TARGETING STEM CELLS AND ERADICATING CML Pr A

TARGETING STEM CELLS AND ERADICATING CML Pr A. G. Turhan, MD, Ph. D Division of Laboratory Hematology and Oncology, Inserm U 935 “Models of Malignant and Therapeutic Stem Cells” University of Poitiers / University Paris Sud 11, France ali. turhan@univ-poitiers. fr

FROM « LEUCOCYTHEMIA » TO TARGETED THERAPIES 1996 Imatinib (IM) 1845 Bennett, Virchow 1974 t (9; 22) J. Rowley 1960 Ph Chromosome Nowell & Hungerford 1984 BCR-ABL 1999 -2007 IRIS Complete Remissions Resistances Mutations BCR-ABL Novel TKI Dasatinib Nilotinib

HEMATOPOIETIC STEM CELLS (HSC) HSC Quiescence (G 0) ASYMMETRICAL DIVISION Differentiation Self-renewal HSC Mature Cells: Red blood cells, Platelets, Granulocytes OU Progenitors. P

HSC: MAJOR PROLIFERATIVE ACTIVITY HSC Granulocytes Red blood cells # of RBC produced / day (Adult 70 kg)= 210. 000 Platelets ~ 10 Billion cells / hour

CML: A STEM CELL DISEASE WITH CLONAL EVOLUTION Ph+ Quiescent Acquisition Of BCR-ABL Ph+ Normal HSC Clonal Evolution Ph+/ACA Differentiation Arrest Blast Crisis Tough et al, Lancet 1963 Whange et al, Blood 1963 Fialkow et al PNAS 1967 Differentiation to terminal Progeny (Granulocytes, Erythro-Mega, B cells)

NORMAL OR CML HSC CAN BE ASSAYED ONLY USING INDIRECT METHODS Method 1: Long-term Culture Human CD 34+ cells 6 Weeks culture: LTC-IC assay 2 -3 months: NOG-IC assay Method 2: Engraftment In immunodeficient mouse Bone marrow Spleen of mice Detection Of Human cells

DYNAMICS OF STEM CELL PROLIFERATION IN CML Marrow Stem cells and Progenitors in permanent cycle Persistence of normal HSC Small numbers of leukemic stem cells Presence of a highly quiescent fraction At diagnosis Coulombel et al NEJM 1983 Turhan et al, Blood 1989 Udomsakdi et al PNAS 1990 Holyoake et al, Blood 1999 Blood Mobilization of high numbers of leukemic progenitors and LTC-IC Into blood

CML STEM CELLS IN THE ERA OF TARGETED THERAPIES WHY SOME PATIENTS IN COMPLETE MOLECULAR RESPONSE RELAPSE UPON IM DISCONTINUATION? Mahon et al, Lancet Oncol 2010

COEXISTENCE OF MULTIPLE MECHANISMS OF IMATINIB RESISTANCE IN Ph 1+ STEM CELLS CD 34+ Ph 1+ Cell Separation DIVIDING QUIESCENT + GLEEVEC Leukemic Cell death Leukemic cell Survival+++ Graham et al, Blood 2002 CD 34+ Ph 1+ Diagnosis Primitive HSC Cell Separation Committed cells Expression BCR-ABL Expression MDR (Drug Resistance) Jiang et al, Leukemia 2007

KINETICS OF LEUKEMIC CELL REDUCTION IN CML ON TKI THERAPY BCR-ABL/ABL RATIO % 1012 Hematological Response Cycling Amplified / Cells & Progenitors 100 Ph 1+ 10 1 MMR 0. 1 Active Stem RQ-PCR+ 0. 01 0. 001 CMR TKI-S 106 0. 0001 Quiescent TKI-R / Primitive Stem RQ-PCR 0 1?

EVALUATION OF BCR-ABL EXPRESSING LSC IN COMPLETE MOLECULAR RESPONSE Bone Marrow Aspirate in CMR Induced by: IFN (n=3) (OFF Rx) IFN , IM (n=2) (OFF Rx) IM, then DASA (n=1) (ON Rx) 220 colonies / patient Purification CD 34+Cells LTC-IC MS 5 MED CHANGE Hox. B 4/MS 5 (Amplification Of HSC) LTC-IC DERIVED CFU-C Analysis of Stem cell-derived colonies by PCR Chomel et al, Blood 2011

VARIABLE AMOUNTS OF BCR-ABL EXPRESSING STEM CELLS PERSIST IN COMPLETE REMISSION D 0 : Clonogenic Pt Previous Therapies CFU-Cs Pools de 10 CFU-Cs Week 5: LTC-IC-derived CFU Individual MS-5 MS 5/HOXB 4 1 IFN-a 0/20 1/18 4/31 2 IFN-a 0/20 1/19 2/39 4/40 * 3 IFN-a 9/19 11/16 1/30 9/30 * 4 IFN-a; IM 2/20 0/19 0/20 5 IFN-a; IM 1/20 0/20 2/17 * 6 IM, DASA 1/17 23/24 12/43 Pools of 10 LTC-ICs MS-5 MS 5/HOXB 4 1/8 0/19 0/20 Chomel et al, Blood 2011 0/20

DO BCR-ABL EXPRESSING STEM CELLS PERSIST IN PATIENTS IN CMR ON IM vs TKI 2 ? IMATINIB Vs DASATINIB / NILOTINIB « PERSISTEM » STUDY

PERSISTEM STUDY: (Jan 2012) CMR > 1 -2 YEARS INDUCED BY IM : 17 patients INDUCED BY DASATINIB 2 patients Goal: DETECTION & QUANTIFICATION OF BCR-ABL EXPRESSING LSC WHAT IS THE FREQENCY OF LSC MRI POST- IM VS TKI 2 ? POST NILOTINIB VS DASATINIB ?

MECHANISMS OF SURVIVAL OF Ph 1+ STEM CELLS IN THE PRESENCE OF TKI? ABL-K mutations Inefficent drug delivery (transport) « Oncogene. Independence » Alternate mechanisms of survival Survival signals Quiescence signals « LEUKEMIC NICHE »

PRIMITIVE HSC IN CML SURVIVE DESPITE INHIBITION OF ACTIVITY OF BCR-ABL CELL DEATH « Oncogene Addiction » + TKI BCR-ABL + HSC + TKI GROWTH FACTORSSURVIVAL CELLS DO NOT NEED BCR-ABL TO SURVIVE: « Oncogene Independence » HOW ? Corbin et al, JCI 2010

CML STEM CELLS: HOW TO BECOME « ONCOGENE-INDEPENDENT ? » -Compensation of TK-induced signalling by other pathways -Niche -Intrinsic signalling -Down-regulation of BCR-ABL expression

BCR-ABL EXPRESSION IN PROGENITORS AND STEM CELLS AT DIAGNOSIS VS REMISSION 1000 CFU-Cs LTC-ICs n=163 n=58 n=13 n=55 median=52. 1 Median=51 Median=12 Median=0. 7 p<0. 0001 BCR-ABL/ABL IS (%) p=0. 0024 100 10 1 0. 01 Kumari et al Blood 2011 p<0. 0001 BCR-ABL-positive hematopoietic colonies TKI-resistant patient with a T 315 I mutation CML patients in UMRD > 3 years Chomel et al, Blood 2012

CML STEM CELL TARGETING CONDITIONS 1 - IDENTIFICATION OF TARGETS WITH PREFERENTIAL EXPRESSION ON LEUKEMIC STEM CELLS (NO OR LITTLE EXPRESSION ON NORMAL CELLS) 2 - DEVELOPMENT OF NOVEL COMPOUNDS STRATEGIES 1 - WAKE-UP of DORMANT CELLS ? -G-CSF -PLERIXAFOR 2 - TARGET QUIESCENCE / SELF-RENEWAL PATHWAYS 3 -IMMUNOMODULATION

HOW TO TARGET SELF-RENEWAL / QUIESCENCE PATHWAYS CML STEM CELLS ? SHH /SMO Gli, Ptch Self-renewal Expression Increased in CML TGF-b FOXO Wnt/b-catenin Maintenance Of LSC Alox 5 a Expression induced In CML HSC DRUGGABLE TARGETS Naka et al, Nature 2010 ; Zhao et al, Cancer Cell 2007; Dierks et al, Cancer Cell 2008; Ito et al, Nature 2008; Chen Y et al, Nat Genet 2009; Zhao et al, Nature 2009 Zhou et al, J Exp Med 2008; PML FOXO Quiescence AHI-1 Jak 2 STAT 5 SIRT Complex BCR-ABL

TARGETING STAT 5 IN CML Pimozide Differential effect On Normal Versus Leukemic progenitors EFFECTS ON STEM CELLS ? Nelson et al, Blood 2011

TARGETING AUTOPHAGY IN CML STEM CELLS Autophagy: A cell survival mechanism Under stress CD 34+ CML cells (n=3) Leukemic cell TKI Majority Of cells: DEATH TKI SOME CELLS: SURVIVAL BY AUTOPHAGY Can we Inhibit Autophagy To kill CML stem cells ? Bellodi; JCI (2009): 1109 -1123

NOVELS DRUGS TARGETING POTENTIALLY TARGETING CML STEM CELLS TARGET DRUG CLINICAL STUDY SHH/ SMO INHIBITORS BMS ALOX 5 a ZILEUTON UNIV MASS HDAC LBH 589 NOVARTIS PML ARSENIC TRIOXIDE ? STAT 5 PIMOZIDE ?

CONCLUSIONS & QUESTIONS 1 - CML STEM CELLS: LONG-TERM PERSISTENCE AND QUIESCENCE -Low BCR-ABL in persistent cells: Potential Protective Factor from genetic instability and progression -Elucidation of the mechanisms of « dormancy » : -Novel strategies of targeting residual cells are in development

QUESTIONS (2) 2 -WHAT ARE THE EFFECTS OF TKI 2 AS A FIRST LINE THERAPY ON LSC ? 3 - WHAT IS THE DYNAMICS OF Ph+ LSC: -On TKI therapy ? -Off TKI therapy? Stem cell monitoring is required as a decisional tool in TKI discontinuation trials

PERSPECTIVES 2010 - 2020 DECADE: CURATIVE STRATEGIES TKI alone -Single agent -Multi-target/Anti Mutants (T 315 I) TKI + IFN-a Anti-SMO ? Anti-PML « Niche » therapy Arsenic TO ? Autophagy Heterogenity of the disease Predictive tests Stem cell Monitoring and follow-up in CMR

UNIVERSITY OF POITIERS DIVISION OF LABORATORY HEMATOLOGY-/ INSERM U 935 A. G. Turhan JC Chomel ML Bonnet N Sorel A Le Corf MC Meunier D Aggoune C. Bouneau M. Voldoire CIC U 802 Poitiers F. Guilhot J Guilhot L. Roy UNIVERSITY PARIS 11 DEPARTMENT of HEMATOLOGY INSERM U 935 Human IPS /ES Core Facility A. Bennaceur-Griscelli M. Mitjavila F Griscelli COLLABORATIONS E. Oberlin P. Rousselot Versailles I Sloma A. Guerci CHU Nancy S. Flamant D Rea St Louis L Tosca S. Ame Strasbourg O. Feraud A. Marfaing, CHU Paris Sud 11 E. Gobbo JH Bourhis IGR D. Divers S. El Marsafy N Oudrhiri G Tachdjian