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Liver lineage confers sensitivity of cancer cells to sulfonation-dependent alkylators: PDX and Immobilized Drug Enrichment

ABSTRACT: The small molecule YC-1 was identified as identified as a selectively active agent against subsets of the two major live cancer subtypes, intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Response to YC-1 is dependent on the expression of the sulfotransferase SULT1A1, which activates YC-1 through sulfonation. The contributions of mass spectrometry to the study were: (i) identifying SULT1A1 as the key player in YC-1 response through observing the protein in a cell line with acquired resistance to the small molecule treatment, (ii) corroborating the role of SULT1A1 in the YC-1 activity by correlating protein expression profiles and drug responses across 37 biliary cancer cell lines, (iii) showing that SULT1A1 expression was associated with IDH mutations, FGFR2 fusion, and BAP1 loss in vivo studying patient-derived xenograft (PDX) models, and (iv) identifying protein targets in a protein enrichment experiment with an immobilized drug. (iii) PDX samples were analyzed using TMT multiplexing on an Orbitrap Fusion mass spectrometer using the SPS-MS3 method. Prior to analysis, samples were fractionated offline using high pH reversed chromatography (12 fractions per TMT set). Three TMT sets were analyzed (ShiBardeesy_PDX_TMT1, ShiBardeesy_PDX_TMT2, and ShiBardeesy_PDX_TMT3), and samples were labeled as follows with channel (bridge channel, 130c): ShiBardeesy_PDX_TMT1: SS101 (126), MG12 (127c), SS106 (127n), MG62 (128c), MG26 (128n), LCCH13 (129c), LCCH3 (129n), Q12050 (130n). ShiBardeesy_PDX_TMT2: SS102 (126), SS110 (127n), MG34 (128c), MG17 (128n), LCCH4 (129c), MG68 (129n), HBT110 (130n). ShiBardeesy_PDX_TMT3: SS103 (126), MG10 (127n), MG59 (128c), MG25 (128n), LCCH10 (129c), MG73 (129n), HBT211 (130n). (iv) Samples from experiments using immobilized small molecules to determine direct interactors of YC-1 were performed using TMT10-barcoding and the SPS-MS3 method on an Orbitrap Lumos mass spectrometer. No off-line fraction was performed. Samples were grouped into two TMT sets (ShiBardeesy_ImmobilizedDrug_TMT1 and ShiBardeesy_ImmobilizedDrug_TMT2). Channel 131N was used as the bridge channel. Samples were labeled as follows: ShiBardeesy_ImmobilizedDrug_TMT1: immobilized_inactiveYC-1_rep_1 (126), immobilized_inactiveYC-1_rep_2 (128n), immobilized_inactiveYC-1_rep_3 (128c), immobilized_YC-1_rep_1 (129n), immobilized_YC-1_plus_soluble_YC-1_competitor_rep_1 (130c). ShiBardeesy_ImmobilizedDrug_TMT2: immobilized_YC-1_rep_2 (127n), immobilized_YC-1_plus_soluble_YC-1_competitor_rep_2 (128n), immobilized_YC-1_plus_soluble_YC-1_competitor_rep_3 (129c), immobilized_YC-1_rep_3 (130c).

