The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

  1. Jacob T Polaski
  2. Dylan B Udy
  3. Luisa F Escobar-Hoyos
  4. Gokce Askan
  5. Steven D Leach
  6. Andrea Ventura
  7. Ram Kannan  Is a corresponding author
  8. Robert K Bradley  Is a corresponding author
  1. Computational Biology Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, United States
  2. Basic Sciences Division, Fred Hutchinson Cancer Research Center, United States
  3. Molecular and Cellular Biology Graduate Program, University of Washington, United States
  4. David M. Rubenstein Center for Pancreatic Cancer Research, Memorial Sloan Kettering Cancer Center, United States
  5. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, United States
  6. Department of Pathology, Stony Brook University, United States
  7. Yale University School of Medicine, United States
  8. Department of Surgery, Memorial Sloan Kettering Cancer Center, United States
  9. Dartmouth Norris Cotton Cancer Center, United States
  10. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, United States
3 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
UPF1 mutations do not result in the acquisition of squamous histological features or confer a growth advantage to mutant cells in vivo.

(A) Schematic of UPF1 gene structure and corresponding encoded protein domains. Intron 10 (I10) contains the bulk of the mutations reported by Liu et al. Scissors indicate the sites targeted by the …

Figure 1—figure supplement 1
Validation experiments in mouse KPC cells.

(A) Titration of adenovirus expressing Cas9 only (AdCas9) or Cas9 and guide RNAs targeting intron 9 and intron 11 of mouse Upf1 (AdUpf1) in KPC cells. Gel corresponds to PCR using genomic DNA with …

Figure 2 with 1 supplement
Mutations in UPF1 intron 10 do not inhibit nonsense-mediated mRNA decay (NMD) or cause exon skipping.

(A) Box plot of NMD efficiency in 293 T cells engineered to contain wild-type (WT) or mutant (P1, P9) UPF1. P1 and P9 correspond to the IVS10+31G>A and IVS10-17G>A mutations reported by Liu et al. …

Figure 2—figure supplement 1
Validation experiments in human 293 T cells and raw gel images for western blot and RT-PCR.

(A) Sanger sequencing of genomic DNA from engineered 293 T cells verifying introduction of the mutation IVS10+31G>A in a homozygous state, as reported by Liu et al. for patient P1, as well as a …

Many reported UPF1 mutations are identical to genetic variants.

