ONCOGENETICS „Origin, evolution and treatment of cancer“ Assoc. Prof. Martin Trbušek, Ph.D. Department of Internal Medicine – Hematology and Oncology University Hospital Brno Faculty of Medicine, Masaryk University CANCER: definition and basic classification CANCER is an abnormal cell growth with subsequent spreading throughout the body creating metastases Basic division follows the cell (tissue) of origin: Carcinomas derive from an epithelial tissue - e.g. breast, lung, colon or pancreatic cancer Sarcomas originate from mesenchymal cells (conective tissue) – e.g. bone tumors Cancer of blood cells or hematopoietic system – leukemias and lymphomas Germ cell tumors – e.g. ovarian cancer or seminomas Somatic mutation theory (SMT) vs. Tissue organization field theory (TOFT) Sonnenschein and Soto, Progress Biophys Mol Biol, 2016 Šmardová and Koptíková, Klinická onkologie 2016 Origin of cancer: conceptual theories SMT: Default setting of a cell is quiescence and cancer represents „an escape“ from it. Malignant cell manifests a selective growth advantage over healty counterparts. TOFT: Default setting of a cell is infinite proliferation (phylogenetically) These are tissues what keep our cells in a resting stage and prevent their unlimited proliferation Inherited tumors (incl. hereditary cancer syndromes) 5-10% of all cancer cases e.g. Li-Fraumeni syndrome associated with TP53 mutations or xeroderma pigmentosum involving mutations in DNA repair genes Sporadic tumors – the rest, originate in a somatic tissue Genetic defects are underlying cause in both cases; In addition, 15-20% of cancer involve an infectious agent (causality) e.g. high risk HPVs in cervical carcinoma Origin of cancer: role of heredity (Specific) aetiology of childhood leukemia Greaves , Nat Rev Cancer 2016 https://media.springernature.com/m685/nature-assets/nrc/journal/v6/n3/images/nrc1816-f1.jpg Analysis of „Guthrie cards“ or cord blood cells …in monozygotic twins Contribution of leukemia and lymphoma research to the SMT Greaves , Nat Rev Cancer 2016 Leukemias and lymphomas represent up to 10% of all cancers worldwide Leuk and Lymp: hallmark aberrations enable molecular classification •Blood cancers have got quite clear „accomplices“ •Typical translocations •Chronic myelogenous leukemia; t(9;22) BCR-ABL •Mantle cell lymphoma; t(11;14) Cyclin D1/IgH •Folicullar lymphoma; t(14;18) Bcl-2/IgH •Burkitt lymphoma; t(8;14) c-Myc/IgH • •… or other characteristic aberrations •Chronic lymphocytic leukemia; del 13q, del 11q, del 17p, trisomy 12 • Classic hallmarks of cancer Hanahann and Weinberg, Cell 2001 Emerging (additional) hallmarks of cancer Hanahann and Weinberg, Cell 2011 Reprogramming of a cellular metabolism and an escape from the immune system Role of cancer stem cells (CSC) in tumor initiation and progression Human body contains ~1014 cells ~1011 cells are renewed every day from the stem cells Only a proportion of cancer cells (CSC) in a given tumor population is able to self-renew (proliferate) infinitely Adopted from Batlle and Clevers Nature Med 2017 Cellular origin of cancer vs. therapy Tumors originate from stem cells or progenitor cells, the development of which is skewed by favoring self-renewal over differentiation This phenomenon hardly aggravates successfull (curable) therapy through a minimal residual disease presence and subsequent relapse based on a resistant clone proliferation mutTP53 mutTP53 wtTP53 wtTP53 BEFORE THERAPY AFTER THERAPY • Clonal evolution and a narrow throat of therapy: •case of AML Ding et al.,Nature 2012 Gene mutations as a hallmark of cancer Výsledek obrázku pro sanger sequencing gel Classic PAGE 35S labelling Výsledek obrázku pro frederick sanger Frederick Sanger Cambridge University Stratton et al., Nature 2009 Breath-taking technological advancements in DNA sequencing Simon et al., Nat Rev Drug Discovery 2013 State-of-the-art: custom-directed NGS Genome Exome Amplicon Transcriptome Cancer Genome Landscapes Vogelstein et al. Science 2013 Driver mutations vs. Passenger mutations Driver genes: ~125 71 TS/54 ONC PRINCIPALS OF DARWINIAN SELECTION Additional cancer genome modifications •Epigenetic silencing of tumor-suppressor genes (promoter methylation) •Global (whole-genome) hypomethylation Výsledek obrázku pro bisulfite sequencing Recurrent mutations in cancer – CLL as an example The most frequent mutations in the genes: SF3B1, ATM, TP53 Landau et al., Nature 2015 Count Coverage