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Nabeel Bardeesy

PROVIDER: MSV000090808 | MassIVE | Tue Nov 29 13:26:00 GMT 2022

REPOSITORIES: MassIVE

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Project description:We used mass spectrometry-based proteomics to perform a phosphoproteomics analysis of selinexor-treated ex vivo AML patient samples (n=20) and 4 AML cell lines (PL-21, NOMO-1, GDM-1 and MV4-11). For quantitative phosphoproteomics, we used a tandem mass tag (TMT)-based approach. Conditions for the TMT 11-plex (Ex vivo samples) and TMT 8-plex (AML cell lines) setups are specified below. Cells were treated with 1 M selinexor (also known as KPT-330) or 0.1% DMSO for 6 hours. The experimental treatment conditions and TMT11-plex (ex vivo samples) and TMT8-plex (cell lines) labeling schemes are specified below: TMT-11-plex TMT channel Patient no Treatment Set 1 126 Ex_24 DMSO Set 1 127N Ex_24 Selinexor Set 1 127C Ex_25 DMSO Set 1 128N Ex_25 Selinexor Set 1 128C Ex_34 DMSO Set 1 129N Ex_34 Selinexor Set 1 129C Ex_41 DMSO Set 1 130N Ex_41 Selinexor Set 1 130C Ex_44 DMSO Set 1 131N Ex_44 Selinexor Set 1 131C Mix pool Mix pool Set 2 126 Ex_9 DMSO Set 2 127N Ex_9 Selinexor Set 2 127C Ex_18 DMSO Set 2 128N Ex_18 Selinexor Set 2 128C Ex_27 DMSO Set 2 129N Ex_27 Selinexor Set 2 129C Ex_29 DMSO Set 2 130N Ex_29 Selinexor Set 2 130C Ex_36 DMSO Set 2 131N Ex_36 Selinexor Set 2 131C Mix pool Mix pool Set 3 126 Ex_8 DMSO Set 3 127N Ex_8 Selinexor Set 3 127C Ex_13 DMSO Set 3 128N Ex_13 Selinexor Set 3 128C Ex_14 DMSO Set 3 129N Ex_14 Selinexor Set 3 129C Ex_19 DMSO Set 3 130N Ex_19 Selinexor Set 3 130C Ex_40 DMSO Set 3 131N Ex_40 Selinexor Set 3 131C Mix pool Mix pool Set 4 126 Ex_6 DMSO Set 4 127N Ex_6 Selinexor Set 4 127C Ex_15 DMSO Set 4 128N Ex_15 Selinexor Set 4 128C Ex_28 DMSO Set 4 129N Ex_28 Selinexor Set 4 129C Ex_39 DMSO Set 4 130N Ex_39 Selinexor Set 4 130C Ex_42 DMSO Set 4 131N Ex_42 Selinexor Set 4 131C Mix pool Mix pool AML cell lines: TMT-8-plex TMT channel Treatment Replicate GDM-1 126 DMSO 1 GDM-1 127N DMSO 2 GDM-1 127C DMSO 3 GDM-1 128N DMSO 4 GDM-1 128C Selinexor 1 GDM-1 129N Selinexor 2 GDM-1 129C Selinexor 3 GDM-1 130N Selinexor 4 MV4-11 126 DMSO 1 MV4-11 127N DMSO 2 MV4-11 127C DMSO 3 MV4-11 128N DMSO 4 MV4-11 128C Selinexor 1 MV4-11 129N Selinexor 2 MV4-11 129C Selinexor 3 MV4-11 130N Selinexor 4 NOMO-1 126 DMSO 1 NOMO-1 127N DMSO 2 NOMO-1 127C DMSO 3 NOMO-1 128N DMSO 4 NOMO-1 128C Selinexor 1 NOMO-1 129N Selinexor 2 NOMO-1 129C Selinexor 3 NOMO-1 130N Selinexor 4 PL-21 126 DMSO 1 PL-21 127N DMSO 2 PL-21 127C DMSO 3 PL-21 128N DMSO 4 PL-21 128C Selinexor 1 PL-21 129N Selinexor 2 PL-21 129C Selinexor 3 PL-21 130N Selinexor 4

