DOCSLIB.ORG

Clinical, Molecular, and Immune Analysis of Dabrafenib-Trametinib

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Supplementary Online Content Chen G, McQuade JL, Panka DJ, et al. Clinical, molecular and immune analysis of dabrafenib-trametinib combination treatment for metastatic melanoma that progressed during BRAF inhibitor monotherapy: a phase 2 clinical trial. JAMA Oncology. Published online April 28, 2016. doi:10.1001/jamaoncol.2016.0509. eMethods. eReferences. eTable 1. Clinical efficacy eTable 2. Adverse events eTable 3. Correlation of baseline patient characteristics with treatment outcomes eTable 4. Patient responses and baseline IHC results eFigure 1. Kaplan-Meier analysis of overall survival eFigure 2. Correlation between IHC and RNAseq results eFigure 3. pPRAS40 expression and PFS eFigure 4. Baseline and treatment-induced changes in immune infiltrates eFigure 5. PD-L1 expression eTable 5. Nonsynonymous mutations detected by WES in baseline tumors This supplementary material has been provided by the authors to give readers additional information about their work. © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 eMethods Whole exome sequencing Whole exome capture libraries for both tumor and normal samples were constructed using 100ng genomic DNA input and following the protocol as described by Fisher et al.,3 with the following adapter modification: Illumina paired end adapters were replaced with palindromic forked adapters with unique 8 base index sequences embedded within the adapter. In-solution hybrid selection was performed using the Illumina Rapid Capture Exome enrichment kit with 38Mb target territory (29Mb baited). The targeted region includes 98.3% of the intervals in the Refseq exome database. Dual-indexed libraries were pooled into groups of up to 96 samples prior to hybridization. The enriched library pools were quantified via PicoGreen after elution from streptavadin beads and then normalized to a range compatible with sequencing template denature protocols. Libraries were sequenced using 76-bp paired-end reads. Output from Illumina software was processed by the Broad Picard data-processing pipeline to yield BAM files containing well-calibrated, aligned reads. Cross-contamination between samples from other individuals was monitored with the ContEst algorithm.4 Somatic single-nucleotide variants in targeted exons were identified with the MuTect algorithm,5 and small insertions or deletions with Indelocator (http://www.broadinstitute.org/cancer/cga/indelocator). Alterations were annotated using Oncotator (http://www.broadinstitute.org/cancer/cga/oncotator) and manually reviewed in the Integrative Genomics Viewer (IGV).6 Statistical analysis of recurrently mutated genes was performed using MutSig.7 Copy number aberrations were quantified using ReCapSeg (http://gatkforums.broadinstitute.org/discussion/5640/recapseg-overview), and reported for each gene as the segmented normalized log2-transformed copy ratio between each tumor sample and a panel of normal samples. Transcriptome sequencing Total RNA was quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 5ng/ul. To generate transcriptome libraries, an automated variant method of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit was used, which preserves strand orientation of the RNA transcript. Briefly, 200ng of total RNA were used for each tumor sample. Poly(A)+ mRNA was isolated using oligo(dT) beads, followed by heat fragmentation and cDNA synthesis. cDNA libraries were then prepared (end repair, base ‘A’ addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment, the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. Pooled libraries were normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using either the HiSeq 2000 or HiSeq 2500. Each run was a 101bp paired-end with an eight-base index barcode read. Data was analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation. Abundances of transcripts, expressed as fragments per kilobase of exon per million (FPKM, upper quartile normalized), were determined using RSEM.8 Immunohistochemistry IHC on FFPE tumor tissues was performed using antibodies against BRAFV600E (Ventana 790-4855), pMAPK(ERK1/2) (Cell Signaling 4376), pS6 (Cell Signaling 4858), pPRAS40 (Cell Signaling 13175), CD8 (Leica PA0183), and PD-L1 (Cell Signaling 13684). © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 Circulating BRAF Peripheral blood mononuclear cells (PBMC) isolated from blood were subjected to total RNA isolation using mirVana™ kit (AM1560). Amount of circulating BRAFV600 was determined from PBMC-derived RNA using RT-PCR as previously described.9 Briefly, wildtype BRAF sequence was digested away by a restrictive endonuclease (TspR1) that preferably digests wildtype BRAF at codon V600 but not BRAFV600, allowing for the detection of BRAFV600 with a BRAFV600-specific RT-PCR assay. © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 eReferences 1. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). European journal of cancer. Jan 2009;45(2):228-247. 2. Trotti A, Colevas AD, Setser A, et al. CTCAE v3.0: development of a comprehensive grading system for the adverse effects of cancer treatment. Seminars in radiation oncology. Jul 2003;13(3):176-181. 3. Fisher S, Barry A, Abreu J, et al. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome biology. 2011;12(1):R1. 4. Cibulskis K, McKenna A, Fennell T, Banks E, DePristo M, Getz G. ContEst: estimating cross-contamination of human samples in next-generation sequencing data. Bioinformatics. Sep 15 2011;27(18):2601-2602. 5. Cibulskis K, Lawrence MS, Carter SL, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature biotechnology. Mar 2013;31(3):213-219. 6. Robinson JT, Thorvaldsdottir H, Winckler W, et al. Integrative genomics viewer. Nature biotechnology. Jan 2011;29(1):24-26. 7. Lawrence MS, Stojanov P, Polak P, et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature. Jul 11 2013;499(7457):214-218. 8. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics. 2011;12:323. 9. Panka DJ, Buchbinder E, Giobbie-Hurder A, et al. Clinical utility of a blood-based BRAF(V600E) mutation assay in melanoma. Molecular cancer therapeutics. Dec 2014;13(12):3210-3218. © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 eTable 1 Clinical efficacy Confirmed Response No. % CR 0 0 PR 2 9 SD 7 30 PD 11 48 NE 3 13 © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 eTable 2 Adverse events All Grades Grade III/IV AE Number % Number % Any event 24 10 17 71 Fever 12 50 0 0 Nausea/vomiting 12 50 0 0 Fatigue 9 38 0 0 Chills 7 29 0 0 Dry mouth 6 25 0 0 Headache 6 25 0 0 Arthralgias 6 25 1 4 Anorexia 5 21 0 0 Constipation 5 21 0 0 Rash 5 21 0 0 Hot flashes 4 17 0 0 Actinic keratosis 0 0 0 0 Cutaneous SCC 0 0 0 0 Decreased ejection fraction 2 8 1 4 Blurred vision 1 4 0 0 Chorioretinopathy 0 0 0 0 Note: Adverse events occurring in >15% of patients and events of special interest © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 eTable 3 Correlation of baseline patient characteristics with treatment outcomes Variable PFS P-Value P-Value Hazard Ratio Disease Control Odds Ratio Age 0.99 0.36 1.32 0.72 Male Sex 0.99 0.98 6.67 0.12 Duration of Prior BRAFi 0.29 0.01 21.33 0.015 >6 months ECOG Performance Score 1- 1.05 0.92 0.60 0.58 2 Disease Stage 0.96 0.93 1.40 0.71 IIIC-M1B Mutation 0.99 0.99 2.86 0.42 V600K/R Baseline LDH 1.75 0.21 0.50 0.50 Elevated Time Lapse from Prior BRAFi 0.70 0.53 1.29 0.82 >0 months BRAFi Best Response 2.10 0.16 0 0.74 SD/PD MORs: Only MAPK Pathway 0.42 0.19 3.75 0.32 Reactivation © 2016 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 eTable 4 Patient responses and baseline IHC results Nu Pati PFS MAPK MAPK pPRAS4 pPRAS4 pS6 pS6 DC? mbe ent (weeks score class 0 score 0 class score class (1=yes, r ) 0=no) 1 Pt0 33.3 -0.06 Low 0.8 Low -1.0 Low 1 1 2 Pt0 12.7 0.30 High 0 Low -1.2 Low 1 2 3 Pt0 8.6 0 3 4 Pt0 19.4 5 5 Pt0 7.9 -0.61 Low 1.2 High 0.4 High 0 8 6 Pt0 3.9 0.67 High 2 High -0.7 Low 0 9 7 Pt1 2.3 1.07 High 2.7 High 1.5 High 0 8 Pt1 5.4 0 1 9 Pt1 15.0 1.31 High 0.2 Low 1.1 High 1 2 10 Pt1 7.6 1.51 High 1.6 High 0 3 11 Pt1 12.9 -0.55 Low 1 4 12 Pt1 32.0 1 5 13 Pt1 15.9 0.71 High 1 7 14 Pt1 35.9 0.51 High 1 8 15 Pt1 3.4 -0.47 Low 0.05 Low -0.8 Low 0 9 16 Pt2 15.9 0.67 High 0.8 High 0 0 17 Pt2 15.9 0.67 High -0.9 Low 0 1 18 Pt2 16.3 1 2 19 Pt2 23.6 1 3 20 Pt2 0.6 4 21 Pt2 7.9 0.48 High 1.1 High 0 6 22 Pt2 11.9 1.51 Low 1.3 High 0 7 23 Pt2 15.9 0 8 Note: Missing value= not evaluable, DC=Disease control © 2016 American Medical Association.
Recommended publications
  • Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling

    Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling

    G C A T T A C G G C A T genes Review Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling Aoife A. Nolan 1, Nourhan K. Aboud 1, Walter Kolch 1,2,* and David Matallanas 1,* 1 Systems Biology Ireland, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland; [email protected] (A.A.N.); [email protected] (N.K.A.) 2 Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland * Correspondence: [email protected] (W.K.); [email protected] (D.M.) Abstract: Oncogenic RAS (Rat sarcoma) mutations drive more than half of human cancers, and RAS inhibition is the holy grail of oncology. Thirty years of relentless efforts and harsh disappointments have taught us about the intricacies of oncogenic RAS signalling that allow us to now get a pharma- cological grip on this elusive protein. The inhibition of effector pathways, such as the RAF-MEK-ERK pathway, has largely proven disappointing. Thus far, most of these efforts were aimed at blocking the activation of ERK. Here, we discuss RAF-dependent pathways that are regulated through RAF functions independent of catalytic activity and their potential role as targets to block oncogenic RAS signalling. We focus on the now well documented roles of RAF kinase-independent functions in apoptosis, cell cycle progression and cell migration. Keywords: RAF kinase-independent; RAS; MST2; ASK; PLK; RHO-α; apoptosis; cell cycle; cancer therapy Citation: Nolan, A.A.; Aboud, N.K.; Kolch, W.; Matallanas, D. Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling.
  • ACVR1 Antibody Cat

    ACVR1 Antibody Cat

    ACVR1 Antibody Cat. No.: 4791 Western blot analysis of ACVR1 in A549 cell lysate with ACVR1 antibody at 1 μg/mL in (A) the absence and (B) the presence of blocking peptide. Specifications HOST SPECIES: Rabbit SPECIES REACTIVITY: Human, Mouse HOMOLOGY: Predicted species reactivity based on immunogen sequence: Bovine: (100%), Rat: (93%) ACVR1 antibody was raised against a 14 amino acid synthetic peptide near the amino terminus of the human ACVR1. IMMUNOGEN: The immunogen is located within the first 50 amino acids of ACVR1. TESTED APPLICATIONS: ELISA, WB ACVR1 antibody can be used for detection of ACVR1 by Western blot at 1 μg/mL. APPLICATIONS: Antibody validated: Western Blot in human samples. All other applications and species not yet tested. At least four isoforms of ACVR1 are known to exist. This antibody is predicted to have no SPECIFICITY: cross-reactivity to ACVR1B or ACVR1C. POSITIVE CONTROL: 1) Cat. No. 1203 - A549 Cell Lysate Properties October 1, 2021 1 https://www.prosci-inc.com/acvr1-antibody-4791.html PURIFICATION: ACVR1 Antibody is affinity chromatography purified via peptide column. CLONALITY: Polyclonal ISOTYPE: IgG CONJUGATE: Unconjugated PHYSICAL STATE: Liquid BUFFER: ACVR1 Antibody is supplied in PBS containing 0.02% sodium azide. CONCENTRATION: 1 mg/mL ACVR1 antibody can be stored at 4˚C for three months and -20˚C, stable for up to one STORAGE CONDITIONS: year. As with all antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures. Additional Info OFFICIAL SYMBOL: ACVR1 ACVR1 Antibody: FOP, ALK2, SKR1, TSRI, ACTRI, ACVR1A, ACVRLK2, Activin receptor type-1, ALTERNATE NAMES: Activin receptor type I, ACTR-I ACCESSION NO.: NP_001096 PROTEIN GI NO.: 4501895 GENE ID: 90 USER NOTE: Optimal dilutions for each application to be determined by the researcher.
  • The Mineralocorticoid Receptor Leads to Increased Expression of EGFR

