CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

Role of the Invariant Peptide Fragment Forming NH.S Hydrogen Bonds in the Active Site of Cytochrome P-450 and Chloroperoxidase: Synthesis and Properties of Cys-Containing Peptide Fe(III) and Ga(III) (Octaethylporphinato) Complexes as Models.

The primary sequence of Cys-X-Gly-Y- (X, hydrophobic residue; Y, hydrophilic residue) is highly conserved in cytochrome P-450s. The amide NHs of Leu, Gly, and X are assumed to form NH.S hydrogen bonds which are also found in the active site fragment, Cys-Pro-Ala-Leu, of chloroperoxidase (CPO). [Fe(III)(OEP)(Z-cys-Leu-Gly-Leu-OMe)] (OEP, octaethylporphinato; Z, benzyloxycarbonyl) and [Fe(III)(OEP)(Z-cys-Pro-Ala-Leu-OMe)] were synthesized as P-450 and CPO model complexes containing the invariant amino acid fragment of the active site. The corresponding gallium(III) complexes were also synthesized to investigate the solution structures using two-dimensional (2D) NMR experiments because the Ga(III) ion is similar to the Fe(III) ion in the ionic radii and in the coordination geometry. The solution structures of the peptide part of the gallium complexes indicate that the invariant fragments maintain a beta I-turn-like conformation and then form NH.S hydrogen bonds between S(gamma)Cys and NH of the third and fourth amino acid residues. The hydrogen bonds have also been confirmed by the (2)H NMR spectra of N(2)H-substituted Fe(III) peptide complexes. The Fe(III)/Fe(II) redox potentials of the Fe(III) complexes indicate that the NH.S hydrogen bonds in the fragments causes a slight positive shift of the redox potential. The tri- and tetrapeptide Fe(III) complexes containing the invariant fragments of P-450 are kinetically stable at 30 degrees C in CH(2)Cl(2). In contrast, [Fe(III)(OEP)(Z-cys-Leu-OMe)] decomposed to give [Fe(II)(OEP)] (22%) and the corresponding disulfide immediately in CD(2)Cl(2) at 30 degrees C for 1 h. These results indicate that the invariant fragments involving the hydrogen bonds cause the stabilization of the high-spin Fe(III) resting state rather than the positive shift of Fe(III)/Fe(II) redox potential.

Crystal structures and 77Se NMR spectra of molybdenum(IV) areneselenolates having intramolecular NH...Se hydrogen bonds.

Salts of the monooxomolybdenum(IV,V) areneselenolates having intramolecular NH...Se hydrogen bonds, [Mo(IV)O(Se-2-RCONHC6H4)4]2- (R = t-Bu, CH3, CF3) and [Mo(V)O(Se-2-t-BuCONHC6H4)4]-, were synthesized and characterized by 1H nuclear magnetic resonance (NMR), 77Se NMR, electron spin resonance (ESR), UV-visible spectra, X-ray analysis, and electrochemical measurements. 77Se-1H correlated spectroscopy (COSY) indicated a significant correlation between amide 1H and selenolate 77Se atoms through an NH...Se hydrogen bond with 1J(77Se-1H) = 5.4 Hz coupling. The hydrogen bonds contribute to the positive shift in the Mo(V)/Mo(IV) redox potential. In the crystal structure of (PPh4)2[Mo(IV)O(Se-2-CH3CONHC6H4)4], an NH...O=Mo hydrogen bond was found. Ab inito calculations support the presence of intramolecular NH...O=Mo and NH...Se hydrogen bonds.

Switching of turn conformation in an aspartate anion peptide fragment by NH . . . O- hydrogen bonds.

Aspartic acid protease model peptides Z-Phe-Asp(COOH)-Thr-Gly-Ser-Ala-NHCy (1) and AdCO-Asp(COOH)-Val-Gly-NHBzl (3), and their aspartate anions (NEt4)[Z-Phe-Asp(COO-)-Thr-Gly-Ser-Ala-NHCy] (2) and (NEt4)[AdCO-Asp(COO-)-Val-Gly-NHBzl] (4), having an invariant primary sequence of the Asp-X(Thr,Ser)-Gly fragment, were synthesized and characterized by 1H-NMR, CD, and infrared (IR) spectroscopies. NMR structure analyses indicate that the Asp O(delta) atoms of the aspartate peptide 2 are intramolecularly hydrogen-bonded with Gly, Ser, Ala NH, and Ser OH, supporting the rigid beta-turn-like conformation in acetonitrile solution. The tripeptide in the aspartic acid 3 forms an inverse gamma-turn structure, which is converted to a beta-turn-like conformation because of the formation of the intramolecular NH . . . O- hydrogen bonds with the Asp O(delta) in 4. Such a conformational change is not detected between dipeptides AdCO-Asp(COOH)-Va-NHAd (5) and (NEt4)[AdCO-Asp(COO-)-Val-NHAd] (6). The pK(a) value of side-chain carboxylic acid (5.0) for 3 exhibits a lower shift (0.3 unit) from that of 5 in aqueous polyethyleneglycol lauryl ether micellar solution. NMR structure analyses for 3 in an aqueous micellar solution indicate that the preorganized turn structure, which readily forms the NH . . . O- hydrogen bonds, lowers the pK(a) value and that resulting hydrogen bonds stabilize the rigid conformation in the aspartate anion state. We found that the formation of the NH . . . O- hydrogen bonds involved in the hairpin turn is correlated with the protonation and deprotonation state of the Asp side chain in the conserved amino acid fragments.