Ras Initiative Update Frank McCormick and Levi Garraway DEPARTMENT OF HEALTH AND HUMAN SERVICES • National Institutes of Health • National Cancer Institute The Frederick National Laboratory is a Federally Funded Research and Development Center operated by Leidos Biomedical Research, Inc., for the National Cancer Institute RAS Initiative Accomplishments: Evaluating Ras dependency 2 SiREN assay for Ras dependency SiREN assay for Ras dependency 70% KD siEGFP 0-10% KD 50% KD 100% KD SiREN assay for Ras dependency RAS Initiative Accomplishments: Biophysical and structural analysis 7 Ras proteins 1 -166 167-185,6 Raf, PI 3’ kinase RalGDS, GAPs Raf, PI 3’ kinase RalGDS, GAPs Raf, PI 3’ kinase RalGDS, GAPs Raf, PI 3’ kinase RalGDS, GAPs Palmitoyl Farnesyl Fully processed KRAS4b A. Gorfe, U-Texas Houston Engineering baculovirus for improved production of processed KRAS • recombineering used to insert FNTA/FNTB genes into the baculovirus genome • eliminated issues with coinfection of multiple viruses • maltose-binding protein (MBP) fusion for greater yield and solubility • Trichoplusia ni (Hi5) insect cells for increased yield Carissa Grose, Dom Esposito, Bill Gillette Processed KRAS4b characterization • Extensive protein characterization – Purified to homogeneity; yield >7mg/L – Intact mass – Predominantly monomeric – Secondary structure equivalent to non-processed KRAS4b KRAS4b- FME – Lower thermal stability Intact mass analysis Analytical ultracentrifugation Secondary structure by CD 2.14 S 25.7 kDa 10 Bill Gillette, Zhaojing Meng, Shelley Perkins, Peter Frank, Pat Alexander, Rodolfo Ghirlando Processed KRAS4b binds to Nanodiscs in a phosphotidylserine-dependent manner Nanodiscs containing DMPS 1,2-dimyristoyl-sn-glycero-3- phospho-L-serine 11 Lakshman Bindu, Que Van FNL; Steve Sligar and Mike Gregory UI-UC Processed KRAS enables assays, screens and structural analysis in the context of membrane Lipid Nanodiscs Liposomes and Processed KRAS tethered bilayers Structures of Characterize Assays and Structural KRAS and reagents Screens analysis effectors on membranes Protein QC Intrinsic GTP hydrolysis NMR (NMRFAM) Biophysical properties GAP-stimulated hydrolysis Hi-res Cryo-EM (NCI) Membrane interactions Effector binding Crystallography (FNL) RBD-KRAS Alpha assays Sligar lab (U-Illinois) Groves lab (UC-Berkeley) Heinrich Lab (NIST) Processed KRAS4b workshop – collaborative opportunities • Frank Heinrich (Neutron reflectivity of protein/membranes; Carnegie Mellon University) – Determine orientation of KRAS4b-FME GDP/GMP-PNP on membrane – Impact of Calmodulin on HVR • Alemayehu Gorfe (Modeling of RAS on membranes; University of Texas Health Science Center) – Interest in testing predicted dimer interactions – Molecular dynamics analysis of FNL extended switch 1 KRAS4b-GDP structure • Mitsuhiko Ikura (NMR of KRAS on membranes; University of Toronto) – Analysis of processed KRAS4b by NMR • Jeff Perry (Small angle X-ray scattering; University of California Riverside) – Screening of crystallography conditions of challenging targets • Vadim Cherezov (Crystallography of membrane proteins; University of Southern California) – Attachment of transmembrane helix for anchoring of processed KRAS4b for crystallography • Jay Groves (RAS on tethered bilayers; University of California at Berkley) – Effector interactions – Development of screenable assays on tethered bilayers 13 Full-length KRAS in complex with GDP Full-length Wild-type KRAS-GDP Wild-type KRAS(1-166)-GMPPNP complex at 1.6 Ang complex at 1.35 Ang N C N C Switch-I Switch-II P-loop Extended switch-I conformation in KRAS • Validate presence of extended switch-I conformation in solution by NMR. Electrostatic surface – Dynamic studies in collaboration with National Magnetic Resonance Facility at Madison. Que Van at FNLCR – High-pressure NMR studies in collaboration with Dr. Kalbitzer, University of Regensburg, Germany. • Virtual compound screening to target the groove present at the base of switch-I region – in collaboration with Dr. Brian Shoichet’s group at Red - negative charge White - neutral UCSF. Blue - positive charge 15 Processed KRAS in complex with PDEδ N C KRAS PDEδ N Resolution: 2.0 Ang 16 RAS Initiative Accomplishments: Inhibitor Screens and Assays 17 Isogenic Screen for RAS Selective Inhibitors -/- -/- lox/lox HRAS NRAS KRAS MEFs Untreated MEFs G1 arrest (day 19*) Drosten M, Dhawahir A, Sum EY, Urosevic J, +4-OHT Lechuga CG, Esteban LM, Castellano E, Guerra C, Santos E, Barbacid M. EMBO J. 2010 + drugs +KRAS G12D Re-enter cell cycle + drugs +HRAS 18 HRASWT vs KRASG12D Pilot Screen HRAS-WT “selective” KRAS-G12D “selective” • Compound library was provided by NCATS (National Center for the Advancement of Translational Sciences) • The library is enriched for “tool” compounds, but also contains FDA approved drugs 19 Kanika Sharma (FNLCR), Kyle Brimacombe (NCATS) KRAS-effector Inhibitor Screen Daiichi-Sankyo Protein-Protein Interaction Library Z-factor = 0.65 20 Reproducibility of KRAS-RBD screening assay Assay is highly reproducible at 50 μM The “hit” rate at 50 μM is approximately 4% 13/320 compounds inhibit the alpha signal >25% 21 KRAS4b-FME binds to CRAF-RBD on Nanodiscs 22 Matt Holderfield, Maria Abreu Blanco RAS Localization Assay Overview ~125 Hit 60 Wells Cells/location identification 8 Locations/ Segmentation well and Analysis Nucleus GFP Membrane GFP-KRAS4bG12V Alla Brafman NCI Developmental Therapeutics Program screening set Reconfirmed hits Primary assay: 150 GFP-KRASG12V 2-C 5 Plate 1 (Z' =0.84) 2-F6 4-B3 Plate 2 (Z' =0.77) 100 5-F7 250 Plate 3 (Z' =0.89) 7-B8 Plate 4 (Z' =0.80) 10-B2 % A ctivity 50 200 Plate 5 (Z' =0.79) 11-C5 Plate 6 (Z' =0.68) FOVMean PM GFP 11-E3 150 Plate 7 (Z' =0.64) 0 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 10 1 0 10 1 0 1 0 10 10 1 0 100 Plate 8 (Z' =0.84) 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 % A ctivity Plate 9 (Z' =0.74) [ µ M] 50 FOVMean PM GFP Plate 10 (Z' =0.73) 150 Plate 11 (Z' =0.83) 11-B5 0 Plate 12 (Z' =0.74) 12-D2 13-B7 Plate 13 (Z' =0.83) 100 PlatePlate 1P late2Plate 3 Plate 4 Plate 5Plate 6Plate 7 Plate 8 9 PlatePlate 10Plate 11Plate 12Plate 13Plate 14Plate 15Plate 16 17 13-C7 Plate 14 (Z' =0.79) 14-D6 Plate 15 (Z' =0.88) 14-F7 % A ctivity 50 ~800 small molecules with Plate 16 (Z' =0.74) 15-D9 biological activity Plate 17 (Z' =0.88) FOVMean PM GFP 16-B9 0 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 10 10 10 1 0 1 0 10 10 1 0 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25 [ µ M] Alla Brafman HaloTag-KRASWT driven-MEFs Proliferate -/- -/- lox/lox HRAS NRAS KRAS MEFs Untreated MEFs G1 arrest (day 19*) Drosten M, Dhawahir A, Sum EY, Urosevic J, Lechuga CG, Esteban LM, Castellano E, +4-OHT Guerra C, Santos E, Barbacid M. EMBO J. 2010 HaloTag-KRAS4b can be HaloTag-KRAS4b rescues imaged in live cells. RASless MEF proliferation. +HaloTagKRAS Cell permeant, super bright, fluorescent Halo ligand from Janelia Farms Scale bar 20 µm TIRF Image: membrane Transmitted light image Nikki Fer and De Chen Characterization of RAS molecules in live cell membranes WT Jump squared displacement analysis HaloTag-KRAS driven-MEFs Three components Fraction Diffusion Mean Const. Rad. 2 Model (um /s) (SDev) Rc (nm) 0.505 0.73 - 1 Normal (0.0193) 0.233 0.1805 44.2 2 Constrained (0.021) 0.2624 0.0178 1.2 3 Constrained (0.026) De Chen and Prabhakar Gudla Single molecule tracking analysis suggests three RAS states in live cell membranes. Information extracted Hypothesis: states represent from individual different complexes in trajectories membrane. P is the probability of the transition from one state to another per second is the dwell time D is the diffusion rate Fτ is the fraction RASless-MEFs, HaloTag-wtKRAS4b [JF646]=50pM, Serum Starved, 37°C, 22,325 trajectories and average trajectory length 12 frames. De Chen and Prabhakar Gudla RAS Initiative Accomplishments: Towards a “RAS Interactome” 28 Cancer.gov/ras 29 RAS Lab (Basecamp) • Private, by invitation only • All posts and comments publish immediately • Supports uploads of documents, 1:1 interactions RAS Reagents Pathway 2.0 clone collection • Generate stop and nostop Gateway Entry clones for all genes • Isoforms chosen by bioinformatic analysis (R. Stephens) - most common transcript observed across different cell lines - many are NOT “isoform 1” or “longest isoform” • 180 genes - 360 total constructs (stop/nostop) - 17 not available commercially in correct isoform - 32 additional not available without mutations • 98% completion Vanessa Wall Jen Mehalko Collaboration with the RAS Community RAS events Synthetic Lethality Workshop, January 6-7 2014 RAS Pathways Workshop, June 11, 2014 Cell Surfaces Workshop, July 23, 2014 AACR Annual Meeting, April 21, 2015 Seminars at FNLCR RAS Structures Workshop, July 21-22, 2015 Channing Der, UNC RAS Immunotherapy Workshop, November 3, 2015 Ken Westover, UTSW RAS Immunotherapy Workshop SpeakersCarla Mattos Include:, Northeastern RAS Symposium, December 15-16, 2015 Mark Philips, NYU Vadim Gaponenko, U-Chicago RAS Symposium Confirmed Speakers Include:Jay Berzofsky , NCI Josh Salafsky, Biodesy, Inc. Jamie Chaft, Sloan-Kettering Calvin Kuo, Stanford Harold Varmus, Weill Cornell Medical College Kris Wood, Duke Lisa Coussens, Oregon Health SciencesMariano Barbacid, CNIO, Madrid Kevan Shokat, University of California, San Francisco Tom Dubensky, Aduro Biotech Cyril Benes, Mass General Allan Balmain, University of California, San Francisco Victor Engelhard, University of VirginiaCarolyn Buser, GlaxoSmithKline Mariano Barbacid, Spanish National Cancer Research Center (CNIO), Madrid Jay Groves, UC-Berkeley