    The Mineralocorticoid Receptor Leads to Increased Expression of EGFR

    www.nature.com/scientificreports OPEN The mineralocorticoid receptor leads to increased expression of EGFR and T‑type calcium channels that support HL‑1 cell hypertrophy Katharina Stroedecke1,2, Sandra Meinel1,2, Fritz Markwardt1, Udo Kloeckner1, Nicole Straetz1, Katja Quarch1, Barbara Schreier1, Michael Kopf1, Michael Gekle1 & Claudia Grossmann1* The EGF receptor (EGFR) has been extensively studied in tumor biology and recently a role in cardiovascular pathophysiology was suggested. The mineralocorticoid receptor (MR) is an important efector of the renin–angiotensin–aldosterone‑system and elicits pathophysiological efects in the cardiovascular system; however, the underlying molecular mechanisms are unclear. Our aim was to investigate the importance of EGFR for MR‑mediated cardiovascular pathophysiology because MR is known to induce EGFR expression. We identifed a SNP within the EGFR promoter that modulates MR‑induced EGFR expression. In RNA‑sequencing and qPCR experiments in heart tissue of EGFR KO and WT mice, changes in EGFR abundance led to diferential expression of cardiac ion channels, especially of the T‑type calcium channel CACNA1H. Accordingly, CACNA1H expression was increased in WT mice after in vivo MR activation by aldosterone but not in respective EGFR KO mice. Aldosterone‑ and EGF‑responsiveness of CACNA1H expression was confrmed in HL‑1 cells by Western blot and by measuring peak current density of T‑type calcium channels. Aldosterone‑induced CACNA1H protein expression could be abrogated by the EGFR inhibitor AG1478. Furthermore, inhibition of T‑type calcium channels with mibefradil or ML218 reduced diameter, volume and BNP levels in HL‑1 cells. In conclusion the MR regulates EGFR and CACNA1H expression, which has an efect on HL‑1 cell diameter, and the extent of this regulation seems to depend on the SNP‑216 (G/T) genotype.
  • Aquaporin Channels in the Heart—Physiology and Pathophysiology

    Aquaporin Channels in the Heart—Physiology and Pathophysiology

    International Journal of Molecular Sciences Review Aquaporin Channels in the Heart—Physiology and Pathophysiology Arie O. Verkerk 1,2,* , Elisabeth M. Lodder 2 and Ronald Wilders 1 1 Department of Medical Biology, Amsterdam University Medical Centers, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; [email protected] 2 Department of Experimental Cardiology, Amsterdam University Medical Centers, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; [email protected] * Correspondence: [email protected]; Tel.: +31-20-5664670 Received: 29 March 2019; Accepted: 23 April 2019; Published: 25 April 2019 Abstract: Mammalian aquaporins (AQPs) are transmembrane channels expressed in a large variety of cells and tissues throughout the body. They are known as water channels, but they also facilitate the transport of small solutes, gasses, and monovalent cations. To date, 13 different AQPs, encoded by the genes AQP0–AQP12, have been identified in mammals, which regulate various important biological functions in kidney, brain, lung, digestive system, eye, and skin. Consequently, dysfunction of AQPs is involved in a wide variety of disorders. AQPs are also present in the heart, even with a specific distribution pattern in cardiomyocytes, but whether their presence is essential for proper (electro)physiological cardiac function has not intensively been studied. This review summarizes recent findings and highlights the involvement of AQPs in normal and pathological cardiac function. We conclude that AQPs are at least implicated in proper cardiac water homeostasis and energy balance as well as heart failure and arsenic cardiotoxicity. However, this review also demonstrates that many effects of cardiac AQPs, especially on excitation-contraction coupling processes, are virtually unexplored.
  • Structural Characterization of Polysaccharides from Cordyceps Militaris and Their Hypolipidemic Effects Cite This: RSC Adv.,2018,8,41012 in High Fat Diet Fed Mice†