Frequency Gene_function RefGene Exon_number cDNA Codon 1752 1752 100 exonic ATM exon40 c.5948A>G p.N1983S 2261 2452 92,21 exonic ATM exon22 c.3161C>G p.P1054R 690 2962 23,3 exonic ATM exon50 c.7311C>A p.Y2437X 100 1203 8,31 exonic ATM exon24 c.3433_3435del p.1145_1145del 74 1433 5,16 exonic ATM exon30 c.4578C>T p.P1526P 46 1281 3,59 exonic ATM exon43 c.6258T>A p.Y2086X 243 8231 2,95 splicing ATM exon19 c.2921+1G>A p.P962Q 19 699 2,72 exonic ATM exon25 c.3705_3709del p.P1235fs 25 1087 2,3 exonic ATM exon5 c.480delT p.S160fs 24 1046 2,29 exonic ATM exon5 c.483G>C p.Q161H 67 3357 2 exonic ATM exon26 c.3837G>A p.W1279X 73 5626 1,3 exonic ATM exon26 c.3952_3960del p.1318_1320del 64 5151 1,24 exonic ATM exon49 c.7181C>T p.S2394L 11 904 1,22 exonic ATM exon63 c.9022C>T p.R3008C 42 3514 1,2 exonic ATM exon10 c.1402_1403del p.K468fs Intraclonal heterogeneity wthinin tumor population (12) affected biochemical pathways in cancer Vogelstein et al., Science 2013 99.9% of all alterations in cancer cells provides no selective growth advantage Mutability of human genome is normal; However, normal is also to avoid aberrant, dangerous cells through continuously operating apoptosis…. Model of tumor initiation and progression Bartkova et al., Nature 2005 Interfence with DNA replication results in apoptosis induction in tumor cells Cleaved proteins PARP and Caspase-3 demonstrate a presence of advanced apoptosis after the Chk1 inhibition; cells: MEC-1, TP53-mutated CLL C:\Users\3755\Downloads\MEC-1 48h EN1, ATRi vše.jpg •Physical cell distruction • • • • • • • •„Trash“ elimination (recycling) Apoptosis: „optimal cell death“ in cancer therapy Kerr et al., Br J Cancer 1972 Discovery of p53 protein: a milestone in oncology research David P. Lane Imperial Cancer Research Fund, London Arnold J. Levine Princeton University, New Jersey Lloyd John Old Memorial Sloan-Kettering Cancer Center, New York Reported in 1979, interaction with a T-antigen of SV40 virus http://www.a-star.edu.sg/portals/2/people/David_Lane.jpg http://www.sns.ias.edu/~alevine/PR-Levine.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/3/3d/Lloyd_J._Old%2C_M.D.%2C_c._1995.jpg/220px -Lloyd_J._Old%2C_M.D.%2C_c._1995.jpg The p53 research from the historical perspective Eliyahu D et al. Participation of p53 cellular tumour antigen in transformation of normal embryonic cells. Nature 1984; 312: 646-9. Parada LF et al. Cooperation between gene encoding p53 tumour antigen and ras in cellular transformation. Nature 1984; 312: 649-51. Jenkins JR et al. Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53. Nature 1984; 312: 651-4. Oncogene or tumor-suppressor? Impact of the TP53 gene disruption on tumor development Donehower et al., Nature 1992 Adopted from: IARC TP53 database Elefants have low cancer rates (Peto paradox) This is (among others) owing to ~20 copies of the TP53 gene DNA damage Hypoxia Telomere shortening Cell cycle checkpoints p21WAF 14-3-3s GADD 45 Reprimo Apoptosis PUMA NOXA Bax KILLER/DR5 p53AIP1 Fas/Apo1/ CD95 PIGs Senescence p21WAF Inhibition of pathological angiogenesis Tsp1 BAI1 Maspin GD-AIF p14ARF ? p53 MDM2 ATM ATR DNA PK Oncogene activation DNA repair GADD45 p48 p53R2 p53 ubiquitination and degradation ? Post-translational modification of p53 activation and stabilisation Transcription- dependent role of p53 Podrobněji k dráze p53: poškozeni (…) - signalizováno dalšími proteiny - modifikace p53, aktivace a stabilizace. Aktivovaný p53¨- aktivace transkripce genů zahrnutých v… i represe a protein-protein interakce Přísná regulace hladiny p53 v normální buňce - přes MDM2. p53 - tr. faktor, funguje jako tetramer, 393 aminokyselin, 50 kDa • • • • • • •DNA Cancer from the point of view of the cell cycle G1 → S → G2 → M G1/S S G2/M Universal inactivation Loss of p53, Rb, p16 etc. Checkpoints Ability to continue with the cell division Analysis of the TP53 gene in CLL patients in the University Hospital Brno Del(17p) using I-FISH Mut TP53 using FASAY and DNA sequencing Wt Mut 17p- TP53 defects impair a therapeutic response Malcikova J et al., Blood 2009 Test of cellular viability in vitro Treatment FLU 48 h DNA damage induces p53-dependent response Real time PCR, treatment 24 h Herndon et al., Leukemia 2017 2H2O accumulation in leukemic cells located in LNs TP53 defects support tumor cells´ proliferation p53 mutations associate with poor survival in CLL patients A: wt-p53/mut-IgVH MS: not reached B: mut-p53/mut-IgVH MS: not reached C: wt-p53/unmut-IgVH MS: 69 months D: mut-p53/unmut IgVH MS: 23 months (A)vs. (B) P=0.016 (B)vs. (D) P=0.018 (C)vs. (D) P<0.001 (A) vs.(C) P<0.001 Note: survival assessed from time of p53 defect identification / investigation showing wt-p53 Individual p53 mutations differ in their impact Fig. A: all mutations Fig. B: mutation + del(17p) A: wt-p53 MS: 69 months B: nonmissense p53 mutations MS: 36 months C: p53 missense out of DBMs MS: 41 months D: p53 missense in DBMs MS: 12 months (D) vs. (C) P=0.009 (D) vs. (B) P=0.002 Trbusek et al., J Clin Oncol 2011 Prognostic impact of TP53 mutations in cancer Adopted from: Robles AI, Harris CC: Clinical outcomes and correlates of TP53 mutations and cancer. Cold Spring Harb Perspect Biol 2010; 2: a001016 p53 activation: breaking a loop with MDM2 Adopted from: IARC TP53 database Impact of ATM defects on p53 activation Odd columns: controls Even columns: IR (5Gy) 1,2 – wt 3,4 – sole 11q- 5,6 – ATM-mut-1 7,8 – ATM-mut-2 Navrkalova et al., Haematologica 2013 Onocogenes: driving cancer cell´s proliferation CLL patients WB c-Myc Frequently TFs Cooperation ONC/TS Treatment of cancer •Surgery (primary site, localized matastases); local radiotherapy • Systemic therapies • •(Combination) chemotherapy; total body irradiation • •Stem cell transplantation (hematopoiteic and solid tumors) • •Immunotherapy , including „CAR T-lymphocytes“ • •„Differentiation“ therapy (e.g. ATRA in APML) • •Use of monoclonal antibodies • •Targeted therapy (small molecule inhibitors) Progress in the treatment of cancer •Satisfactory outcomes • •Chronic myeloid leukemia • •Some childhood leukemias (e.g. ALL, ETV6-RUNX1-positive) • •Hodgkin´s lymphoma • •Testicular tumor in young men Favorable genetic features: ØHallmark abnormality, low genomic instability ØLow pressure to inactivate the TP53 tumor-suppressor gene Progress in the treatment of cancer •Unsatisfactory outcomes • •Malignant melanoma (metastatic variant, OS ˂10% at 5 years) • •TP53-mutated chronic lymphocytic leukemia (median OS ~3 years) • •Cervical carcinoma (high-risk HPVs, direct p53 inactivation) Unfavorable genetic features: ØGenetic heterogeneity of tumor cell population ØInactivation of genes responding to therapeutic intervention within the DNA damage response (DDR) pathway Treatment „by differentiation“: APL Greaves, Nat Rev Cancer 2016 Therapy using monoclonal antibodies Výsledek obrázku pro ofatumumab mechanism of action •Targeting to a cell surface epitope (specificity vs.effectivity) •1st mAb in clinic: rituximab, 1997 • •Available also fo solid cancers (e.g. trastuzumab in breast cancer) •Complex machanisms of action (CDC, ADCC, apoptosis) Protein targeting (inhibition) using small molecules Adopted from: Vogelstein B et al. Cancer Genome Landscapes, Science 2013 ØKinases: relatively „easy“inhibition of enzymatic activity All clinically approved small molecule drugs target kinases ØOncogenes: only minority of them have enzymatic activity In contrast, many oncogenes have multiple interactions ØTumor-suppressors: very difficult replacement of the lost function. An option is to target a complementary activated pathway (e.g. BRCA loss → addiction to PARP activity). Ø Synthetic lethality within DNA damage response Adopted from: Fang B, J Med Chem 2014 Specific targeting may lead to distinct outcomes Mutation V600E in BRAF protein is detected in malignant melanoma (MM) as well as in metastatic colorectal cancer (CRC) However, a specific inhibitor of BRAF signalling (Vemurafenib) is hihgly effective in MM, but not in CRC The reasons is an activation of the PI3K/AKT pathway eliminating the effect of the inhibition in the latter cancer Current portfolio of specific molecular targeting Summary •Cancer is a „disease of genes“, regardless of the presence or absence of a heritable predisposition •Genetic background of different cancers have some common features, but overall variability is huge and requires „the cancer-specific“ approach • •Major obstacle of effective therapy represent in many cancers defects in the TP53 gene (or the p53 pathway in general) •Technologial advancements in tumor cell analyses are enormous (e.g. NGS), however the data interpretation remains sometimes (frequently?) elusive •Molecular therapy seems to be directed to a patient-specific „coctail“ of several drugs with accompanying mechanisms of action (no „one pill“ at horizon…..) THANK YOU VERY MUCH FOR YOUR ATTETION! m.trbusek@volny.cz