title: Melphalan-flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma.
abstract: Chemotherapy options in advanced urothelial carcinoma (UC) remain limited. Here we evaluated the peptide-based alkylating agent melphalan-flufenamide (mel-flufen) for UC.
  
  
  
  UC cell lines J82, RT4, TCCsup and 5637 were treated with mel-flufen, alone or combined with cisplatin, gemcitabine, dasatinib or bestatin. Cell viability (MTT assay), intracellular drug accumulation (liquid chromatography) apoptosis induction (apoptotic cell nuclei morphology, western blot analysis of PARP-1/caspase-9 cleavage and Bak/Bax activation) were evaluated. Kinome alterations were characterized by PathScan array and phospho-Src validated by western blotting. Aminopeptidase N (ANPEP) expression was evaluated in UC clinical specimens in relation to patient outcome.
  
  
  
  In J82, RT4, TCCsup and 5637 UC cells, mel-flufen amplified the intracellular loading of melphalan in part via aminopeptidase N (ANPEP), resulting in increased cytotoxicity compared to melphalan alone. Mel-flufen induced apoptosis seen as activation of Bak/Bax, cleavage of caspase-9/PARP-1 and induction of apoptotic cell nuclei morphology. Combining mel-flufen with cisplatin or gemcitabine in J82 cells resulted in additive cytotoxic effects and for gemcitabine also increased apoptosis induction. Profiling of mel-flufen-induced kinome alterations in J82 cells revealed that mel-flufen alone did not inhibit Src phosphorylation. Accordingly, the Src inhibitor dasatinib sensitized for mel-flufen cytotoxicity. Immunohistochemical analysis of the putative mel-flufen biomarker ANPEP demonstrated prominent expression levels in tumours from 82 of 83 cystectomy patients. Significantly longer median overall survival was found in patients with high ANPEP expression (P = 0.02).
  
  
  
  Mel-flufen alone or in combination with cisplatin, gemcitabine or Src inhibition holds promise as a novel treatment for UC.
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden. Electronic address: kristina.viktorsson@ki.se.
authors.forename: Kristina
authors.initials: K
authors.lastname: Viktorsson
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden; Department of Oncology, Radiumhemmet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
authors.forename: Carl-Henrik
authors.initials: CH
authors.lastname: Shah
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden.
authors.forename: Therese
authors.initials: T
authors.lastname: Juntti
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden.
authors.forename: Petra
authors.initials: P
authors.lastname: Hååg
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Zielinska-Chomej
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden.
authors.forename: Adam
authors.initials: A
authors.lastname: Sierakowiak
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden; Department of Oncology, Radiumhemmet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Holmsten
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden.
authors.forename: Jessica
authors.initials: J
authors.lastname: Tu
authors.affiliation: InSpira Medical AB, SE-135 53 Tyresö, Sweden.
authors.forename: Jack
authors.initials: J
authors.lastname: Spira
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Kanter
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden; Department of Oncology, Radiumhemmet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
authors.forename: Rolf
authors.initials: R
authors.lastname: Lewensohn
authors.affiliation: Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden; Department of Oncology, Radiumhemmet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Ullén
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D000069439:Dasatinib / Q000494:pharmacology*; D006801:Humans; D008558:Melphalan / Q000031:analogs & derivatives* / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D014571:Urologic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D019459:Urothelium / Q000187:drug effects / Q000473:pathology; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
chemical_list: D018906:Antineoplastic Agents, Alkylating; C494772:BAK1 protein, human; D047428:Protein Kinase Inhibitors; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C585069:melflufen; D010649:Phenylalanine; D019061:src-Family Kinases; D008558:Melphalan; D000069439:Dasatinib
keywords: Aminopeptidase N; Apoptosis; Cisplatin; Dasatinib; Gemcitabine; Melphalan-flufenamide; Src; Urothelial carcinoma
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