    Structural Characterization of Polysaccharides from Cordyceps Militaris and Their Hypolipidemic Effects Cite This: RSC Adv.,2018,8,41012 in High Fat Diet Fed Mice†

    RSC Advances View Article Online PAPER View Journal | View Issue Structural characterization of polysaccharides from Cordyceps militaris and their hypolipidemic effects Cite this: RSC Adv.,2018,8,41012 in high fat diet fed mice† Zhen-feng Huang, ‡ Ming-long Zhang,‡ Song Zhang,* Ya-hui Wang and Xue-wen Jiang Cordyceps militaris is a crude dietary therapeutic mushroom with high nutritional and medicinal values. Mushroom-derived polysaccharides have been found to possess antihyperglycemic and antihyperlipidemic activities. This study aimed to partially clarify the structural characterization and comparatively evaluate hypolipidemic potentials of intracellular- (IPCM) and extracellular polysaccharides of C. militaris (EPCM) in high fat diet fed mice. Results indicated that IPCM-2 is a-pyran polysaccharide with an average molecular weight of 32.5 kDa, was mainly composed of mannose, glucose and galactose with mass percentages of 51.94%, 10.54%, and 37.25%, respectively. EPCM-2 is an a-pyran Creative Commons Attribution 3.0 Unported Licence. polysaccharide with an average molecular weight of 20 kDa that is mainly composed of mannose, glucose and galactose with mass percentages of 44.51%, 18.33%, and 35.38%, respectively. In in vivo study, EPCM-1 treatment (100 mg kgÀ1 dÀ1) showed potential effects on improving serum lipid profiles of hyperlipidemic mice, reflected by decreasing serum total cholesterol (TC), triglyceride (TG) and low density lipoprotein-cholesterol (LDL-C) levels by 20.05%, 45.45% and 52.63%, respectively, while IPCM-1 treatment
  • Table S1 the Four Gene Sets Derived from Gene Expression Profiles of Escs and Differentiated Cells

    Table S1 the Four Gene Sets Derived from Gene Expression Profiles of Escs and Differentiated Cells

    Table S1 The four gene sets derived from gene expression profiles of ESCs and differentiated cells Uniform High Uniform Low ES Up ES Down EntrezID GeneSymbol EntrezID GeneSymbol EntrezID GeneSymbol EntrezID GeneSymbol 269261 Rpl12 11354 Abpa 68239 Krt42 15132 Hbb-bh1 67891 Rpl4 11537 Cfd 26380 Esrrb 15126 Hba-x 55949 Eef1b2 11698 Ambn 73703 Dppa2 15111 Hand2 18148 Npm1 11730 Ang3 67374 Jam2 65255 Asb4 67427 Rps20 11731 Ang2 22702 Zfp42 17292 Mesp1 15481 Hspa8 11807 Apoa2 58865 Tdh 19737 Rgs5 100041686 LOC100041686 11814 Apoc3 26388 Ifi202b 225518 Prdm6 11983 Atpif1 11945 Atp4b 11614 Nr0b1 20378 Frzb 19241 Tmsb4x 12007 Azgp1 76815 Calcoco2 12767 Cxcr4 20116 Rps8 12044 Bcl2a1a 219132 D14Ertd668e 103889 Hoxb2 20103 Rps5 12047 Bcl2a1d 381411 Gm1967 17701 Msx1 14694 Gnb2l1 12049 Bcl2l10 20899 Stra8 23796 Aplnr 19941 Rpl26 12096 Bglap1 78625 1700061G19Rik 12627 Cfc1 12070 Ngfrap1 12097 Bglap2 21816 Tgm1 12622 Cer1 19989 Rpl7 12267 C3ar1 67405 Nts 21385 Tbx2 19896 Rpl10a 12279 C9 435337 EG435337 56720 Tdo2 20044 Rps14 12391 Cav3 545913 Zscan4d 16869 Lhx1 19175 Psmb6 12409 Cbr2 244448 Triml1 22253 Unc5c 22627 Ywhae 12477 Ctla4 69134 2200001I15Rik 14174 Fgf3 19951 Rpl32 12523 Cd84 66065 Hsd17b14 16542 Kdr 66152 1110020P15Rik 12524 Cd86 81879 Tcfcp2l1 15122 Hba-a1 66489 Rpl35 12640 Cga 17907 Mylpf 15414 Hoxb6 15519 Hsp90aa1 12642 Ch25h 26424 Nr5a2 210530 Leprel1 66483 Rpl36al 12655 Chi3l3 83560 Tex14 12338 Capn6 27370 Rps26 12796 Camp 17450 Morc1 20671 Sox17 66576 Uqcrh 12869 Cox8b 79455 Pdcl2 20613 Snai1 22154 Tubb5 12959 Cryba4 231821 Centa1 17897
  • Iron Depletion Reduces Abce1 Transcripts While Inducing The