Project description:Proteome maps of biochemically enriched synaptic tissue from human Alzheimers Disease (AD) and control angular gyrus samples to highlight the synapse as a site of integration for pathophysiological processes active in AD. The samples were combined into eleven TMTpools (01 to 11). Samples pools were fractionated using basic pH reversed-phase chromatography (bRPLC) and at least 12 fractions were analyzed by LC-MS2/MS3 per pool. The RAW files are: Carlyle_TMTpool_01_fraction_01 to Carlyle_TMTpool_01_fraction_12 Carlyle_TMTpool_02_fraction_01 to Carlyle_TMTpool_02_fraction_12 Carlyle_TMTpool_03_fraction_01 to Carlyle_TMTpool_03_fraction_12 Carlyle_TMTpool_04_fraction_01 to Carlyle_TMTpool_04_fraction_12 Carlyle_TMTpool_05_fraction_01 to Carlyle_TMTpool_05_fraction_12 Carlyle_TMTpool_06_fraction_01 to Carlyle_TMTpool_06_fraction_12 Carlyle_TMTpool_07_fraction_01 to Carlyle_TMTpool_07_fraction_12 Carlyle_TMTpool_08_fraction_01 to Carlyle_TMTpool_08_fraction_08, Carlyle_TMTpool_08_fraction_09A, Carlyle_TMTpool_08_fraction_09B, Carlyle_TMTpool_08_fraction_10 to Carlyle_TMTpool_08_fraction_12 Carlyle_TMTpool_09_fraction_01 to Carlyle_TMTpool_09_fraction_12 Carlyle_TMTpool_10_fraction_01 to Carlyle_TMTpool_10_fraction_12 Carlyle_TMTpool_11_fraction_01 to Carlyle_TMTpool_11_fraction_12 The labeling scheme is: TMTpool_01: RUSH_0592 (126), RUSH_0034 (127n), RUSH_0755 (127c), RUSH_0143 (128n), RUSH_1442 (128c), RUSH_0570 (129n), Pooled_sample (129c), Pooled_sample (130n), RUSH_0992 (130c), RUSH_0857 (131n), bridge (131c). TMTpool_02: RUSH_0974 (126), RUSH_1440 (127n), RUSH_0240 (127c), RUSH_0032 (128n), RUSH_0406 (128c), RUSH_0065 (129n), RUSH_1376 (129c), RUSH_0684 (130n), RUSH_0269 (130c), RUSH_1159 (131n), bridge (131c). TMTpool_03: RUSH_0136 (126), Pooled_sample (127n), RUSH_0537 (127c), RUSH_1250 (128n), RUSH_0795 (128c), RUSH_0001 (129n), RUSH_0404 (129c), RUSH_0606 (130n), RUSH_0229 (130c), RUSH_0465 (131n), bridge (131c). TMTpool_04: RUSH_0734 (126), RUSH_0873 (127n), RUSH_0743 (127c), Pooled_sample (128n), RUSH_0712 (128c), RUSH_0751 (129n), RUSH_0302 (129c), RUSH_0499 (130n), RUSH_1218 (130c), RUSH_1421 (131n), bridge (131c). TMTpool_05: RUSH_1392 (126), RUSH_0890 (127n), RUSH_1006 (127c), RUSH_1063 (128n), RUSH_0128 (128c), RUSH_0590 (129n), RUSH_0674 (129c), RUSH_0572 (130n), RUSH_0413 (130c), RUSH_1288 (131n), bridge (131c). TMTpool_06: RUSH_1487 (126), RUSH_1348 (127n), RUSH_0007 (127c), RUSH_1323 (128n), RUSH_1498 (128c), RUSH_0976 (129n), RUSH_0859 (129c), RUSH_0544 (130n), RUSH_0067 (130c), RUSH_1384 (131n), bridge (131c). TMTpool_07: RUSH_0212 (126), RUSH_1474 (127n), Pooled_sample (127c), RUSH_0010 (128n), RUSH_0931 (128c), RUSH_1205 (129n), RUSH_1200 (129c), RUSH_0206 (130n), RUSH_0376 (130c), RUSH_0460 (131n), bridge (131c). TMTpool_08: RUSH_0927 (126), RUSH_0963 (127n), RUSH_0055 (127c), RUSH_0085 (128n), RUSH_0780 (128c), RUSH_0327 (129n), RUSH_0354 (129c), RUSH_0123 (130n), RUSH_1358 (130c), RUSH_1424 (131n), bridge (131c). TMTpool_09: RUSH_1334 (126), RUSH_0921 (127n), Pooled_sample (127c), RUSH_1093 (128n), RUSH_1499 (128c), RUSH_1419 (129n), Pooled_sample (129c), RUSH_0555 (130n), RUSH_0618 (130c), RUSH_0732 (131n), bridge (131c). TMTpool_10: RUSH_1176 (126), Pooled_sample (127n), RUSH_0958 (127c), RUSH_0447 (128n), RUSH_1485 (128c), RUSH_0158 (129n), RUSH_0252 (129c), RUSH_0078 (130n), RUSH_0415 (130c), Pooled_sample (131n), bridge (131c). TMTpool_11: RUSH_1313 (126), RUSH_0989 (127n), RUSH_0295 (127c), RUSH_0177 (128n), RUSH_0064 (128c), RUSH_0919 (129n), RUSH_0847 (129c), RUSH_0266 (130n), RUSH_0933 (130c), Pooled_sample (131n), bridge (131c).

Project description:Part I - SI-NET tumors High Resolution Isoelectric Focusing (HiRIEF) LC-MS and relative quantification by iTRAQ 8-plex was used to analyze 14 small intestinal neuroendocrine tumors (SI-NETs). The data was obtained from two separate TMT10plex sets and linked by a single internal pooled standard. The internal pooled standard was made by combining equal aliquots of the tryptic peptide mixtures from each of the 14 tissue samples. iTRAQ set1 was composed of 7 SI-NET samples, all from different individuals, and internal pooled standard labelled as follows: Channel 113 (sample Screen-1 with liver metastasis), Channel 114 (sample Screen-8 with liver metastasis), Channel 115 (sample Screen-3 with liver metastasis), Channel 116 (sample Screen-2 with liver metastasis), Channel 117 (sample Screen-5 no liver metastasis), Channel 118 (sample Screen-4 no liver metastasis), Channel 119 (sample Screen-10 no liver metastasis), Channel 121 (internal pooled standard). iTRAQ set2 was composed of 7 SI-NET samples, all from different individuals, and internal pooled standard labelled as follows: Channel 113 (sample Screen-7 with liver metastasis), Channel 114 (sample Screen-11 with liver metastasis), Channel 115 (sample Screen-13 with liver metastasis), Channel 116 (sample Screen-6 no liver metastasis), Channel 117 (sample Screen-12 no liver metastasis), Channel 118 (sample Screen-9 no liver metastasis), Channel 119 (sample Screen-14 no liver metastasis), Channel 121 (internal pooled standard). Part II - time course profiling in cell lines High Resolution Isoelectric Focusing (HiRIEF) LC-MS and relative quantification by TMT 10-plex was used to analyze cellular response to the neddylation inhibitor pevonedistat (MLN4924) at different timepoints in two SI-NET (small intestinal neuroendocrine tumors) cell lines. The data was obtained from two separate TMT 10-plex experiments. TMT set1 includes a time course experiment upon pevonedistat treatment of CNDT2 cells with harvests at 2h, 6h, 12h and 24h after treatment as well as of untreated control cells. Isobaric tag labelling of peptide samples with TMT10plex was used as follows. Biological duplicate controls (TMT channels 126, 127N), duplicate 2h (127C, 128N), duplicate 6h (128C, 129N), duplicate 12h (129C, 130N) and duplicate 24h (130C, 131) samples were employed. TMT set2 includes a time course experiment upon pevonedistat treatment of HC45 cells with harvests at 2h, 6h, 12h and 24h after treatment as well as of untreated control cells. Isobaric tag labelling of peptide samples with TMT10plex was used as follows. Biological duplicate controls (TMT channels 126, 127N), duplicate 2h (127C, 128N), duplicate 6h (128C, 129N), duplicate 12h (129C, 130N) and duplicate 24h (130C, 131) samples were employed.

Project description:The small molecule YC-1 was identified as identified as a selectively active agent against subsets of the two major live cancer subtypes, intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Response to YC-1 is dependent on the expression of the sulfotransferase SULT1A1, which activates YC-1 through sulfonation. The contributions of mass spectrometry to the study were: (i) identifying SULT1A1 as the key player in YC-1 response through observing the protein in a cell line with acquired resistance to the small molecule treatment, (ii) corroborating the role of SULT1A1 in the YC-1 activity by correlating protein expression profiles and drug responses across 37 biliary cancer cell lines, (iii) showing that SULT1A1 expression was associated with IDH mutations, FGFR2 fusion, and BAP1 loss in vivo studying patient-derived xenograft (PDX) models, and (iv) identifying protein targets in a protein enrichment experiment with an immobilized drug. (i) and (ii): Cell line samples from datasets (i) and (ii) were analyzed together in a set of 12 TMT sets (ShiBardeesy_CellLines_TMT1 to TMT12) using TMT11-barcoding. The samples were analyzed on an Orbitrap Lumos mass spectrometer using the SPS-MS3 method. Prior to analysis, samples were fractionated offline using high pH reversed chromatography (12 fractions per TMT set). Across all TMT sets, channel 131c was used as the bridge channel (pooled digests of all samples). Samples were labeled as follows (rep = replicate: (i) Cell lines samples with acquired resistance to YC-1 treatment: ShiBardeesy_CellLines_TMT1: RBE_rep_1 (126), RBE YC1 Resistant 6_rep_1 (129c). ShiBardeesy_CellLines_TMT2: RBE YC1 Resistant 3_rep_1 (129n), RBE YC1 Resistant 1_rep_1 (130n). ShiBardeesy_CellLines_TMT3: RBE YC1 Resistant 2_rep_1 (126), RBE YC1 Resistant 5_rep_1 (130c). ShiBardeesy_CellLines_TMT4: RBE YC1 Resistant 4_rep_1 (126). ShiBardeesy_CellLines_TMT5: RBE YC1 Resistant 1_rep_2 (127n), RBE YC1 Resistant 4_rep_2 (128c), RBE YC1 Resistant 3_rep_2 (130c). ShiBardeesy_CellLines_TMT6: RBE YC1 Resistant 2_rep_2 (126), RBE YC1 Resistant 6_rep_2 (127c). ShiBardeesy_CellLines_TMT7: RBE_rep_2 (128n), RBE YC1 Resistant 5_rep_2 (130n). (ii) Proteomes of untreated biliary cell lines (baseline): ShiBardeesy_CellLines_TMT1: RBE_rep_1 (126), GB2_rep_1 (127n), HuCCT1_rep_1 (127c), ECC3_rep_1 (128n), ICC10_rep_1 (128c), KMCH-1_rep_1 (129n), SNU-1196_rep_1 (130n). ShiBardeesy_CellLines_TMT2: ICC13-7_rep_1 (126), ICC8_rep_1 (127n), ICC9_rep_1 (127c), SNU-478_rep_1 (128n), CC-SW-1_rep_1 (128c), CC-LP-1_rep_1 (129c), SSP-25_rep_1 (130c), ICC10-8_rep_1 (131n) ShiBardeesy_CellLines_TMT3: SNU-1079_rep_1 (127n), ICC5_rep_1 (127c), ICC7_rep_1 (128c), COR-L105_rep_1 (129n), ICC17_rep_1 (129c), GBC1_rep_1 (131n). ShiBardeesy_CellLines_TMT4: ICC3_rep_1 (127n), ICC2_rep_1 (127c), ICC106_rep_1 (128n), ICC11_rep_1 (128c), HKGZ-CC_rep_1 (129n), SNU-869_rep_1 (129c), HuH28_rep_1 (130n), ICC12_rep_1 (130c), SNU-308_rep_1 (131n). ShiBardeesy_CellLines_TMT5: ICC2_rep_2 (126), ECC3_rep_2 (127c), GB2_rep_2 (128n), ICC11_rep_2 (129n), ICC7_rep_2 (129c), SNU-869_rep_2 (130n), SSP-25_rep_2 (131n). ShiBardeesy_CellLines_TMT6: CC-LP-1_rep_2 (127n), COR-L105_rep_2 (128n), HuCCT1_rep_2 (129n), ICC12_rep_2 (129c), ICC3_rep_2 (131n), ICC5_rep_2 (130n), SNU-478_rep_2 (130c). ShiBardeesy_CellLines_TMT7: ICC10_rep_2 (126), ICC10-6_rep_2 (127n), ICC10-8_rep_2 (127c), RBE_rep_2 (128n), ICC9_rep_2 (128c), ICC17_rep_2 (129n), ICC8_rep_2 (129c), CC-SW-1_rep_2 (130c), GBC1_rep_2 (131n). ShiBardeesy_CellLines_TMT8: ICC13-7_rep_2 (127c), HuH28_rep_2 (128n), HKGZ-CC_rep_2 (128c), SNU-1079_rep_2 (129n), KMCH-1_rep_2 (129c), SNU-1196_rep_2 (130n), SNU-308_rep_2 (131n). ShiBardeesy_CellLines_TMT9: SG231_rep_1 (126), KKU-100_rep_1 (127n), YSCCC_rep_1 (127c), ICC4_rep_1 (128n), TKKK_rep_1 (128c), ECC2_rep_1 (129n), KKU-M213/M214 (129c). ShiBardeesy_CellLines_TMT10: YSCCC_rep_2 (126), KKU-M213/M214_rep_2 (127n), SG231_rep_2 (127c), TKKK_rep_2 (129n), ECC2_rep_2 (129c), ICC4_rep_2 (130n), KKU-100_rep_2 (131n). ShiBardeesy_CellLines_TMT11: ICC13-7_rep_1 (129n). ShiBardeesy_CellLines_TMT12: ICC13-7_rep2 (131n)

Project description:Mass spectrometry-based proteomics was applied to unravel Erk signaling in mouse embryonic stem cells (ESCs). A previously described engineered ESCs system for Erk induction via a drug-inducible cRAF-ErT2 fusion (Hamilton, W. B. & Brickman, J. M., Cell Rep 2014) was used in two different setups measuring the phosphoproteome in an 11-plex tandem mass tag (TMT)-based approach as described below. Fgf4 stimulation and MEK inhibition: ESCs were stimulated with Fgf4 (40 nM, recombinant, mouse, 5 minutes) following pretreatment (30 minutes) with either DMSO (1:10000) or MEK inhibition (MEKi: 1 microM PD0325901): The experimental treatment conditions and TMT11-plex labeling are specified below: 126: DMSO, unstimulated control replicate 1 127N: DMSO, unstimulated control replicate 2 127C: DMSO, unstimulated control replicate 3 128N: DMSO + Fgf4 replicate 1 128C: DMSO + Fgf4 replicate 2 129N: DMSO + Fgf4 replicate 3 129C: MEKi + Fgf4 replicate 1 130N: MEKi + Fgf4 replicate 2 130C: MEKi + Fgf4 replicate 3 131N: MEKi replicate 1 131C: MEKi replicate 2 Analog sensitive Erk2 setup: In the ESC system the ERK2-/- ESCs (Hamilton et al., PloS ONE, 2013) were engineered to stably express an inducible cRAF-ErT2 fusion protein expressed from the same transgene as a FLAG-tagged analogue sensitive Erk2Q103A. Erk1 was subsequently removed by CRISPR-Cas9 mutagenesis using sgRNAs flanking exon 2. Cells were pre-treated for 30 minutes with either the ATP-mimetic compound 1-NM-PP1 (2 microM) (Endo et al., Biochem Biophys Res Commun 2006), the Mek1 inhibitor PD0325901 (1 microM), or DMSO (0.01%), followed by induction or not of the cRAF-ErT2 fusion by (Z)-4-Hydroxytamoxifen (4OHT) (250 nM) for 2 hours as indicated. The experimental treatment conditions and TMT11-plex labeling are specified below: 126: DMSO pretreatment, non-induced control, replicate 1 127N: DMSO pretreatment, non-induced control, replicate 2 127C: DMSO pretreatment, non-induced control, replicate 3 128N: DMSO pretreatment, 4OHT-induction, replicate 1 128C: DMSO pretreatment, 4OHT-induction, replicate 2 129N: DMSO pretreatment, 4OHT-induction, replicate 3 129C: PP1 pretreatment, 4OHT-induction, replicate 1 130N: PP1 pretreatment, 4OHT-induction, replicate 2 130C: PP1 pretreatment, 4OHT-induction, replicate 3 131N: MEKi pretreatment, 4OHT-induction, replicate 1 131C: MEKi pretreatment, 4OHT-induction, replicate 2

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Liver lineage confers sensitivity of cancer cells to sulfonation-dependent alkylators: PDX and Immobilized Drug Enrichment

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