Getting started in research

Transcription

1 Getting started in research Assoc. Prof. Steven Lane MBBS (Hons) PhD FRACP FRCPA Team Head: Translational Leukaemia Research Laboratory Queensland Institute of Medical Research Clinical Haematologist: Royal Brisbane and Women s Hospital Queensland Institute of Medical Research 1

2 First up, a short introduction My career path (to date) MBBS at University of Queensland ( ) Clinical training ( including 2002 in the UK) Clinical Haematologist (FRACP) and Haematopathologist (FRCPA) PhD UQ Research Fellowship Harvard Medical School ( ) Team Head, QIMR (B) Consultant haematologist, RBWH 2011-

3 Myeloid blood cancers Myeloid malignancy Acute Myeloid Leukaemia (AML) Myeloproliferative neoplasm (MPN) Myelodysplastic syndromes Approximately 2000 patients diagnosed/yr Limited treatment options Supportive care (transfusions etc) Chemotherapy Bone marrow transplantation Targeted therapy (last 10 years)

4 A brief note about clinical research Audits, case series, retrospective reviews A great way to understand the process of scientific rigor, communication and eventually publication Generally low impact, few citations however may be important findings for your field Clinical research is possible in some areas Limited by competition Conflicting interests of drug companies Difficulties with recruitment The BIG changes in clinical medicine come from basic scientific reearch Imatinib in CML and other targeted therapies Vaccine for cervical cancer Your enthusiasm and self-motivation will be obvious These are key indicators to future employers/ colleges/ fellowships

5 Getting started in research Better get yourself a mentor (son), better get a REAL good one (or more) I have been very fortunate to have great mentors along the way Peter Mollee PAH Haematology Andrew Boyd RBWH/ QIMR Gary Gilliland Harvard Professor Geoff Hill Haematologist/ Bone Marrow Transplant Physician Director of Research, RBWH Cancer Care Director of Cancer Program, QIMR Berghofer NHMRC Australia Fellow Tragic NZ rugby fan Professor David Williams Director Hematology/ Oncology Children s Hospital Boston, DFCI Harvard Medical School, Boston President American Soc. Hematology All round good guy

6 I decided to go overseas to do a research fellowship Do something different Others were doing clinical fellowships What can you bring back that makes you unique? Travel somewhere new Go to the best place you can possibly imagine/ think of Do not give up Professor D. Gary Gilliland Harvard Medical School, Boston

7 Myeloproliferative neoplasms (MPN) Group of malignant blood disorders Proliferation of mature blood cells and accumulation in the blood, bone marrow and spleen jakstatsignaling.com

8 Introducing the myeloproliferative neoplasms (MPN) Chronic myeloid leukaemia Too many white blood cells BCR-ABL Stem cells Polycythemia vera Too many red blood cells Transformation Myeloid progenitors Essential thrombocythemia Too many platelets Acute leukaemia Myelofibrosis Mastocytosis Graphics: M.Flynn Adapted from Levine and Gilliland, Nat Rev Cancer 2007

9 CML and imatinib: the silver bullet Druker, Blood 2008

10 CML and imatinib: the silver bullet Druker, NEJM 2001 Druker, NEJM 2006

11 Non-CML myeloproliferative neoplasms (MPN) Chronic myeloid leukaemia Too many white blood cells Stem cells Myeloid progenitors Polycythemia vera Too many red blood cells JAK2 V617F MPL W515L Essential thrombocythemia Transformation JAK2 V617F MPL W515L Acute leukaemia Myelofibrosis Too many platelets Mastocytosis Graphics: M.Flynn Adapted from Levine and Gilliland, Nat Rev Cancer 2007

12 The discovery of JAK2 V617F in human myeloproliferative neoplasms (MPN) A recurrent mutation in JAK2 tyrosine kinase from patients with MPN, JAK2 V617F Causes ligand independent activation of JAK2 signaling Most common mutation in polycythemia vera (95%), essential thrombocythemia (~50%) and myelofibrosis (~50%) Present in hematopoietic stem cells Mutations in exon 12 of JAK2 also found in erythrocytosis Kaushansky, JCI 2005 James, et al. Nature 2005 Levine, et al. Cancer Cell 2005 Kralovics, et al. N Engl J Med 2005 Baxter, et al. Lancet 2005