    Iron Depletion Reduces Abce1 Transcripts While Inducing The

    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 October 2019 doi:10.20944/preprints201910.0252.v1 1 Research Article 2 Iron depletion Reduces Abce1 Transcripts While 3 Inducing the Mitophagy Factors Pink1 and Parkin 4 Jana Key 1,2, Nesli Ece Sen 1, Aleksandar Arsovic 1, Stella Krämer 1, Robert Hülse 1, Suzana 5 Gispert-Sanchez 1 and Georg Auburger 1,* 6 1 Experimental Neurology, Goethe University Medical School, 60590 Frankfurt am Main; 7 2 Faculty of Biosciences, Goethe-University Frankfurt am Main, Germany 8 * Correspondence: [email protected] 9 10 Abstract: Lifespan extension was recently achieved in Caenorhabditis elegans nematodes by 11 mitochondrial stress and mitophagy, triggered via iron depletion. Conversely in man, deficient 12 mitophagy due to Pink1/Parkin mutations triggers iron accumulation in patient brain and limits 13 survival. We now aimed to identify murine fibroblast factors, which adapt their mRNA expression 14 to acute iron manipulation, relate to mitochondrial dysfunction and may influence survival. After 15 iron depletion, expression of the plasma membrane receptor Tfrc with its activator Ireb2, the 16 mitochondrial membrane transporter Abcb10, the heme-release factor Pgrmc1, the heme- 17 degradation enzyme Hmox1, the heme-binding cholesterol metabolizer Cyp46a1, as well as the 18 mitophagy regulators Pink1 and Parkin showed a negative correlation to iron levels. After iron 19 overload, these factors did not change expression. Conversely, a positive correlation of mRNA levels 20 with both conditions of iron availability was observed for the endosomal factors Slc11a2 and Steap2, 21 as well as for the iron-sulfur-cluster (ISC)-containing factors Ppat, Bdh2 and Nthl1.
  • CDK4/6 Inhibitors in Melanoma: a Comprehensive Review

    CDK4/6 Inhibitors in Melanoma: a Comprehensive Review

    cells Review CDK4/6 Inhibitors in Melanoma: A Comprehensive Review Mattia Garutti 1,*, Giada Targato 2 , Silvia Buriolla 2 , Lorenza Palmero 1,2 , Alessandro Marco Minisini 2 and Fabio Puglisi 1,2 1 CRO Aviano National Cancer Institute IRCCS, 33081 Aviano, Italy; [email protected] (L.P.); [email protected] (F.P.) 2 Department of Medicine (DAME), University of Udine, 33100 Udine, Italy; [email protected] (G.T.); [email protected] (S.B.); [email protected] (A.M.M.) * Correspondence: [email protected] Abstract: Historically, metastatic melanoma was considered a highly lethal disease. However, recent advances in drug development have allowed a significative improvement in prognosis. In particular, BRAF/MEK inhibitors and anti-PD1 antibodies have completely revolutionized the management of this disease. Nonetheless, not all patients derive a benefit or a durable benefit from these therapies. To overtake this challenges, new clinically active compounds are being tested in the context of clinical trials. CDK4/6 inhibitors are drugs already available in clinical practice and preliminary evidence showed a promising activity also in melanoma. Herein we review the available literature to depict a comprehensive landscape about CDK4/6 inhibitors in melanoma. We present the molecular and genetic background that might justify the usage of these drugs, the preclinical evidence, the clinical available data, and the most promising ongoing clinical trials. Keywords: CDK4/6; CDK4; CDK6; melanoma; Palbociclib; Ribociclib; Abemaciclib Citation: Garutti, M.; Targato, G.; Buriolla, S.; Palmero, L.; Minisini, A.M.; Puglisi, F. CDK4/6 Inhibitors in Melanoma: A Comprehensive 1. Introduction Review. Cells 2021, 10, 1334.
  • Human Periprostatic Adipose Tissue: Secretome from Patients With

