The preclinical efficacy of a novel telomerase inhibitor, imetelstat, in AML: A randomized trial in patient-derived xenografts

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1 The preclinical efficacy of a novel telomerase inhibitor, imetelstat, in AML: A randomized trial in patient-derived xenografts Claudia Bruedigam, Ph.D Gordon and Jessie Gilmour Leukaemia Research Laboratory Headed by A/Professor Steven Lane

2 Telomerase is activated to maintain the long-term replicative potential in most cancers including AML Telomerase is overexpressed in most AML Roth et al., Leukemia 23 AML oncogenes activate telomerase Gessner et al., Leukemia 21 Calado and Young, N Engl J Med 29 LSC have shortened telomeres and increased telomerase activity Drummond et al., Leukemia 25, Bernard et al., Leukemia 29 Genetic depletion of telomerase eradicates LSC upon enforced replication via cell cycle arrest and apoptosis Bruedigam et al., Cell Stem Cell 214 Bruedigam et al., Cell Stem Cell 214- QIMR Berghofer Medical Research Institute 2

3 Imetelstat (JNJ ) is a competitive inhibitor of telomerase activity Imetelstat is a covalently lipidated 13- mer oligonucleotide that binds the RNA template of telomerase Herbert et al., Oncogene 25 Imetelstat induced molecular and complete hematological responses in essential thrombocythemia (89%) Baerlocher et al., NEJM 215 Ruden et al, Cancer Treatment Reviews 213 Imetelstat showed efficacy in myelofibrosis (complete or partial remission in 21%) Tefferi et al., NEJM 215 Phase II / III trial to evaluate imetelstat in low or intermediate-1 risk myelodysplastic syndrome NCT QIMR Berghofer Medical Research Institute 3

4 Generating an AML patient-derived xenograft inventory Primary AML patient sample Ficoll separation CD3 depletion Blast morphology (Wright-Giemsa: peripheral blood) Donor chimerism [%] Engraftment (Peripheral blood donor chimerism) engrafted not engrafted ongoing Sublethal irradiation (2.8 Gy) AML marker expression (Bone marrow and spleen) Weeks post-transplant NSGS Engraftment AML symptoms hcd45 hgpr mcd hcd45 hcd34 hcd hcd hcd45 Percent survival 1 5 Survival Individual samples tested (#): 35 Engraftment success rate (%): 72 Median survival (days): Days post-transplant QIMR Berghofer Medical Research Institute 4

5 Preclinical testing of imetelstat in AML PDX Age at diagnosis Gender Primary or serial AML patient sample 1 15 Age at diagnosis [years] # AML patient samples 1 5 male female Sublethal irradiation (2.8 Gy) NSGS (CD3 depletion) # AML patient samples ELN prognosis (Doehner et al., Blood 21 Alpermann et al., Blood 211) adverse intermediate 2 intermediate 1 favorable (Check peripheral blood donor chimerism) Randomize Imetelstat (15 mg / kg bw) or PBS tiw ip Engraftment AML symptoms QIMR Berghofer Medical Research Institute 5

6 Imetelstat prolongs overall survival in AML PDX Overall survival Percent survival 1 5 PBS Imetelstat Days post-start of treatment Median survival: PBS: 83 Imetelstat: 153 p <.1 QIMR Berghofer Medical Research Institute 6

7 Imetelstat suppresses AML expansion in 14 out of 15 PDX Overall survival AML expansion Percent survival Days post-start of treatment Median survival: PBS: 83 Imetelstat: 153 PBS Imetelstat Peripheral blood donor chimerism [%] PBS Imetelstat p < Days post-start of treatment QIMR Berghofer Medical Research Institute 7

8 AML PDX can be separated into two groups with distinct response to imetelstat therapy Sustained responders Poor responders Percent survival 1 5 p <.1 Percent survival 1 5 p =.3351 PBS Imetelstat Days post-start of treatment Days post-start of treatment # AML patient samples ELN prognosis (Sustained responders) favorable adverse intermediate 2 intermediate 1 # AML patient samples ELN prognosis (Poor responders) QIMR Berghofer Medical Research Institute 8

9 Next generation sequencing reveals baseline mutations in AML patient samples # AML patient samples NPM1 DNMT3A IDH2 FLT3 NRAS KRAS TET2 ETV6 EZH2 FANCM MKI67 PTPN11 RAD21 SRSF2 WT1 APC ARID1B ASXL1 AXL BCOR BCORL1 BRCA1 BRD4 CD36 CXCR4 ECT2L ESR1 ETS1 FANCF JAK3 KDM2B MAGED1 MSH2 MSH3 PARK2 PLCG2 PTPRD RAD54L RUNX1 SMC1A HemePACT assay in collaboration with Stanley Chun-Wei Lee and Omar Abdel-Wahab, MSKCC SMO STAG1 TLL2 TRAF2 U2AF1 ZNF73 QIMR Berghofer Medical Research Institute 9

10 # AML patient samples The identity and distribution of mutations in selected PDX reflects larger AML cohorts NPM1 DNMT3A IDH2 A No. of Unique Patients with Driver Mutation FLT3 NRAS KRAS TET2 ETV6 EZH2 FANCM MKI67 PTPN11 RAD21 SRSF2 WT1 APC ARID1B ASXL1 AXL BCOR BCORL1 FLT3 NPM1 ELN favorable risk ELN intermediate-i risk ELN intermediate-ii risk ELN adverse risk DNMT3A NRAS complex TET2 7/7q +8/8q IDH2 CEBPA RUNX1 PTPN11 5/5q IDH1 TP53 SRSF2 inv(16) MLL WT1 KRAS 17/17p /11q t(6;9) ELN risk not available ASXL1 KIT STAG2 t(15;17) t(8;21) t(11q23;x) RAD21 9q abn3q 12/12p EZH2 PHF6 Y SF3B1 +21CBL Papaemmanuil et al., NEJM 216 BRCA1 BRD4 CD36 CXCR4 ECT2L ESR1 ETS1 FANCF JAK3 KDM2B MAGED1 MSH2 MSH3 PARK2 PLCG2 PTPRD RAD54L RUNX1 HemePACT assay in collaboration with Stanley Chun-Wei Lee and Omar Abdel-Wahab, MSKCC KDM5A MLL2 ZRSR2 JAK2 CREBBP KDM6A MLL3 BRAF FBXW7 ATRX CUX1 RB1 MPL PRPF4B PTEN U2AF1 BCOR GATA2 NF1 2/2q CDKN2A GNAS MLL5 SF1 U2AF2 CBLB IKZF1 SF3A1 SH2B3 4/4q MYC /18q inv(3)/t(3;3) EP3 ETV6 +13 SMC1A SMO STAG1 TLL2 TRAF2 U2AF1 ZNF73 QIMR Berghofer Medical Research Institute 1

11 Imetelstat response is correlated with a distinct mutational landscape Mutations occuring in both sustained and poor responders NPM1 DNMT3A IDH2 KRAS EZH2 PTPN11 SRSF2 Poor responders Sustained responders QIMR Berghofer Medical Research Institute 11

12 Imetelstat response is correlated with a distinct mutational landscape Mutations occuring in both sustained and poor responders Mutations occuring exclusively in sustained responders NPM1 DNMT3A IDH2 KRAS EZH2 PTPN11 SRSF2 FLT3 NRAS TET2 RAD21 ARID1B CD36 ECT2L JAK3 KDM2B MAGED1 MSH2 MSH3 PARK2 PTPRD RAD54L RUNX1 SMC1A STAG1 ZNF73 Poor responders Sustained responders QIMR Berghofer Medical Research Institute 12

13 Imetelstat response is correlated with a distinct mutational landscape Mutations occuring in both sustained and poor responders Mutations occuring exclusively in sustained responders Mutations occuring exclusively in poor responders NPM1 DNMT3A IDH2 KRAS EZH2 PTPN11 SRSF2 FLT3 NRAS TET2 RAD21 ARID1B CD36 ECT2L JAK3 KDM2B MAGED1 MSH2 MSH3 PARK2 PTPRD RAD54L RUNX1 SMC1A STAG1 ZNF73 ETV6 FANCM MKI67 WT1 APC ASXL1 AXL BCOR BCORL1 BRCA1 BRD4 CXCR4 ESR1 ETS1 FANCF PLCG2 SMO TLL2 TRAF2 U2AF1 Poor responders Sustained responders QIMR Berghofer Medical Research Institute 13

14 Gene ontology analysis of mutually exclusive mutations reveals distinct molecular pathways postreplication repair double-strand break repair response to ionizing radiation DNA recombination chromosome segregation response to radiation DNA repair response to DNA damage stimulus response to abiotic stimulus DNA metabolic process cell cycle process cellular response to stress apoptosis programmed cell death cell death death cell cycle Sustained responders Fold enrichment regulation of myeloid cell differentiation positive regulation of cell differentiation pattern specification process Pathways in cancer immune system development positive regulation of developmental process chromosome organization regulation of programmed cell death regulation of cell death Poor responders Fold enrichment QIMR Berghofer Medical Research Institute 14

15 Gene ontology analysis of mutually exclusive mutations reveals distinct molecular pathways Sustained responders 1. DNA repair postreplication repair double-strand break repair response to ionizing radiation DNA recombination chromosome segregation response to radiation DNA repair response to DNA damage stimulus response to abiotic stimulus DNA metabolic process cell cycle process cellular response to stress apoptosis programmed cell death cell death death cell cycle Fold enrichment Poor responders regulation of myeloid cell differentiation positive regulation of cell differentiation pattern specification process Pathways in cancer immune system development positive regulation of developmental process chromosome organization regulation of programmed cell death regulation of cell death Fold enrichment QIMR Berghofer Medical Research Institute 15

16 Gene ontology analysis of mutually exclusive mutations reveals distinct molecular pathways Sustained responders 1. DNA repair 2. Cell cycle postreplication repair double-strand break repair response to ionizing radiation DNA recombination chromosome segregation response to radiation DNA repair response to DNA damage stimulus response to abiotic stimulus DNA metabolic process cell cycle process cellular response to stress apoptosis programmed cell death cell death death cell cycle Fold enrichment Poor responders regulation of myeloid cell differentiation positive regulation of cell differentiation pattern specification process Pathways in cancer immune system development positive regulation of developmental process chromosome organization regulation of programmed cell death regulation of cell death Fold enrichment QIMR Berghofer Medical Research Institute 16

17 Gene ontology analysis of mutually exclusive mutations reveals distinct molecular pathways Sustained responders 1. DNA repair 2. Cell cycle postreplication repair double-strand break repair response to ionizing radiation DNA recombination chromosome segregation response to radiation DNA repair response to DNA damage stimulus response to abiotic stimulus DNA metabolic process cell cycle process cellular response to stress apoptosis programmed cell death cell death death cell cycle Fold enrichment 3. Development and differentiation regulation of myeloid cell differentiation positive regulation of cell differentiation pattern specification process Pathways in cancer immune system development positive regulation of developmental process chromosome organization regulation of programmed cell death regulation of cell death Poor responders Fold enrichment QIMR Berghofer Medical Research Institute 17

18 Gene ontology analysis of mutually exclusive mutations reveals distinct molecular pathways Sustained responders 1. DNA repair 2. Cell cycle postreplication repair double-strand break repair response to ionizing radiation DNA recombination chromosome segregation response to radiation DNA repair response to DNA damage stimulus response to abiotic stimulus DNA metabolic process cell cycle process cellular response to stress apoptosis programmed cell death cell death death cell cycle Fold enrichment 3. Development and differentiation 4. Pathways in cancer regulation of myeloid cell differentiation positive regulation of cell differentiation pattern specification process Pathways in cancer immune system development positive regulation of developmental process chromosome organization regulation of programmed cell death regulation of cell death Poor responders Fold enrichment QIMR Berghofer Medical Research Institute 18

19 Imetelstat induces DNA damage and loss of quiescence in LSC in vivo AML patient sample Sublethal irradiation (2.8 Gy) NSGS H2AX MFI in G1 LSC [normalized to PBS] H2AX p <.1 LSC in G [%] G p =.2 AML-16 AML-5 AML-18 (Check peripheral blood donor chimerism) Randomize H2AX. PBS Imetelstat PBS Imetelstat PBS Imetelstat Imetelstat (15 mg / kg bw) or PBS tiw ip Endpoint analysis at disease onset of PBS group QIMR Berghofer Medical Research Institute 19

20 Modelling normal human hematopoiesis Cord blood donor sample Sublethal irradiation (2.8 Gy) CD34 enrichment NSG Imetelstat (15 mg / kg bw) or PBS tiw ip 1 weeks Human hematopoiesis phenotype endpoint analysis QIMR Berghofer Medical Research Institute 2

21 Imetelstat primarily depletes B lymphocytes Cord blood donor sample Donor chimerism (Spleen) B cells (Spleen) Myeloid cells (Spleen) Sublethal irradiation (2.8 Gy) NSG CD34 enrichment Imetelstat (15 mg / kg bw) or PBS tiw ip 1 weeks Donor chimerism [%] ** **** Donor 1: PBS Donor 1: Imetelstat Donor 2: PBS Donor 2: Imetelstat CD19+ [% of viable CD45+] * p =.8 Donor 1: PBS Donor 1: Imetelstat Donor 2: PBS Donor 2: Imetelstat CD33+ [% of viable CD45+] * p =.667 Donor 1: PBS Donor 1: Imetelstat Donor 2: PBS Donor 2: Imetelstat Human hematopoiesis phenotype endpoint analysis QIMR Berghofer Medical Research Institute 21

22 Human cord blood - derived stem cells are preserved during imetelstat treatment Cord blood donor sample Sublethal irradiation (2.8 Gy) NSG CD34 enrichment Imetelstat (15 mg / kg bw) or PBS tiw ip 1 weeks Donor chimerism [%] Donor chimerism (Bone marrow) ** **** Donor 1: PBS Donor 1: Imetelstat Donor 2: PBS Donor 2: Imetelstat CD34+CD38- from CD45+ % HSC (Bone marrow) Donor 1: PBS Donor 1: Imetelstat Donor 2: PBS Donor 2: Imetelstat MFI H2AX in G1 CD34+CD H2AX in HSC (Bone marrow) Donor 1: PBS Donor 1: Imetelstat Donor 2: PBS Donor 2: Imetelstat Human hematopoiesis phenotype endpoint analysis QIMR Berghofer Medical Research Institute 22

23 Summary: Preclinical efficacy of imetelstat in AML PDX Imetelstat is effective in a subgroup (6%) of AML patient samples Imetelstat prevents expansion and prolongs overall survival in AML PDX (PBS: 83 days; Imetelstat: 153 days post-start of treatment) Sustained responses to imetelstat are correlated with favorable cytogenetics, mutational profiles of DNA damage and activation of DNA damage response pathways This study has generated preclinical data to inform clinical trials and provide a precision approach to targeted therapies in patients with AML QIMR Berghofer Medical Research Institute 23

24 Acknowledgements QIMR Berghofer Medical Research Institute Gordon and Jessie Gilmour Leukaemia Research Lab - Steven Lane - Brad Wackrow - Axia Song - Amy Porter - Joanne Sutton - Sebastien Jacquelin - Therese Vu - Rebecca Austin - Emma Dishington - Solene Guignes - Jasmin Straube - FACS Core: Grace, Paula, Michael - Animal House: Sue, Jonathan, Dave - Statistics: Leesa, Louise, Mandy, Peter Royal Brisbane and Women s Hospital - Patients and health professionals Memorial Sloan Kettering Institute - Omar Abdel-Wahab - Stanley Chun-Wei Lee The University of Queensland Diamantina Institute, Translational Research Institute - Andrew Moore The University of New South Wales - Richard Lock The University of South Australia - Hamish Scott Funding NHMRC Leukaemia Foundation of Australia Cure Cancer Australia Rio Tinto Ride to Conquer Cancer Janssen (research funding agreement) QIMR Berghofer Medical Research Institute 24