ESMO ADVANCED COURSE ON NTRK GENE FUSION Mechanisms of gene fusion, fusion partners and consequences in oncogenesis Description, Structure and function of TRK and NTRK in ontogenesis Mehdi Brahmi Lyon, 13-14 September 2019 DISCLOSURE OF INTEREST No conflict of interest 1. Mechanisms of gene fusion 2. Gene fusion partners 3. Consequences in oncogenesis INTRODUCTION • Gene fusions (chromosomal rearrangements) • Juxtapositioning of 2 previously independent genes • From 2 nonhomologous chromosomes • Hybrid genes Translation of deregulated and/or chimeric protein • Most common type of structural chromosomal abnormalities • Not exclusive to cancer cells • Screening of cells from developing embryos • Translocations in 0.7 per 1000 live births = de novo • +/- associated to developmental abnormalities in some cases (unbalanced translocations) • Particularly common in cancer cells • Total number of gene fusions > 10 000 • > 90% identified in the past 5 years (deep-sequencing) • Listed in databases • COSMIC; http://www.sanger.ac.uk/genetics/CGP/cosmic/; dbCRID; http://dbcrid.biolead.org GENOMIC ALTERATIONS IN CANCER Drivers vs Passengers • High rate of chromosomal rearrangements in cancer • Most solid tumors • Changes in chromosome number (aneuploidy) • Deletions, inversions, translocations • Other genetic abnormalities • Drivers or Passengers • Passengers > Drivers • Alterations in non coding regions • Easy survival because mutations (TP53, etc…) • Statistical methods to identify “driver genes” • Frequency, Reccurence • Predicted effects « DRIVER » GENE FUSION AND CANCER • Frequency of recurrent gene fusions varies depending on the specific type of cancer • Currently-known to drive ~20% of cancer cases • All major types of tumors • Benign tumors • Myolipoma and HMGA2-C9orf92 fusion transcript • Malignant tumors • Epithelial origins • NSCLC and EML4-ALK fusion transcript • But mostly Hematological malignancies and Mesenchymal malignancies • CML and BCR-ABL fusion transcript • Ewing sarcomas and EWSR1-FLI1 fusion transcript « DRIVER » GENE FUSION AND CANCER • Frequency of recurrent gene fusions varies depending on the specific type of cancer • Currently-known to drive ~20% of cancer cases • All major types of tumors • Benign tumors • Myolipoma and HMGA2-C9orf92 fusion transcript • Malignant tumors • Epithelial origins • NSCLC and EML4-ALK fusion transcript • But mostly Hematological malignancies and Mesenchymal malignancies • CML and BCR-ABL fusion transcript • Ewing sarcomas and EWSR1-FLI1 fusion transcript GENE FUSIONS AND CONJONCTIVE TUMORS • Gene fusions have been described in approximately 1/3 of soft tissue tumors • 142 different fusions have been reported • > 50% recurrent in the same histologic subtype • Pivotal driver mutations • Detection of gene fusions highly important • Knowledge about pathogenetic mechanisms • Strongly associated with a particular histotype = molecular diagnostic markers • Some chimeric proteins, directly or indirectly, constitute excellent treatment targets CONJONCTIVE TUMORS WHO classification th • The 4 edition of the WHO Classification of Tumours of Soft Tissue and Bone “blue book” was published in February 2013 • > 100 subtypes : complex diagnostic CLASSIFICATION BASED ON LOCATION CLASSIFICATION BASED ON BIOPATHOLOGY REFINEMENTS IN SARCOMA CLASSIFICATION Specific translocations (FUS-DDIT3) Myxoïd LPS (15%) Gene amplification (MDM2 + CDK4) Liposarcomas WD/DD LPS (75-80%) Complex genetic alterations Pleomorpic LPS (5-10%) HISTORICAL : GENE FUSIONS DISCOVERY 1st specific chromosome change 1st characteristic translocation in a Discovery of EWSR1–FLI1 in neoplasia: Philadelphia malignant mesenchymal tumour: in Ewing sarcoma chromosome detected in CML t(2;13)(q36;q14) in ARMS 1960 1976 1982 1986 1992 1st characteristic translocation in lymphoma: 1st characteristic translocation in t(8;14)(q24;q32) in Burkitt lymphoma malignant epithelial tumour: t(X;1)(p11;q21) in kidney cancer HISTORICAL : TREATMENTS First FDA-approved TKI treatment for First FDA-approved TKI treatment for a a neoplasia with specific gene fusion: carcinoma with a specific gene fusion: Imatinib in CML (BCR-ABL) Crizotinib in NSCLC (EML4-ALK) 2001 2006 2013 2018 First FDA-approved TKI treatment for a First FDA-approved TKI treatment for a specific mesenchymal tumour with a specific gene fusion: gene fusion regardless tumor origin: Imatinib in DFSP (COL1A1-PDGFB) Larotrectinib in NTRK-fused cancers 1. Mechanisms of gene fusion 2. Gene fusion partners 3. Consequences in oncogenesis FUSION TRANSCRIPT • Rearrangements of chromosomes • Structural rearrangements • Translocations, Inversions, Deletion, Insertion, Duplication • Formation of gene fusions • Translation into fusion transcripts and proteins • Alternative splicing • Splicing of precursor mRNA into different isoforms • Resulting in so-called transcription-induced gene fusions (TIGFs) • Neighbouring genes located on the same DNA strand : cis-TIGFs • Genes located far apart or on different chromosomes : trans-TIGFs • Example of prostate cancer • t(1;1)(q32;q32) : SLC45A3–ELK4 • t(10;10)(q11;q11) : MSMB–NCOA4 BALANCED VS UNBALANCED REARRANGEMENTS Alternative splicing Gene rearrangements FUSION GENE NH3 COOH chimeric protein SPONTANEOUS EVENT • Recurrent translocations in tumors generally thought to arise spontaneously • Very low-frequency events • Strongly selected at the cellular level via their contributions to oncogenesis. • Formation of recurrent reciprocal chromosomal translocations in tumors • During mitosis (DNA replication) • 2 double-strand breaks (DSB) occur at 2 non homologous chromosomes • Escape from normal DSB repair • 4 broken ends synapsed and ligated to form chromosomal rearrangements • NHEJ : Non homologous endogenous End-joining pathway • Error-prone mechanism that does not use any DNA sequence homology to repair the DSB SIMPLE VS COMPLEX REARRANGEMENTS • Complex DNA rearrangements > Simple DNA rearrangements • Events • Chromotripsis • Chromoplexy • Poorer prognosis PROMOTING FACTORS • Translocation breakpoint positions/location : Non-random and recurrent • Spatial proximity of chromosomes in the nucleus • Features of the DNA sequence (repeats, fragile sites and endonuclease misrecognition sites) • Usually conserve reading-frame compatibility • Intrinsic promoting factors • Oxidative metabolism • Replication stress • Extrinsic factors Thyroid cancer associated with radiation exposure from Chernobyl accident • Ionizing radiation • Chemotherapeutic agents Radiation-associated Ewing sarcoma IMPORTANCE OF LOCATION • Chromosomal position of 2 relatively equivalent oncogenes influence ability to contribute to oncogenic translocations • Example of c-Myc and n-Myc • Similar functional activity in vivo including oncogenic potential • Expressed in developing lymphocytes cells • Translocations in B cell and Burkitt’s lymphomas involve c-Myc (but not n-Myc) • Insertion of n-Myc coding sequence in place of c-Myc coding sequence • Competition with normal c-Myc allele to generate recurrent translocations leading to pro-B cell lymphomas • In this context, chromosomal environment might influence • Frequency of DSB around oncogene • Spatial proximity to translocation partner • Alter transcriptional regulation or DNA repair. 1. Mechanisms of gene fusion 2. Gene fusion partners 3. Consequences in oncogenesis TRANSCRIPTION FACTORS (TF) • TF typically occur as C-terminal partners in the fusions • Where they retain their DNA-binding domain(s) in the chimeric protein • Some TF specifically associated with only some neoplasia • Hypothesis • TF implicated in cancers when target genes are in relevance to cells in which tumors originate • Permissive for neoplastic transformation • Example • Congenital spindle cell rhabdomyosarcoma • VGLL2, TEAD1, and SRF • Transcriptional activators of muscle-specific genes PROTEIN KINASES (PK) AND GROWTH FACTORS (GF) • Tyrosine kinases receptors • C-terminal partner in all cases • Results at a protein level : Constitutive activation of the kinase domain • Large variety of different N-terminal partners • Main role : Providing more active promoter and ensure high level of transcription • Other roles • Loss of regulatory elements from the wild-type PK • Contributing oligomerization domains • Ensuring a particular subcellular localization of the chimeric protein • Growth factors • C-terminal partners +++ • Contribution N-ter partner restricted to providing enhanced transcription CHROMATIN REGULATORS • Cellular effects of such fusion proteins are more global than for fusions involving TF • Undifferentiated tumors +++ • Example : URCS with BCOR-CCNB3 fusion DILEMMA • Most human genes and encoded proteins • ≥ 1 function • Depending on how, when and where they are expressed Difficulty to classify gene fusions into distinct, separate subgroups • Examples • EWSR1 protein • RNA-binding +++ but interacts also with other proteins as well as with DNA • NCOA2 protein • Transcription factor but DNA-binding part of the protein never included in the fusion proteins • Complexity of the pathogenetic impact of the fusions • Several fusions listed among TF probably exert pathogenetic impact by affecting chromatin configuration. 1. Mechanisms of gene fusion 2. Gene fusion partners 3. Consequences in oncogenesis DRIVER VS PASSENGERS • Pathogenetic impact of many of the newly detected gene fusions = inherent dilemma with deep sequencing • Strong driver alterations vs passenger mutations caused by the increased genetic instability that is a hallmark of many malignant neoplasms • Arguments