    Human Periprostatic Adipose Tissue: Secretome from Patients With

    CANCER GENOMICS & PROTEOMICS 16 : 29-58 (2019) doi:10.21873/cgp.20110 Human Periprostatic Adipose Tissue: Secretome from Patients With Prostate Cancer or Benign Prostate Hyperplasia PAULA ALEJANDRA SACCA 1, OSVALDO NÉSTOR MAZZA 2, CARLOS SCORTICATI 2, GONZALO VITAGLIANO 3, GABRIEL CASAS 4 and JUAN CARLOS CALVO 1,5 1Institute of Biology and Experimental Medicine (IBYME), CONICET, Buenos Aires, Argentina; 2Department of Urology, School of Medicine, University of Buenos Aires, Clínical Hospital “José de San Martín”, Buenos Aires, Argentina; 3Department of Urology, Deutsches Hospital, Buenos Aires, Argentina; 4Department of Pathology, Deutsches Hospital, Buenos Aires, Argentina; 5Department of Biological Chemistry, School of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires, Argentina Abstract. Background/Aim: Periprostatic adipose tissue Prostate cancer (PCa) is the second most common cancer in (PPAT) directs tumour behaviour. Microenvironment secretome men worldwide. While most men have indolent disease, provides information related to its biology. This study was which can be treated properly, the problem consists in performed to identify secreted proteins by PPAT, from both reliably distinguishing between indolent and aggressive prostate cancer and benign prostate hyperplasia (BPH) disease. Evidence shows that the microenvironment affects patients. Patients and Methods: Liquid chromatography-mass tumour behavior. spectrometry-based proteomic analysis was performed in Adipose tissue microenvironment is now known to direct PPAT-conditioned media (CM) from patients with prostate tumour growth, invasion and metastases (1, 2). Adipose cancer (CMs-T) (stage T3: CM-T3, stage T2: CM-T2) or tissue is adjacent to the prostate gland and the site of benign disease (CM-BPH). Results: The highest number and invasion of PCa.
  • Protein Identities in Evs Isolated from U87-MG GBM Cells As Determined by NG LC-MS/MS

    Protein Identities in Evs Isolated from U87-MG GBM Cells As Determined by NG LC-MS/MS