13 JAK2 signaling is turned on by cytokines EPO/ TPO and IL3 TPO EPO IL3 EpoR, Mpl, Il3R Jak2 Stat p p Jak2 Stat PI3K/AKT/mTOR Stat 1,3,5 p Ras/MEK/ERK Survival Proliferation Graphics: M.Flynn Adapted from Levine and Gilliland, Nat Rev Cancer 2007

14 JAK2 V617F causes ligand independent pathway activation TPO EPO IL3 EpoR, Mpl, Il3R Jak2 V617F Stat p p Jak2 V617F Stat PI3K/AKT/mTOR Stat 1,3,5 p Ras/MEK/ERK Survival Proliferation Graphics: M.Flynn Adapted from Levine and Gilliland, Nat Rev Cancer 2007

15 A new model of Jak2V617F MPN Jak2 exon14 WT E2ACre exon14 V617F Mx1Cre exon14 V617F Jak2 WT configuration Jak2 VF configuration Jak2 WT Jak2 VF G1849T Ann Mullally, Harvard Medical School

16 Red blood cells Jak2V617F causes a myeloproliferative neoplasm (MPN) resembling human polycythemia vera Jak2 WT Jak2 VF Reference range Spleen Mullally A *, Lane SW *, et al. Cancer Cell 2010 *equal contribution

17 Jak2V617F-induced myeloproliferative neoplasm (MPN) is lethal thrombotic complications

18 FcγR ckit Which cells are responsible for initiating Jak2V617F MPN LKS + LKS+ CD Gy + Support BM Recipient CD45.1 Sca-1 MEP GMP CMP Jak2 V617F+ Progenitors CD Support BM 11Gy Recipient CD45.1 CD34

19 Hematocrit (%) The Jak2V617F MPN-initiating cell is contained within the HSC-enriched LKS+ compartment Weeks Post BMT 2º Jak2 +/+ LKS + Reference range

20 Hematocrit (%) The Jak2V617F MPN-initiating cell is contained within the HSC-enriched LKS+ compartment Weeks Post BMT 2º Jak2 +/VF MEP 2º Jak2 +/VF GMP 2º Jak2 +/+ LKS + Reference range

21 Hematocrit (%) The Jak2V617F MPN-initiating cell is contained within the HSC-enriched LKS+ compartment Weeks Post BMT 2º Jak2 +/VF LKS + 2º Jak2 +/VF MEP 2º Jak2 +/VF GMP 2º Jak2 +/+ LKS + Reference range

22 Fortuitously, clincial trials were rolling on: Ruxolitinib in MPN COMFORT I Patients with MF (N = 309) Randomized 1:1 USA, Canada, Australia INC424 (oral) 15 mg BID or 20 mg BID Placebo (oral) BID COMFORT I Primary Endpoint Number of subjects achieving 35% reduction in spleen volume from baseline to week 24* COMFORT II Patients with MF (N = 219) Randomized 2:1 INC424 (oral) 15 mg BID or 20 mg BID Best available therapy COMFORT II Primary Endpoint Number of subjects achieving 35% reduction in spleen volume from baseline to week 48* EUROPE: Austria, Belgium, France, Germany, Italy, Netherlands, Spain, Sweden, UK

23 JAK2 V617F levels by QPCR Patients treated with JAK2 inhibitors have reduction in spleen size but only minor effect on disease burden (by QPCR) Spleens get smaller! Minor (and variable) reduction in disease burden See also Harrison et al, Verstovek et al. NEJM, 2012 and commentary by Tefferi Verstovek, NEJM 2010, 363: Pardanani, J Clin Oncol, 2011, 29:

24 Can we treat MPN by simply turning off Jak2 signaling? TPO EPO IL3 EpoR, Mpl, Il3R Jak2 V617F Stat p p Jak2 V617F Stat PI3K/AKT/mTOR Stat 1,3,5 p Ras/MEK/ERK Survival Proliferation Graphics: M.Flynn Adapted from Levine and Gilliland, Nat Rev Cancer 2007

25 Jak2 inhibitors are effective in reducing spleen size Jak2VF Vehicle Primary Jak2 V617F mice Jak2VF JAK2 inhibitor Jak2 Inhibitor TG101348

26 Jak2 inhibitor treatment does not eradicate MPN stem cells Jak2 +/VF donor JAK2 Inhibitor (6wks) or Vehicle Control (6wks) Jak2 +/VF LKS 11Gy JAK2 Inhibitor (10wks) or Vehicle Control (10wks) Reference range Jak2 +/VF BM 11Gy Examine disease phenotype Jak2 Inhibitor TG Cancer Cell, 2010

27 What s the point of working on stem cells if you can t do anything to target them? Using clinical questions to guide your research

28 Interferon-alpha (IFNα) in the treatment of patients with MPN IFNα has clinical efficacy in JAK2 V617F polycythemia vera Most patients achieve complete hematological responses Reduced JAK2V617F allelic burden Complete molecular responses in15% (30% by 4 years) Mechanism of IFNα in JAK2 V617F MPN unknown Immunomodulatory Anti-proliferative Direct cytotoxicity Kiladjian, Blood 2008, 2006 Larsen, Hematology, 2009 Quintas-Cadama, J Clin Oncol 2009 Turlure, ASH 2011 Kiladjian, Leukemia 2009 Lu, Blood 2010 Platanias, Nat Rev Immunol 2005 Activation of normal HSC leading to impaired function Essers, Nature 2009 Sato, Nat Med 2009 Hartner, Nat Immunol 2009

29 HCT % WCC (*10 3 /ml) Spleen weight (mg) IFNα is effective in treating murine Jak2 V617F MPN Jak2 VF donor CD45.2 engraftment Interferon alpha 4 weeks Jak2 WT donor CD45.1 Recipient Reduction in polycythemia Reduction in leukocytosis Reduction in spleen size 100 p< p< p< Vehicle IFNa 0 Vehicle IFNa 0 Vehicle IFNa Blood, 2013

30 CD150 LT-HSC #/ hind limbs CD150 IFNα depletes Jak2 V617F long-term disease-initiating HSC Jak2 VF donor CD45.2 Jak2 WT donor CD45.1 Recipient engraftment Interferon alpha 4 weeks Gated on lineage low ckit high Sca1 + Long-term HSC CD WT % 5 Vehicle 6.3 % 10 4 Jak2 VF Vehicle p= p= WT % 5 IFNa 0.55 % 10 4 Jak2 VF IFNa WT CD Jak2 VF Vehicle IFNα

31 Targeting LSCs in myeloproliferative neoplasms 1. Identified leukaemia stem cells in Jak2V617F-driven Myeloproliferative neoplasm (MPN) MPN stem cells Myeloid progenitor cells Myeloproliferation Elevated blood counts Extramedullary hematopoiesis Thrombotic complications 2. MPN stem cells are LT-HSC? 3. MPN stem cells are resistant to targeted inhibitors 4. MPN stem cells can be eradicated by interferon alpha Self-renewal Quiescence HSC Differentiation Transformation Bone marrow fibrosis Acute leukemia Figure1: MPN stem cells are responsible for disease initiation and propagation in vivo. 5. Using murine models to understand mechanism of IFNα depletion of MPN stem cells Cancer Cell 2010 Blood 2012 Blood 2013 Oncotarget 2013

32 Returning to Australia forging an identity

33 Risk to age 75 years 1 in in in in in in 473 Risk to age 85 years 1 in in in in in in 218 Mean age Acute Myeloid Leukaemia (AML) Rate (per 100,000) 6 Rate (per 100,000) 45 ~900 patients diagnosed with AML per 5 year in Australia ~800 AML deaths/ year in Australia Despite 3 initial response to chemotherapy, most patients will 2 relapse 1 Relapsed disease is incurable with standard chemotherapy AML relapse is mediated by leukaemia Incidence stem males cells (LSCs) Incidence females Incidence males Incidence 85+ females Mortality males Mortality females Mortality males Mortality females Figure B.2a: Acute myeloid leukaemia incidence and mortality rates (b, c), Figure B.2b: Acute myeloid leukaemia incidence and mortality rates (d) by age at diagnosis, 2007 (a) The estimates were based on incidence/mortality data for 1998 to See Appendix F for further details on the methodology used. The estimates for males and females may not add to the estimates for persons due to rounding. (b) The rates were age-standardised to the Australian population as at 30 June 2001 and are expressed per 100,000 population. (c) The first year for which national mortality data for acute myeloid leukaemia are available is 1997.

34 Telomerase is expressed in AML and is required for efficient disease onset Telomerase expression Telomerase activity Telomere length Haematopoietic stem cells Oncogenic Retrovirus Leukaemia stem cells MLLAF9-induced AML AML1ETO + KRas G12C -induced AML WT or Terc-/- 5.5Gy Hemaexplorer (

35 Percent survival Percent survival Functional depletion of LSC populations by Telomerase deletion Leukaemic mouse Leukaemia cells 5.5Gy WT WT or Terc-/- Limiting dilution 100K, 10K, 1K, 100 cells Terc -/ wt 100k wt 10k wt 1k wt 0.1k Days post-transplant terc -/- 100k terc -/- 10k terc -/- 1k terc -/- 0.1k Days post-transplant

36 Pharmacological targeting of telomerase with imetelstat impairs human AML LSC function and prolongs survival in primary AML xenografts Human AML NSGS recipient Imetelstat 15mg/kg 3x/wk Doxorubicin 1.5mg/kg x3

37 We have shown that telomerase inhibition effectively targets AML stem cells Telomerase deficiency eradicates LSC function upon enforced replication Terc-/- LSCs are eradicated via p53 dependent apoptosis and cell cycle arrest A Terc-/- gene expression signature correlates with favourable prognosis in AML (i.e. paralysed LSCs) Imetelstat impairs the expansion of human AML LSCs in patient derived xenografts Bruedigam C., et al. Cell Stem Cell 2014

38 Where are we going? Our pre-clinical trials unit AML patient sample inventory 1' injected 1' >1% 1' AML 2' >1% 2' AML GRN DOXO RBWH 5 Human AML Imetelstat 15mg/kg 3x/wk Doxorubicin 1.5mg/kg x3 No Response Temporary Response Sustained Response Pre-treatment Post-treatment Successful Not Successful Ongoing RBWH 6 RBWH 7 RBWH 8 RBWH 12 RBWH 14 RBWH 16 RBWH 17 RBWH 19 RBWH 20 RBWH 21 RBWH 23 RBWH 24 RBWH 25 RBWH 26 RBWH 32 RBWH 33 RBWH 35 RBWH 36 RBWH 37 RBWH 38 RBWH 39 RBWH 40 RCH 11 RCH 13 RCH 18 RCH 67 UNSW 4 UNSW 5 UNSW 9 UNSW 16 UNSW 17 UNSW 18 Monomac6 NB4 THP1

39 Where are we going? New models of AML Peripheral Blood films Genotype/ Phenotype Features Flt3 ITD/ITD or Flt3 ITD/+ Myeloproliferative Chronic myelomonocytic leukaemia (CMML) Increased mature white cells Increased platelets Increased monocytes Sclcre:Cdx2 x Flt3 ITD/+ Myelodysplastic features Without Cre LoxP sites Hypersegmented neutrophils Reticulocytosis Anisopoikilocytosis Red cell fragmentation Reduced platelets CAG STOP Cdx2 T2A mcherry After Cre expression Sclcre:Cdx2 x Flt3 ITD/ITD Acute myeloid leukaemia CAG Cdx2 T2A mcherry Circulating blasts Reduced/ absent platelets Anaemia

40 Getting started in research Find yourself a great mentor (or 2, or more) Participate seminars, presentations, visiting speakers Be inquisitive, self discipline is key Follow your passion Take your opportunities

41 Acknowledgements QIMR Berghofer Medical Research Institute Translational Leukaemia Research Lab - Claudia Bruedigam - Telomerase - Therese Vu Cdx2 - Catherine Paine-Kuhn, Solene Guignes, Axia Song - Lucie Leveque, Sebastien Jacquelin - Rebecca Austin, Stacey Lowe - Geoff Hill - Sarbjit Ryas, Catherine McCarthy, RBWH - Andrew Moore, RCH - Richard Lock, UNSW - Ross Dickins, WEHI University of Copenhagen, Denmark Frederik Bagger Harvard Medical School David Williams, Gary Gilliland Ann Mullally Scott Armstrong Florian Heidel (Magdeburg, Germany) Ulm University, Germany Lars Bullinger DKFZ, Heidelberg, Germany Mick Milsom Funding NHMRC Career Development Fellowship (2014-8) NHMRC Project: (2012-4), (2014-6) Leukaemia Foundation Cure Cancer Australia Rio Tinto Ride to Conquer Cancer QIMR Berghofer Medical Research Institute 41