(A) Illustration of the mutations in UPF1 intron 10 (I10) reported by Liu et al. Each row indicates the wild-type (WT) sequence from the reference human genome or mutations reported by Liu et al. …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (H. sapiens)UPF1EnsemblENSG00000005007
Gene (M. musculus)Upf1EnsemblENSMUSG00000058301.8
Strain, strain background (M. musculus)MouseNCI Charles RiverCharles River: C57BL/6 albinoMice for pancreatic injections
Strain, strain background (E. coli)NEB Stable Competent E. coli (High efficiency)New England BiolabsC3040Chemically competent
Genetic reagent (M. musculus)AdUpf1This paperN/AAdenovirus expressing CRISPR gRNAs targeting mouse Upf1 introns 9-11
Cell line (H. sapiens)HEK 293TATCCCRL-11268Human cell line to model patient mutations
Cell line (M. musculus)KPCGenerated from the PDX-1-Cre; LSL-KrasG12D/+; LSL Trp53R172H/+N/AMurine cell line to model Upf1 exon skipping mutations (Hingorani et al., 2005)
Provided by Robert Vonderheide
AntibodyAnti-human UPF1
(rabbit monoclonal)
AbcamCat No. ab10936WB: (1:1000)
AntibodyAnti-human GAPDH (rabbit polyclonal)AbcamCat No. ab9485WB: (1:1000)
AntibodyAnti-rabbit secondary antibody (goat monoclonal)AbcamCat No. ab216777WB: (1:10000)
IRDye 680RD
AntibodyAnti-mouse UPF1 (rabbit monoclonal)Cell signaling technologyCat No.9435WB: (1:1000)
AntibodyAnti-mouse tubulin (mouse monoclonal)Sigma-AldrichCat No. T9206WB: (1:2000)
AntibodyAnti-rabbit secondary antibody (from donkey)AmershamNA93VWB: (1:5000)
AntibodyAnti-mouse secondary antibody (from sheep)AmershamNA931WB: (1:5000)
AntibodyAnti-mouse p40-ΔNp63 (rabbit polyclonal)AbcamCat No. ab166857WB: (1:100)
Recombinant DNA reagentpX459/Cas9 expression plasmidAddgeneCat No. 48139Ran et al., 2013
Recombinant DNA reagentphCMV-MUPPMID:9671053PlasmidControl for transfection efficiency of pmCMV-GI-Norm and pmCMV-GI-39Ter
Recombinant DNA reagentpmCMV-Gl-NormPMID:9671053PlasmidTransient transfection construct coding for full-length β-globin
Recombinant DNA reagentpmCMV-Gl-39TerPMID:9671053PlasmidTransient transfection construct coding for truncated β-globin with PTC at amino acid 39
Sequence-based reagentmUpf1_FThis paperPCR primerGGTGATGAGATTGCTATTGAGC
Sequence-based reagentmUpf1_RThis paperPCR primerTGTTCCTGATCTGGTTGTGC
Sequence-based reagentmUpf1-intron_9-gDNA_FThis paperGuide DNA OligoCACCGTTGTGAGGGCCATACCCTTG
Sequence-based reagentmUpf1-intron_9-gDNA_RThis paperGuide DNA OligoAAACCAAGGGTATGGCCCTCACAAC
Sequence-based reagentmUpf1-intron_11-gDNA_FThis paperGuide DNA OligoCACCGCCGTTGAGCTGATGGTGGCT
Sequence-based reagentmUpf1-intron_11-gDNA_RThis paperGuide DNA OligoAAACAGCCACCATCAGCTCAACGGC
Sequence-based reagenthUPF1_FThis paperGenomic DNA PCR primerAAAACGTTTGCCGTGGATGAG
Sequence-based reagenthUPF1_RThis paperGenomic DNA PCR primerCACATAGAGAGCGGTAGGCA
Sequence-based reagenthUPF1-gDNA_FThis paperGuide DNA oligoGCGCGCGGGGCCTCGCCCAT
Sequence-based reagenthUPF1-patient_1-HDR_RThis paperDNA HDR ultramerGCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCCGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGTGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG
Sequence-based reagenthUPF1-patient_9-HDR_RThis paperDNA HDR ultramerGCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCTGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGCGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG
Sequence-based reagenthUPF1-PAM-control-HDR_R (wild type)This paperDNA HDR ultramerGCTCAGTGGTCTTTGCAGCACAGTCTTCACGGCATAAACCTTCAATACAAGCGGCCGTTAGGGGCAGCCTCCGCTTGCGTCCCGGGCCATGGGCGAGGCCCCGCGCGCTGAGGACGGCGCGCACCTG
Sequence-based reagenthUPF1-RT-PCR_FThis paperRT-PCR primerGGATGAGATATGCCTGCGGT
Sequence-based reagenthUPF1-RT-PCR_RThis paperRT-PCR primerTTCTCGTCGGCAGACGACAG