    Protein identities in EVs isolated from U87-MG GBM cells as determined by NG LC-MS/MS. No. Accession Description Σ Coverage Σ# Proteins Σ# Unique Peptides Σ# Peptides Σ# PSMs # AAs MW [kDa] calc. pI 1 A8MS94 Putative golgin subfamily A member 2-like protein 5 OS=Homo sapiens PE=5 SV=2 - [GG2L5_HUMAN] 100 1 1 7 88 110 12,03704523 5,681152344 2 P60660 Myosin light polypeptide 6 OS=Homo sapiens GN=MYL6 PE=1 SV=2 - [MYL6_HUMAN] 100 3 5 17 173 151 16,91913397 4,652832031 3 Q6ZYL4 General transcription factor IIH subunit 5 OS=Homo sapiens GN=GTF2H5 PE=1 SV=1 - [TF2H5_HUMAN] 98,59 1 1 4 13 71 8,048185945 4,652832031 4 P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 - [ACTB_HUMAN] 97,6 5 5 35 917 375 41,70973209 5,478027344 5 P13489 Ribonuclease inhibitor OS=Homo sapiens GN=RNH1 PE=1 SV=2 - [RINI_HUMAN] 96,75 1 12 37 173 461 49,94108966 4,817871094 6 P09382 Galectin-1 OS=Homo sapiens GN=LGALS1 PE=1 SV=2 - [LEG1_HUMAN] 96,3 1 7 14 283 135 14,70620005 5,503417969 7 P60174 Triosephosphate isomerase OS=Homo sapiens GN=TPI1 PE=1 SV=3 - [TPIS_HUMAN] 95,1 3 16 25 375 286 30,77169764 5,922363281 8 P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] 94,63 2 13 31 509 335 36,03039959 8,455566406 9 Q15185 Prostaglandin E synthase 3 OS=Homo sapiens GN=PTGES3 PE=1 SV=1 - [TEBP_HUMAN] 93,13 1 5 12 74 160 18,68541938 4,538574219 10 P09417 Dihydropteridine reductase OS=Homo sapiens GN=QDPR PE=1 SV=2 - [DHPR_HUMAN] 93,03 1 1 17 69 244 25,77302971 7,371582031 11 P01911 HLA class II histocompatibility antigen,
  • Heterotrimeric Go Protein Links Wnt-Frizzled Signaling with Ankyrins to Regulate the Neuronal Microtubule Cytoskeleton Anne-Marie Lüchtenborg1,2, Gonzalo P

    Heterotrimeric Go Protein Links Wnt-Frizzled Signaling with Ankyrins to Regulate the Neuronal Microtubule Cytoskeleton Anne-Marie Lüchtenborg1,2, Gonzalo P

    © 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3399-3409 doi:10.1242/dev.106773 RESEARCH ARTICLE Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton Anne-Marie Lüchtenborg1,2, Gonzalo P. Solis1, Diane Egger-Adam2, Alexey Koval1, Chen Lin1,2, Maxime G. Blanchard1, Stephan Kellenberger1 and Vladimir L. Katanaev1,2,* ABSTRACT The evolutionarily conserved Wg pathway is important for Drosophila neuromuscular junctions (NMJs) represent a powerful numerous developmental programs and cellular processes (Logan model system with which to study glutamatergic synapse formation and Nusse, 2004). In the nervous system of Drosophila,Wg and remodeling. Several proteins have been implicated in these signaling is involved in the formation of neuromuscular junctions processes, including components of canonical Wingless (Drosophila (NMJs) (Packard et al., 2002; Miech et al., 2008). Being a Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton glutamatergic synapse, the Drosophila NMJ provides a useful linker Ankyrin 2, but possible interconnections and cooperation experimental model with which to study mammalian central between these proteins were unknown. Here, we demonstrate that nervous system synapses, their formation and remodeling (Collins the heterotrimeric G protein Go functions as a transducer of Wingless- and DiAntonio, 2007). The Drosophila NMJ is a beads-on-a-string- Frizzled 2 signaling in the synapse. We identify Ankyrin 2 as a target like structure that is formed at the axon terminus and is composed of – – of Go signaling required for NMJ formation. Moreover, the Go-ankyrin distinct circular structures the synaptic boutons which contain interaction is conserved in the mammalian neurite outgrowth pathway.
  • Application of a MYC Degradation

    Application of a MYC Degradation

    SCIENCE SIGNALING | RESEARCH ARTICLE CANCER Copyright © 2019 The Authors, some rights reserved; Application of a MYC degradation screen identifies exclusive licensee American Association sensitivity to CDK9 inhibitors in KRAS-mutant for the Advancement of Science. No claim pancreatic cancer to original U.S. Devon R. Blake1, Angelina V. Vaseva2, Richard G. Hodge2, McKenzie P. Kline3, Thomas S. K. Gilbert1,4, Government Works Vikas Tyagi5, Daowei Huang5, Gabrielle C. Whiten5, Jacob E. Larson5, Xiaodong Wang2,5, Kenneth H. Pearce5, Laura E. Herring1,4, Lee M. Graves1,2,4, Stephen V. Frye2,5, Michael J. Emanuele1,2, Adrienne D. Cox1,2,6, Channing J. Der1,2* Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adeno- carcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry–based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, Downloaded from UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage- dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability 62 through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser .