

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry.

J Vis Exp 2020 08 18(162). Epub 2020 Aug 18.

Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital; Center for Proteomics and Metabolomics, St. Jude Children's Research Hospital;

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.
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http://dx.doi.org/10.3791/61684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752892PMC
August 2020

Toxicoproteomic Profiling of Transgenic Mice Treated with Rifampicin and Isoniazid.

Cells 2020 07 9;9(7). Epub 2020 Jul 9.

Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Tuberculosis is a global health threat that affects millions of people every year, and treatment-limiting toxicity remains a considerable source of treatment failure. Recent reports have characterized the nature of -mediated hepatotoxicity and the systemic toxicity of antitubercular drugs. The antitubercular drug isoniazid plays a role in such pathologic states as acute intermittent porphyria, anemia, hepatotoxicity, hypercoagulable states (deep vein thrombosis, pulmonary embolism, or ischemic stroke), pellagra (vitamin B deficiency), peripheral neuropathy, and vitamin B deficiency. However, the mechanisms by which isoniazid administration leads to these states are unclear. To elucidate the mechanism of rifampicin- and isoniazid-induced liver and systemic injury, we performed tandem mass tag mass spectrometry-based proteomic screening of and mice treated with combinations of rifampicin and isoniazid. Proteomic profiling analysis suggested that the liver proteome is affected by antitubercular therapy to disrupt [Fe-S] cluster assembly machinery, [2Fe-2S] cluster-containing proteins, cytochrome P450 enzymes, heme biosynthesis, homocysteine catabolism, oxidative stress responses, vitamin B metabolism, and vitamin B metabolism. These novel findings provide insight into the etiology of some of these processes and potential targets for subsequent investigations. Data are available via ProteomeXchange with identifier PXD019505.
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http://dx.doi.org/10.3390/cells9071654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407182PMC
July 2020

A Cancer-Specific Ubiquitin Ligase Drives mRNA Alternative Polyadenylation by Ubiquitinating the mRNA 3' End Processing Complex.

Mol Cell 2020 03 21;77(6):1206-1221.e7. Epub 2020 Jan 21.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address:

Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.
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http://dx.doi.org/10.1016/j.molcel.2019.12.022DOI Listing
March 2020

Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability.

EMBO Rep 2019 07 24;20(7):e47352. Epub 2019 May 24.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Melanoma antigen genes (MAGEs) are emerging as important oncogenic drivers that are normally restricted to expression in male germ cells but are aberrantly expressed in cancers and promote tumorigenesis. Mechanistically, MAGEs function as substrate specifying subunits of E3 ubiquitin ligases. Thus, the activation of germline-specific genes in cancer can drive metabolic and signaling pathways through altered ubiquitination to promote tumorigenesis. However, the mechanisms regulating MAGE expression and activity are unclear. Here, we describe how the MAGE-A3/6 proteins that function as repressors of autophagy are downregulated in response to nutrient deprivation. Short-term cellular starvation promotes rapid MAGE-A3/6 degradation in a proteasome-dependent manner. Proteomic analysis reveals that degradation of MAGE-A3/6 is controlled by the CRL4-DCAF12 E3 ubiquitin ligase. Importantly, the degradation of MAGE-A3/6 by CRL4-DCAF12 is required for starvation-induced autophagy. These findings suggest that oncogenic MAGEs can be dynamically controlled in response to stress to allow cellular adaptation, autophagy regulation, and tumor growth and that CRL4-DCAF12 activity is responsive to nutrient status.
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http://dx.doi.org/10.15252/embr.201847352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607007PMC
July 2019

Mutant LRRK2 mediates peripheral and central immune responses leading to neurodegeneration in vivo.

Brain 2018 06;141(6):1753-1769

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Blvd, Memphis TN 38105, USA.

Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene result in late-onset Parkinson's disease. The incomplete penetrance of LRRK2 mutations in humans and LRRK2 murine models of Parkinson's disease suggests that the disease may result from a complex interplay of genetic predispositions and persistent exogenous insults. Since neuroinflammation is commonly associated with the pathogenesis of Parkinson's disease, we examine a potential role of mutant LRRK2 in regulation of the immune response and inflammatory signalling in vivo. Here, we show that mice overexpressing human pathogenic LRRK2 mutations, but not wild-type mice or mice overexpressing human wild-type LRRK2 exhibit long-term lipopolysaccharide-induced nigral neuronal loss. This neurodegeneration is accompanied by an exacerbated neuroinflammation in the brain. The increased immune response in the brain of mutant mice subsequently has an effect on neurons by inducing intraneuronal LRRK2 upregulation. However, the enhanced neuroinflammation is unlikely to be triggered by dysfunctional microglia or infiltrated T cells and/or monocytes, but by peripheral circulating inflammatory molecules. Analysis of cytokine kinetics and inflammatory pathways in the peripheral immune cells demonstrates that LRRK2 mutation alters type II interferon immune response, suggesting that this increased neuroinflammatory response may arise outside the central nervous system. Overall, this study suggests that peripheral immune signalling plays an unexpected-but important-role in the regulation of neurodegeneration in LRRK2-associated Parkinson's disease, and provides new targets for interfering with the onset and progression of the disease.
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http://dx.doi.org/10.1093/brain/awy077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190032PMC
June 2018

Extensive Peptide Fractionation and y Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

Anal Chem 2017 03 22;89(5):2956-2963. Epub 2017 Feb 22.

Heilongjiang University of Chinese Medicine , Harbin, Heilongjiang 150040, China.

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.
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http://dx.doi.org/10.1021/acs.analchem.6b04415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467445PMC
March 2017

Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia.

Cancer Cell 2015 Sep 27;28(3):343-56. Epub 2015 Aug 27.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2C4, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5G 1L7, Canada.

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.
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http://dx.doi.org/10.1016/j.ccell.2015.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573904PMC
September 2015

Quantitative protein analysis by mass spectrometry.

Methods Mol Biol 2015 ;1278:281-305

St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

Mass spectrometry is one of the most sensitive methods in analytical chemistry, and its application in proteomics has been rapidly expanded after sequencing the human genome. Mass spectrometry is now the mainstream approach for identification and quantification of proteins and posttranslational modifications, either in small scale or in the entire proteome. Shotgun proteomics can analyze up to 10,000 proteins in a comprehensive study, with detection sensitivity in the picogram range. In this chapter, we describe major experimental steps in a shotgun proteomics platform, including sample preparation in the context of studying protein-protein interaction, mass spectrometric data acquisition, and database search to identify proteins and posttranslational modification analysis. Proteome quantification strategies and bioinformatics analysis are also illustrated. Finally, we discuss the capabilities, limitations, and potential improvements of current platforms.
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http://dx.doi.org/10.1007/978-1-4939-2425-7_17DOI Listing
December 2015

HECT-E3 ligase ETC-1 regulates securin and cyclin B1 cytoplasmic abundance to promote timely anaphase during meiosis in C. elegans.

Development 2013 May 11;140(10):2149-59. Epub 2013 Apr 11.

Department of Molecular Pharmacology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

The anaphase inhibitor securin plays a crucial role in regulating the timing of sister chromatid separation during mitosis. When sister chromatid pairs become bioriented, the E3 ligase anaphase promoting complex/cyclosome (APC/C) ubiquitylates securin for proteolysis, triggering sister chromatid separation. Securin is also implicated in regulating meiotic progression. Securin protein levels change sharply during cell cycle progression, enabling its timely action. To understand the mechanism underlying the tightly regulated dynamics of securin, we analyzed the subcellular localization of the securin IFY-1 during C. elegans development. IFY-1 was highly expressed in the cytoplasm of germ cells. The cytoplasmic level of IFY-1 declined immediately following meiosis I division and remained low during meiosis II and following mitoses. We identified a C. elegans homolog of another type of E3 ligase, UBE3C, designated ETC-1, as a regulator of the cytoplasmic IFY-1 level. RNAi-mediated depletion of ETC-1 stabilized IFY-1 and CYB-1 (cyclin B1) in post-meiosis I embryos. ETC-1 knockdown in a reduced APC function background caused an embryonic lethal phenotype. In vitro, ETC-1 ubiquitylates IFY-1 and CYB-1 in the presence of the E2 enzyme UBC-18, which functions in pharyngeal development. Genetic analysis revealed that UBC-18 plays a distinct role together with ETC-1 in regulating the cytoplasmic level of IFY-1 during meiosis. Our study reports a novel mechanism, mediated by ETC-1, that co-operates with APC/C to maintain the meiotic arrest required for proper cell cycle timing during reproduction.
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http://dx.doi.org/10.1242/dev.090688DOI Listing
May 2013
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