Sequence-based reagentPositive control gBlock to detect UPF1 splice variant (ΔE10-11)This paperDNA gBlockACATGCGGCTCATGCAGGGGGATGAGATATGCCTGCGGTACAAAGGGGACCTTGCGCCCCTGTGGAAAGGGATCGGCCACGTCATCAAGGTCCCTGATAATTATGGCGATGAGATCGCCATTGAGCTGCGGAGCAGCGTGGGTGCACCTGTGGAGGTGACTCACAACTTCCAGGTGGATTTTGTGTGGAAGTCGACCTCCTTTGACAGGCCGGTGCTGGTGTGTGCTCCGAGCAACATCGCCGTGGACCAGCTAACGGAGAAGATCCACCAGACGGGGCTAAAGGTCGTGCGCCTCTGCGCCAAGAGCCGTGAGGCCATCGACTCCCCGGTGTCTTTTCTGGCCCTGCACAACCAGATCAGGAACATGGACAGCATGCCTGAGCTGCAGAAGCTGCAGCAGCTGAAAGACGAGACTGGGGAGCTGTCGTCTGCCGACGAGAAGCGGTACCGGGCCTTGAAGCGCACCGCAGAGAGAGAGCTGCTGATG
Sequence-based reagentmMup1-qPCR_FPMID:25564732qPCR primerGACCTATCCAATGCCAATCG (exon 5/6 junction)
Sequence-based reagentmMup1-qPCR_RPMID:25564732qPCR primerGATGATGGTGGAGTCCTGGT (exon 7)
Sequence-based reagenthβ-globin-qPCR_FPMID:25564732qPCR primerGCTCGGTGCCTTTAGTGATG (exon 2)
Sequence-based reagentmβ-globin-qPCR_RPMID:25564732qPCR primerCCCAGCACAATCACGATCATA (exon 3, mouse specific)
Commercial assay or kitRNeasy Plus Mini KitQiagenCat No. 79654
Commercial assay or kitZero Blunt TOPO PCR cloning systemThermoFisherCat No. K280020
Chemical compound, drugPhosphatase inhibitorThermoFisherCat No. A32959
Chemical compound, drugProtease inhibitorThermofisherCat No. A32963
Chemical compound, drugPenicillin/StreptomycinGIBCOCat No. 15070063
Chemical compound, drugLipofectamine 2000ThermoFisherCat No. 11668030
Chemical compound, drugLipofectamine 3000InvitrogenCat No. L3000001
Chemical compound, drugPuromycinThermoFisherCat No. A1113803
Software, algorithmGuideScan v1.0PMID:28263296http://www.guidescan.com/
Software, algorithmFiji v2.0.0ImageJhttps://imagej.net/Fiji
Software, algorithmRSEM v1.2.4PMID:21816040deweylab.github.io/RSEM/RRID:SCR_013027
Software, algorithmBowtie v1.0.0PMID:19261174github.com/BenLangmead/bowtie/; RRID:SCR_005476
Software, algorithmTopHat v2.0.8bPMID:19289445ccb.jhu.edu/software/tophat/index.shtml
RRID:SCR_013035
Software, algorithmMISO v2.0PMID:21057496genes.mit.edu/burgelab/miso/ RRID:SCR_003124
Software, algorithmIGV v2.3.90Thorvaldsdottirsoftware.broadinstitute.org/software/igv/ RRID:SCR_011793
Software, algorithmPrism v7.0GraphPad Prism v7.0http://www.graphpad.com/
RRID:SCR_002798
OtherSuperScript IV Reverse TranscriptaseThermoFisherCat No. 18090010
OtherQ5 High-Fidelity DNA PolymeraseNew England BiolabsCat No. NEB #M0491
OtherPowerUp SYBER Green Master MixThermoFisherCat No. A25742
OtherRNase-Free DNase SetQiagenCat No. 79254

Additional files

Supplementary file 1

(a) Histopathological classification of pancreatic tumors derived from orthotopic injection of control or Upf1-targeted KPC cells. Tumors displaying >25% squamous differentiation were classified as positive for squamous features. (b) Differentially spliced isoforms identified in 293 T cells engineered to have the ‘patient 1’ UPF1 genotype relative to WT control cells. (c) Differentially spliced isoforms identified in 293 T cells engineered to have the ‘patient 9’ UPF1 genotype relative to WT control cells. (d) Overlap between UPF1 mutations reported by Liu et al. and genetic variants present in the 1000 Genomes, NHLBI Exome Sequencing Project, ExAC, and gnomAD databases. Genomic coordinates are specified with respect to the GRCh37/hg19 genome assembly. (e) Summary statistics of somatic mutations in UPF1 identified in our reanalysis of whole-exome and whole-genome sequencing data from Fang et al. (f) All genetic differences in UPF1 and KRAS from the reference genome, including both somatic mutations and inherited genetic variants, that we identified in our reanalysis of all samples from Fang et al.’s cohorts.

https://cdn.elifesciences.org/articles/62209/elife-62209-supp1-v2.xlsx
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