title: Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM.
abstract: Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Nidhan K
authors.initials: NK
authors.lastname: Biswas
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Vikas
authors.initials: V
authors.lastname: Chandra
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Neeta
authors.initials: N
authors.lastname: Sarkar-Roy
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Tapojyoti
authors.initials: T
authors.lastname: Das
authors.affiliation: AMRI Hospitals, JC-16 Salt Lake City, Kolkata 700098, India.
authors.forename: Rabindra N
authors.initials: RN
authors.lastname: Bhattacharya
authors.affiliation: Medica Superspeciality Hospital, 127 Mukundapur, Kolkata 700099, India.
authors.forename: Laxmi N
authors.initials: LN
authors.lastname: Tripathy
authors.affiliation: Medica Superspeciality Hospital, 127 Mukundapur, Kolkata 700099, India.
authors.forename: Sunandan K
authors.initials: SK
authors.lastname: Basu
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Shantanu
authors.initials: S
authors.lastname: Kumar
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Subrata
authors.initials: S
authors.lastname: Das
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Ankita
authors.initials: A
authors.lastname: Chatterjee
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Ankur
authors.initials: A
authors.lastname: Mukherjee
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Pryiadarshi
authors.initials: P
authors.lastname: Basu
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Arindam
authors.initials: A
authors.lastname: Maitra
authors.affiliation: University of Pittsburgh, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
authors.forename: Ansuman
authors.initials: A
authors.lastname: Chattopadhyay
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Analabha
authors.initials: A
authors.lastname: Basu
authors.affiliation: National Institute of Biomedical Genomics, Kalyani, West Bengal 741251, India.
authors.forename: Surajit
authors.initials: S
authors.lastname: Dhara
mesh_terms: D000483:Alleles; D000813:Anilides / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D003606:Dacarbazine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D059472:Exome; D005260:Female; D005500:Follow-Up Studies; D016195:G2 Phase / Q000187:drug effects / Q000235:genetics; D005787:Gene Frequency; D005909:Glioblastoma / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011859:Radiography; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology; D000077204:Temozolomide
chemical_list: D000813:Anilides; D053823:Hedgehog Proteins; C538724:HhAntag691; D009363:Neoplasm Proteins; D011725:Pyridines; C509239:SHH protein, human; D003606:Dacarbazine; D000077204:Temozolomide