Journal: Cancer Research Communications

Article Title: SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma

doi: 10.1158/2767-9764.CRC-23-0234

Figure Lengend Snippet: ALK-mutant neuroblastoma cell lines are sensitive to SHP2 inhibition. A, Protein expression in a panel of neuroblastoma cell lines with ALK-mutant (ALK mut ) or wild-type (ALK wt ) status. Calculation of IC 50 in neuroblastoma cells following treatment with TNO155 ( B ), ceritinib ( C ), or lorlatinib ( D ) for 72 hours. **, P < 0.01; ***, P < 0.001.

Article Snippet: TNO155 (#HY-136173) and SHP099 (#HY-100388A) were purchased from MedChemExpress, and ceritinib (#S7083), lorlatinib (#S7536), and crizotinib (#S1068) were purchased from Selleck Chemicals.

Techniques: Mutagenesis, Inhibition, Expressing

Journal: Cancer Research Communications

Article Title: SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma

doi: 10.1158/2767-9764.CRC-23-0234

Figure Lengend Snippet: TNO155 synergizes with ALK-TKIs in ALK-mutant neuroblastoma cells. Cell viability (alamarBlue) analysis ( A ) and determination of drug interaction ( B ) in neuroblastoma cells with ALK wild-type (WT) or mutant (MUT) status treated with DMSO control, TNO155, ceritinib, or TNO155 plus ceritinib for 72 hours. C, Western immunoblots of Kelly and SH-SY5Y cells treated with TNO155 (1 µmol/L), ceritinib (0.1 µmol/L), or combination for 24 hours. Cell viability analysis ( D ) and determination of drug interaction ( E ) in neuroblastoma cells treated with TNO155, lorlatinib, or TNO155 plus lorlatinib for 72 hours. F, Western immunoblots of Kelly and SH-SY5Y cells treated with TNO155 (1 µmol/L), lorlatinib (1 µmol/L), or combination for 24 hours. Drug concentrations are shown in . Synergy was calculated using the EOB model. EOB scores > 0, synergistic. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: TNO155 (#HY-136173) and SHP099 (#HY-100388A) were purchased from MedChemExpress, and ceritinib (#S7083), lorlatinib (#S7536), and crizotinib (#S1068) were purchased from Selleck Chemicals.

Techniques: Mutagenesis, Control, Western Blot

Journal: Cancer Research Communications

Article Title: SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma

doi: 10.1158/2767-9764.CRC-23-0234

Figure Lengend Snippet: TNO155 sensitizes ALK-mutant zebrafish xenografts to ALK inhibitors. Zebrafish embryos (2 dpf) were injected with GFP-expressing Kelly ( A ), SH-SY5Y ( B ), or LAN-6 ( C ) cells costained with DiI dye. At 1 day post injection (1 DPI), xenografted larvae were submersed in TNO155, ceritinib, or lorlatinib alone or in combination treatments and incubated at 35°C for 72 hours. Tumor mass was assessed after 72 hours of drug exposure (4 DPI), and GFP fluorescence (area × pixel intensity) was quantified and normalized to pretreatment (1 DPI). Tumor cell invasion within the yolk-sac was visualized (A and D ) and quantified ( E ) in Kelly larval xenografts treated with TNO155, ceritinib or lorlatinib alone or in combination for 72 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: TNO155 (#HY-136173) and SHP099 (#HY-100388A) were purchased from MedChemExpress, and ceritinib (#S7083), lorlatinib (#S7536), and crizotinib (#S1068) were purchased from Selleck Chemicals.

Techniques: Mutagenesis, Injection, Expressing, Incubation, Fluorescence

Journal: Cancer Research Communications

Article Title: SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma

doi: 10.1158/2767-9764.CRC-23-0234

Figure Lengend Snippet: Treatment regimens administered to Kelly xenografts

Article Snippet: TNO155 (#HY-136173) and SHP099 (#HY-100388A) were purchased from MedChemExpress, and ceritinib (#S7083), lorlatinib (#S7536), and crizotinib (#S1068) were purchased from Selleck Chemicals.

Techniques:

Journal: Cancer Research Communications

Article Title: SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma

doi: 10.1158/2767-9764.CRC-23-0234

Figure Lengend Snippet: TNO155 and lorlatinib treatment delays tumor growth in ALK-mutant mouse xenografts. A, Tumor volumes of Kelly xenografts treated orally with high- or low-dose regimens of TNO155, lorlatinib, combination treatment (Combo), or vehicle control for 4 weeks. Tumor growth ( B ) and Kaplan–Meier survival curves ( C ) of Kelly xenografts treated with high-dose vehicle ( n = 5), TNO155 ( n = 4), lorlatinib ( n = 5), or combination (Combo, n = 4) treatments. Tumor growth ( D ) and Kaplan–Meier survival curves ( E ) of Kelly xenografts treated with low-dose vehicle ( n = 3), TNO155 ( n = 3), lorlatinib ( n = 3), or combination (Combo, n = 4) treatments F, Protein expression analyses of tumor lysates treated with vehicle control ( n = 3), TNO155 (7.5 mg/kg twice per day, n = 3), lorlatinib (1 mg/kg twice per day, n = 3), or combination (Combo, n = 3) for 20 days. *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: TNO155 (#HY-136173) and SHP099 (#HY-100388A) were purchased from MedChemExpress, and ceritinib (#S7083), lorlatinib (#S7536), and crizotinib (#S1068) were purchased from Selleck Chemicals.

Techniques: Mutagenesis, Control, Expressing

Journal: Cancer Research Communications

Article Title: SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma

doi: 10.1158/2767-9764.CRC-23-0234

Figure Lengend Snippet: Dual SHP2/ALK inhibition resensitizes lorlatinib-resistant ALK F1174L cells and reduces tumor regrowth. A, Kelly cells were treated with increasing doses of lorlatinib (0.1–5.0 µmol/L) or DMSO over 90 days to select for subpopulations of lorlatinib-resistant (Kelly-LR) or -sensitive (Kelly-S) cells. Cell lines were maintained under chronic exposure to lorlatinib or DMSO. Created with BioRender.com. Cell viability (alamarBlue) and IC 50 was assessed in Kelly-S and Kelly-LR cells treated with increasing concentrations of lorlatinib alone ( B ) or lorlatinib plus TNO155 ( C ) for 72 hours. A maximum of 150 µmol/L of either or both drugs was serially diluted (1:3) to assess wide-range dose efficacy (0–150 µmol/L, doses of 0.0076, 0.0228, 0.0685, 0.205, 0.617, 1.851, 5.555, 16.666, 50, and 150 µmol/L). D, Western immunoblots of Kelly-S and Kelly-LR cells treated with TNO155 (1.5 µmol/L), lorlatinib (1 µmol/L), or combination treatment for 6 hours. Tumor growth ( E ) and Kaplan–Meier survival curves ( F ) of Kelly xenografts treated with two rounds of low-dose vehicle control ( n = 4), TNO155 ( n = 3), lorlatinib ( n = 5), or combination treatment (Combo, n = 4) for 4 weeks each, with a 3 week drug break in between each round. **, P < 0.01; ***, P < 0.001.

Article Snippet: TNO155 (#HY-136173) and SHP099 (#HY-100388A) were purchased from MedChemExpress, and ceritinib (#S7083), lorlatinib (#S7536), and crizotinib (#S1068) were purchased from Selleck Chemicals.

Techniques: Inhibition, Western Blot, Control

Journal: Cancer Research Communications

Article Title: SHP2 Inhibition with TNO155 Increases Efficacy and Overcomes Resistance of ALK Inhibitors in Neuroblastoma

doi: 10.1158/2767-9764.CRC-23-0234

Figure Lengend Snippet: Alterations in RAS-MAPK pathway associate with lorlatinib resistance. A, Abundance of specific BRAF, RAF1, and ERK2 phospho-sites identified by LC/MS-MS in Kelly-LR samples relative to Kelly-S. B, GTP pulldown (PD) of WCE lysates of Kelly-S and Kelly-LR cells. IB, immunoblot. C, Western immunoblots of Kelly-S and Kelly-LR cells treated with TNO155 (1.5 µmol/L), lorlatinib (1 µmol/L) or combination treatment for 6 hours. *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: TNO155 (#HY-136173) and SHP099 (#HY-100388A) were purchased from MedChemExpress, and ceritinib (#S7083), lorlatinib (#S7536), and crizotinib (#S1068) were purchased from Selleck Chemicals.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: bioRxiv

Article Title: SUMM4 complex couples insulator function and DNA replication timing control

doi: 10.1101/2021.10.02.462895

Figure Lengend Snippet: a , Recombinant SUMM4. Mod(Mdg4)-His6, 67.2 (p100, cyan arrowhead) and 59.1 (p75, green arrowhead) splice forms were co-expressed with FLAG-SUUR (red arrowheads, p130 and p65) or separately in Sf9 cells and purified by FLAG or Ni-NTA affinity chromatography. Mod(Mdg4)-67.2 forms a specific complex with SUUR. b , ATPase activities of recombinant FLAG-ISWI (green bars), FLAG-SUUR (red bars) and SUMM4 (FLAG-SUUR + Mod(Mdg4)-67.2-His6, purple bars). Equimolar proteins were analyzed in reactions in the absence or presence of plasmid DNA or equivalent amounts of reconstituted oligonucleosomes, ±H1. rSUUR(KA) and rMMD4, ATPases activities of K59A mutant of SUUR (gray bars) and Mod(Mdg4)-67.2-His6 (cyan bars). Hydrolysis rates were converted to moles ATP per mole protein per minute. All reactions were performed in triplicate, error bars represent standard deviations. p -values for statistically significant differences are indicated (Mann-Whitney test). c , DNA- and nucleosome-dependent stimulation or inhibition of ATPase. The activities were analyzed as in ( b ). Statistically significant differences are shown (Mann-Whitney test). d , Nucleosome sliding activities by EpiDyne ® -PicoGreen™ assay (see Methods ) with 5 nM of recombinant ISWI, SUUR or SUMM4. Reaction time courses are shown for terminally (6-N-66) and centrally (50-N-66) positioned mononucleosomes ( Extended Data Figs. E2g - j ). RFU, relative fluorescence units produced by PicoGreen fluorescence.

Article Snippet: In brief, a recombinant ATPase over a concentration range ( Extended Data Figs. E2g - j ) was mixed with 10 nM EpiDyne biotinylated nucleosome remodeling substrate (EpiCypher) , terminally positioned 6-N-66 (219 bp DNA fragment) or centrally positioned 50-N-66 (263 bp) and 1 mM ATP in 20 µL remodeling buffer, 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 3 mM MgCl2, 0.01% (v/v) Tween-20, 0.01% (w/v) BSA.

Techniques: Recombinant, Purification, Affinity Chromatography, Plasmid Preparation, Mutagenesis, MANN-WHITNEY, Inhibition, Picogreen Assay, Fluorescence, Produced

Journal: bioRxiv

Article Title: SUMM4 complex couples insulator function and DNA replication timing control

doi: 10.1101/2021.10.02.462895

Figure Lengend Snippet: a , Physical interactions of recombinant EGG, SUUR and WDE. Untagged EGG (green arrowhead) was co-expressed with FLAG-SUUR (red arrowheads, p130 and p65) or WDE-FLAG (purple arrowhead) in Sf9 cells and purified by FLAG affinity chromatography. EGG forms a specific complex with WDE but not SUUR. b , Recombinant FLAG-SUUR(K59A) and FLAG-ISWI expressed in Sf9 cells. See legend to . c , Protein composition of in vitro reconstituted chromatin. Oligonucleosomes prepared from plasmid DNA and core histones with (+H1) or without H1 (–H1) were analyzed by SDS-PAGE and Coomassie staining. Positions of BSA, H1 and core histone bands are indicated on the right; molecular mass markers (kDa) are shown on the left. d , Micrococcal nuclease (MNase) analysis of reconstituted chromatin. Partial digestion with five different dilutions of MNase was performed on H1-free (– H1) and H1-containing (+H1) oligonucleosomes. Deproteinated DNA fragments were analyzed by agarose gel electrophoresis and stained with ethidium. Note the increased nucleosome repeat length in (+H1) lanes consistent with H1 incorporation. Triangles at the top indicate increasing MNase concentrations; 123 bp ladder was used as a molecular mass marker. e , Chromatosome stop assay. Oligonucleosomes assembled with or without H1 were subjected to partial MNase digestion, and DNA was analyzed by agarose gel electrophoresis and ethidium bromide staining. Positions of the core particle and chromatosome DNA are indicated by arrowheads. DNA fragment sizes in the 20-bp DNA ladder marker are shown. f , EpiCypher ® EpiDyne ® - PicoGreen™ assay design. EpiDyne nucleosomes encompass a restriction site shielded by the initial nucleosome position but exposed for Dpn II cleavage upon remodeling (sliding or displacement). Biotinylated substrates are immobilized on streptavidin magnetic beads. Digest by Dpn II releases the substrates from beads, and supernatant is quantified by PicoGreen™ (dsDNA detection reagent) fluorescence. g , Titration of Drosophila ISWI remodeling activity using terminally (6-N-66) or centrally (50-N-66) positioned mononucleosomes. Early reaction time points were separately plotted to indicate linear ranges. RFU, relative fluorescence units. h , Early remodeling rates for ISWI were calculated by linear regression analyses of data in respective linear ranges. ISWI exhibits a stronger remodeling activity with a centrally positioned nucleosome substrate. i , Titration of human BRG1 remodeling activity. Data are presented as in ( g ). j , Early remodeling rates for BRG1 were calculated and plotted as in ( h ). BRG1 does not exhibit a bias towards remodeling centrally or terminally positioned nucleosomes.

Article Snippet: In brief, a recombinant ATPase over a concentration range ( Extended Data Figs. E2g - j ) was mixed with 10 nM EpiDyne biotinylated nucleosome remodeling substrate (EpiCypher) , terminally positioned 6-N-66 (219 bp DNA fragment) or centrally positioned 50-N-66 (263 bp) and 1 mM ATP in 20 µL remodeling buffer, 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 3 mM MgCl2, 0.01% (v/v) Tween-20, 0.01% (w/v) BSA.

Techniques: Recombinant, Purification, Affinity Chromatography, In Vitro, Plasmid Preparation, SDS Page, Staining, Agarose Gel Electrophoresis, Marker, Picogreen Assay, Magnetic Beads, Fluorescence, Titration, Activity Assay