title: Clonal inventory screens uncover monoclonality following serial transplantation of MGMT P140K-transduced stem cells and dose-intense chemotherapy.
abstract: Gene transfer of mutant O(6)-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs. However, insertional mutagenesis, high regenerative stress associated with selection, and the genotoxic potential of alkylating drugs represent considerable risk factors for clinical applications of this approach. Therefore, we investigated the long-term effect of MGMT(P140K) gene transfer followed by repetitive, dose-intensive treatment with alkylating agents in a murine serial bone marrow transplant model and assessed clonality of hematopoiesis up to tertiary recipients. The substantial selection pressure resulted in almost completely transduced hematopoiesis in all cohorts. Ligation-mediated PCR and next-generation sequencing identified several repopulating clones carrying vector insertions in distinct genomic regions that were ∼ 9 kb of size (common integration sites). Beside polyclonal reconstitution in the majority of the mice, we also detected monoclonal or oligoclonal repopulation patterns with HSC clones showing vector insertions in the Usp10 or Tubb3 gene. Interestingly, neither Usp10, Tubb3, nor any of the genes located in common integration sites have been linked to clonal expansion in previous preclinical or clinical gene therapy trials. However, a considerable number of these genes are involved in DNA damage response and cell fate decision pathways following cytostatic drug application. Thus, in summary, our study advocates ligation-mediated PCR and next generation sequencing as an effective and reliable method to identify gene products associated with clonal survival in specific experimental settings such as chemoselection using alkylating agents.
authors.affiliation: Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg 69120, Germany.
authors.forename: Frank A
authors.initials: FA
authors.lastname: Giordano
authors.forename: Ursula R
authors.initials: UR
authors.lastname: Sorg
authors.forename: Jens-Uwe
authors.initials: JU
authors.lastname: Appelt
authors.forename: Nico
authors.initials: N
authors.lastname: Lachmann
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bleier
authors.forename: Ingo
authors.initials: I
authors.lastname: Roeder
authors.forename: Veronika
authors.initials: V
authors.lastname: Kleff
authors.forename: Michael
authors.initials: M
authors.lastname: Flasshove
authors.forename: W Jens
authors.initials: WJ
authors.lastname: Zeller
authors.forename: Heike
authors.initials: H
authors.lastname: Allgayer
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Stefan
authors.initials: S
authors.lastname: Fruehauf
authors.forename: Thomas
authors.initials: T
authors.lastname: Moritz
authors.forename: Stephanie
authors.initials: S
authors.lastname: Laufs
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D016026:Bone Marrow Transplantation; D002999:Clone Cells; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006410:Hematopoiesis / Q000187:drug effects / Q000235:genetics*; D006412:Hematopoietic Stem Cells; D051379:Mice; D023421:Models, Animal; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D000477:Alkylating Agents; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes