ESMO ADVANCED COURSE ON NTRK GENE FUSION · • NTRK (Neurotrophic tropomyosin receptor kinase) 1, 2 and 3 : family of 3 genes encoding Trk • Activated by neurotrophins • Nerve

ESMO ADVANCED COURSEON NTRK GENE FUSIONMechanisms of gene fusion, fusion partners and consequences in oncogenesisDescription, 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

http://www.sanger.ac.uk/genetics/CGP/cosmic/http://dbcrid.biolead.org/

• 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

GENOMIC ALTERATIONS IN CANCERDrivers vs Passengers

« 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 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

GENE FUSIONS AND CONJONCTIVE TUMORS

• The 4th edition of the WHO Classification of Tumours of Soft

Tissue and Bone “blue book” was published in February 2013

• > 100 subtypes : complex diagnostic

CONJONCTIVE TUMORSWHO classification

CLASSIFICATION BASED ON LOCATION

CLASSIFICATION BASED ON BIOPATHOLOGY

REFINEMENTS IN SARCOMA CLASSIFICATION

Liposarcomas

Specific translocations (FUS-DDIT3)Myxoïd LPS (15%)

Gene amplification (MDM2 + CDK4)WD/DD LPS (75-80%)

Complex genetic alterationsPleomorpic LPS (5-10%)

HISTORICAL : GENE FUSIONS DISCOVERY

1st specific chromosome change in neoplasia: Philadelphia

chromosome detected in CML

1960 1976 1982 1986

1st characteristic translocation in lymphoma: t(8;14)(q24;q32) in Burkitt lymphoma

1st characteristic translocation in a malignant mesenchymal tumour:

t(2;13)(q36;q14) in ARMS

Discovery of EWSR1–FLI1in Ewing sarcoma

1st characteristic translocation in malignant epithelial tumour:

t(X;1)(p11;q21) in kidney cancer

1992

HISTORICAL : TREATMENTS

First FDA-approved TKI treatment for a neoplasia with specific gene fusion:

Imatinib in CML (BCR-ABL)

20132001 2006

First FDA-approved TKI treatment for a mesenchymal tumour with a specific gene fusion:

Imatinib in DFSP (COL1A1-PDGFB)

First FDA-approved TKI treatment for a carcinoma with a specific gene fusion:

Crizotinib in NSCLC (EML4-ALK)

2018

First FDA-approved TKI treatment for a specific gene fusion regardless tumor origin:

Larotrectinib in NTRK-fused cancers

• 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

FUSION TRANSCRIPT

BALANCED VS UNBALANCED REARRANGEMENTS

Gene rearrangements

Alternative splicing

FUSION GENE

COOHNH3 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

• Ionizing radiation• Chemotherapeutic agents

Thyroid cancer associated with radiation exposure from Chernobyl accident

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.

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.

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• Evaluation in experimental models• Frequency and recurrence• Associated with relatively few other mutations• Restricted to one or a few morphologic subtypes.

CHIMERIC GENES

• Translocations resulting in creation of a chimeric gene• Aberrant gene expression• Coding for a hybrid protein with abnormal activity

• Types of gene involved in the fusions • TF or associated co-factors

• Perturbation (increase or repress) transcription• PK (receptor and non receptor)

• Constitutive activation kinase signaling (dimerization +++)• Nucleoporins (mediates the nucleo-cytoplasmic transport of protein)• Rspondins (involved in WNT signaling)• septins (important role in mitosis)

EXAMPLE OF EWING SARCOMA

• Main fusions

• EWSR1-FLI1 t(11;22)(q24;q12)

• EWSR1-FLI2 t(21;22)(q22;q12)

TRANSCRIPTIONAL DEREGULATION

• Chromosome rearrangements lead to abnormal gene expression• Fusion of strong promoter to proto-oncogene• Example

• Dermatofibrosarcoma protuberans• Fusions of COL1A1 and PDGFRB• Upregulation of the 3ʹ gene by ‘promoter swapping’

• Difficulty of detection• Chromosomal rearrangements that result in the exchange of promoter elements with subsequent

transcriptional upregulation of the complete coding sequence of the two genes could go undetected as there is no chimeric mRNA

Promoter « swapping »

GENE TRUNCATIONS

• Resulting in the inactivation of tumour suppressors

• Example of the BCOR-rearranged sarcomas

• Several chromosome rearrangements described• BCOR-CCNB3• BCOR-ZC3H7B• BCOR-ITD

Description, structure and function of TRK

and NTRK in ontogenesis

HISTORICAL

DEFINITION

• Trk (tropomyosin receptor kinase) A, B and C : family of 3 receptor tyrosine kinases

• NTRK (Neurotrophic tropomyosin receptor kinase) 1, 2 and 3 : family of 3 genes encoding Trk• Activated by neurotrophins

• Nerve growth factor (NGF)• Brain-derived neurotrophic factor (BDNF)• Neurotrophins 3 (NT3)• Neurotrophins 4 (NT4)

• Neurotrophin signaling through these receptors regulates • Cell survival and proliferation• Fate of neural precursors, axon and dendrite growth and patterning• Expression and activity of functionally important proteins (ion channels and neurotransmitter receptors)

DESCRIPTION AND STRUCTUREMature protein and extracellular domain

• Trk are initially synthesized as precursor proteins

• Post-translational glycosylation of the extracellular domains of these precursors• Mature protein products : TrkA (140 kDa), TrkB (145kDa) and TrkC (145kDa)

• Similar structural domains in extracellular region• 2 immunoglobulin-like (Ig1 and Ig2) motifs• 3 leucine-rich 24-residue motifs (LRR1–3)

• Specific to Trk proteins

• Trk A, B and C interacts with different ligands• Predominantly via the Ig2 domain• Proximal transmembrane region

• TRK splice variants with differential affinities for neurotrophins• Isoform TrkAII (non-neuronal tissues) activated by NGF and NT-3• Isoform TrkAI (neuronal tissues) activated only by NGF

• Intracellularly : Tyrosine kinase domain

• Description of isoforms of TrkB and TrkC that lack kinase domain• Expressed at high levels in mature neurons• Dominant-negative isoforms (competing with full-length receptors)

DESCRIPTION AND STRUCTUREInteraction with ligands and intracellular domain

TRK ACTIVATION

MAP-K SIGNALING PATHWAY

• Essential for differentiation of neurons

• Several pathways lead from Trk receptors to activation of Ras

• Intermediates between proximal Trk-induced signaling and activation of MAPK signaling• Various differentiated phenotypes of neurons

PKC SIGNALING

• Phosphorylated TrkA/B/C binds PLCγ• PLCγ is then activated through phosphorylation

• Activated PLCγ plays a crucial role in hippocampal Long-term potentiation (LTP)• Persistent strengthening of synapses based on recent patterns of activity

• ≈ Tetanic stimulation

• Patterns of synaptic activity that produce a long-lasting increase in signal transmission between 2 neurons• 1 of several phenomena underlying synaptic plasticity (ability of chemical synapses to change their strength)

• Memories are thought to be encoded by modification of synaptic strength• LTP widely considered 1 of the major cellular mechanisms that underlies learning and memory

COMPLEXITY

• Wide range of cross-interactions between Trk receptors and neurotrophins

• Trk-neurotrophin interactions depend on many factors

• Binding affinity

• Abundance

• Selectivity

• Extracellular/intracellular modulation by other proteins

P75NTR RECEPTOR

• Alters Trk functions

• Interacts with NT• Influences conformations of Trk• Altering binding specificity and affinity• Altering kinase activity directly in the cytoplasm

• Modulates Trk activity by activation of NF-κB

• Activates distinct signaling pathways antagonistic to those activated by Trk

• Directly induces apoptosis via the JNK-p53-Bax pathway• Suppressed by Trk receptor-initiated signaling

TRK IN DEVELOPMENT AND PHYSIOLOGY

TRK AND ONTOGENESIS

• Trk receptor activation has different consequences depending on• Cell origin and stage of cell differentiation. • Concentration of neurotrophins, type and abundance of neurotrophin receptors

• Neurons• Trk localized on the membrane of the axon end Activated by NT• Transported in vesicles to the body of neurons, by several protein complexes• Promoting neuronal growth, differentiation and survival• Regulation of synaptic strength and plasticity

• Non-neuronal tissues• Vasculature, ovaries and immune system +++

CONSEQUENCES OF TRK INHIBITION

TRK-INDUCED DISEASES

• Various diseases can be caused either by

• NTRK loss-of function mutations

• Impairments synthesis neurotrophins

• Examples

• Loss-of-function NTRK1 mutations• CIPA disease : “congenital insensitivity to pain with anhidrosis”• Hereditary sensory and autonomic neuropathy• Rare inherited disorder• Impaired ability to sense differences in temperature or feel pain.

• Impairments in the BDNF–TRKB axis• Hyperphagia and consequent obesity• Severe ataxia• Defects in memory, learning and nociception

• Cancer genome sequencing has demonstrated that gene fusions are typical features of cancer cells• Consequence of DSB and pathways of non-homologous end-joining

• Fusion genes occur in both hematological malignancies and solid tumors• Passengers >>> Drivers

• Several partners : TF, PK, GF, etc…• Complexity of the pathogenetic impact of the fusions

• Diagnosis and Therapeutic impact

CONCLUSIONSGene fusions

• Oncogene

• Gene expression : nervous system

• Tyrosine kinase receptors

• Ligands : neurotrophins

• Implicated in several gene fusions described in different type of cancer

A changing paradigm for cancer treatment

CONCLUSIONSTrk and NTRK

REFERENCES

• Mertens F, Johansson B, Fioretos T, Mitelman F. The emerging complexity of gene fusions in cancer. Nat Rev Cancer. juin2015;15(6):371-81

• Mertens F, Antonescu CR, Mitelman F. Gene fusions in soft tissue tumors: Recurrent and overlapping pathogenetic themes. Genes Chromosomes Cancer. avr 2016;55(4):291-310

• Mitelman F, Johansson B, Mertens F. Fusion genes and rearranged genes as a linear function of chromosome aberrations in cancer. Nat Genet. avr 2004;36(4):331-4

• Cocco E, Scaltriti M, Drilon A. NTRK fusion-positive cancers and TRK inhibitor therapy. Nat Rev Clin Oncol. 2018;15(12):731-47

• Bunting SF, Nussenzweig A. END-JOINING, TRANSLOCATIONS AND CANCER. Nat Rev Cancer. juill 2013;13(7):443-54

• Lee JJ-K, Park S, Park H, Kim S, Lee J, Lee J, et al. Tracing Oncogene Rearrangements in the Mutational History of Lung Adenocarcinoma. Cell. 13 juin 2019;177(7):1842-1857.e21

• Anderson ND, de Borja R, Young MD, Fuligni F, Rosic A, Roberts ND, et al. Rearrangement bursts generate canonical genefusions in bone and soft tissue tumors. Science. 31 2018;361(6405)

Contacts ESMO

European Society for Medical Oncology Via L. Taddei 4, CH-6962 Viganello – LuganoT. +41 (0)91 973 19 00F. +41 (0)91 973 19 [email protected]

esmo.org

Mehdi BRAHMI, [email protected]

Cancer Reserch Center of Lyon, Team « Biology of rare sarcomas »

Thank you for your attention

Centre Léon Bérard (Lyon), Department of medical oncology

ESMO ADVANCED COURSE�on NTRK Gene fusionDisclosure of interestSlide Number 3INTRODUCTIONGenomic alterations in cancer« Driver » gene fusion and cancer« Driver » gene fusion and cancerGENE FUSIONS AND conjonctive tumorsConjonctive tumorsCLASSIFICATION BASED ON LOCATIONCLASSIFICATION BASED ON BIOPATHOLOGYrefinements in sarcoma classificationSlide Number 13Historical : gene fusions discoveryHistorical : treatmentsSlide Number 16FUSION TRANSCRIPTBalanced vs unbalanced rearrangementsSlide Number 19Fusion GENESpontaneous eventSIMPLE VS COMPLEX REARRANGEMENTSPromoting factorsimportance of location Slide Number 25Transcription Factors (TF)Protein Kinases (PK) and growth factors (gf)Chromatin Regulators dilemmaSlide Number 30DRIVER VS PASSENGERSChimeric genesEXAMPLE OF EWING SARCOMATranscriptional deregulationGene truncationsGene truncationsSlide Number 37historicalDEFINITIONDESCRIPTION AND STRUCTUREDescription and structureSlide Number 42MAP-K signaling pathwayPkc SignalingCOMPLEXITYp75NTR ReceptorTRK in development and physiologyTrk and ontogenesis Slide Number 49Slide Number 50Slide Number 51Trk-induced diseasesconclusionsconclusionsreferencesSlide Number 56

Documents

ESMO ADVANCED COURSE ON NTRK GENE FUSION · • NTRK (Neurotrophic tropomyosin receptor kinase) 1, 2 and 3 : family of 3 genes encoding Trk • Activated by neurotrophins • Nerve