Development of Highly Active Anti-Leukemia Stem Cell Therapy (HALT)

Transcription

1 Development of Highly Active Anti-Leukemia Stem Cell Therapy (HALT) CIRM/CSCC disease team grant CIRM PI: D. Carson co-pi: C. Jamieson CSCC PI: J. Dick co-pi: J. Wang Project leaders: ROR1 mab Development: T. Kipps CML and LSC Niche Modulation: C. Jamieson AML LSC Eradication: J. Dick ALL LSC Eradication: J. Danska

2 LEUKEMIA STEM CELLS (LSC) LSC LSC LSC Adapted from Reya et al Nature 2001 Leukemia stem cells (LSC) evolve from progenitors as Reya, a Morrison, result Clarke & of Weissman. reprogramming Nature 2001 of stem cell pathways that regulate: self-renewal dormancy survival LSC

3

4 THE BONE MARROW NICHE

5 Targeting LSC in the Niche

6 Highly Active Anti-Leukemia Stem Cell Therapy (HALT) LSC HALT Leukemia loses ability to generate new cells Drugs that kill blasts but not LSC LSC Leukemia regenerates Leukemia degenerates LSC Overall Experimental Plan

7 LEUKEMIA STEM CELL RNA SEQUENCING

8 JAK2 CRYSTAL STRUCTURE WITH TG TG co-crystallized with JAK2 JH1 domain produced in SF9 insect cells also binds FLT3 Bound in ATP pocket Key interactions confirmed Hood, Noronha, Soll

9 TG INHIBITS HUMAN JAK2+ STEM CELL ENGRAFTMENT 4 mice/group TG101348: Geron I, Abrahamsson AE, Barroga CF et al. Cancer Cell 2008:13: ) Reduced Bioluminescent Engraftment 2) Reduced Human Red Blood Cell Engraftment 3) Reduced JAK2 V617F expression CIRM SEED, CIRM Training Grant

10 Now in Phase ll and lll Trials for Myeloproliferative Neoplasms in 33 countries

11

12 SYNERGISTIC INHIBITION OF BLAST LSC SELF-RENEWAL Intra-hepatically injected neonates mice Patient Cells CD34 Selection by MACS 1% CD45+ cells 6-8 weeks 14-day oral treatment: Vehicle JAK2 Inhibitor (60 mg/kg BID) Dasatinib (50 mg/kg) Combination FACS, RNA SEQ, NANOPRO AND SERIAL TRANSPLANT LSC engraftment

13 SPLICE ISOFORM BIOMARKERS OF LSC RESPONSE MYC STAT5A

14 NANOPROTEOMIC BIOMARKERS OF LSC RESPONSE VEHICLE TG VEHICLE TG VEHICLE TG VEHICLE TG Court Recart, Sadarangani

15 SYNERGISTIC INHIBITION OF LSC SELF-RENEWAL Serial Transplant

16 Summary (1) CD123, which signals through JAK/STAT, and FLT3 isoforms distinguish myeloid LSC from their chronic phase counterparts. MYC and STAT5A isoforms are key myeloid LSC biomarkers of response. Combined JAK2 and FLT3 inhibition with TG (SAR302503) reduces LSC survival in the niche. SAR and combined with dasatinib reduces LSC survival and self-renewal in the niche (IIT submitted).

17 DORMANT HUMAN LSC ARE RETAINED IN THE BONE MARROW NICHE H&E hcd45 Ki67 hcd45 / DAPI ph-h3 / DAPI

18 SONIC HEDGEHOG ACTIVATION MAINTAINS MOUSE LSC Cyclopamine PF Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Zhao C, Chen A, Jamieson C et al. Nature. 2009;458:776-9 Expansion of Bcr-Abl-positive leukemic stem cells is dependent on Hedgehog pathway activation. Dierks C, Beigl R, Guo GR et al. Cancer Cell (3);

19 SHH PATHWAY ACTIVATION PORTENDS BLASTIC TRANSFORMATION

20 SONIC HEDGEHOG INHIBITION OF HUMAN LSC DAPI CD45 GLI2 MERGE GLI2 GLI2 Shih, Sadarangani

21 SHH INHIBITION ABROGATES LSC DORMANCY IN THE NICHE Shih et al submitted

22 PF AND DASATINIB DECREASE LSC BURDEN Shih, Sadarangani

23 ASH Abstract 424 Phase 1 Dose-Escalation Study of PF , an Oral Hedgehog Inhibitor, in Patients With Select Hematologic Malignancies Catriona Jamieson, 1 Jorge E. Cortes, 2 Vivian Oehler, 3 Michele Baccarani, 4 Hagop M. Kantarjian, 2 Cristina Papayannidis, 4 Kristen N. Rice, 1 Xiaoxi Zhang, 5 Naveed Shaik, 6 Rachel Courtney, 6 Wendy J. Levin, 6 and Giovanni Martinelli 4 1 University of California San Diego, La Jolla, CA; 2 The University of Texas M.D. Anderson Cancer Center, Houston, TX; 3 University of Washington, Seattle, WA; 4 Institute of Hematology and Medical Oncology "L. & A. Seràgnoli, University of Bologna, Bologna, Italy; 5 Pfizer Oncology, New York, NY; 6 Pfizer Oncology, San Diego, CA

24 PF CLINICAL RESPONSES IN AML Disease Response Dose(s) CRi 80 mg AML 50% reduction in bone marrow blasts (6 patients) 10, 10, 80, 80, 80, 270 mg Bone Marrow Blasts (%) in AML Responders SCREENING CYCLE2/DAY1 CYCLE4/DAY1 Abbreviation: CRi, complete remission with incomplete blood count recovery (Progressed from MDS) (M6 Progressed from MDS) (Progressed from CMML) (M2 (De Novo) (M0 (De Novo) (M2 (De Novo) (Unknown)

25 Summary (2) Shh signaling pathway activation predicts progression to blast crisis and LSC generation. Shh inhibition with a selective smoothened (SMO) antagonist, PF , inhibits LSC dormancy in the niche. Selective Shh inhibition promotes LSC cycling and enhances sensitivity to tyrosine kinase inhibitors preclinically and clinically (IIT submitted).

26 ROR1 Embryonic tyrosine kinase-like molecule with high level expression in fetal brain, blood cell precursors, with an extracellular Frizzled-like domain Not expressed in adult tissues High level expression in CLL, AML, ALL Wnt5a is a binding ligand

27 ROR1 is expressed only in CLL, but not normal B-cells or normal adult tissue. To generate anti-ror1 antibodies, we immunized mice using different vaccination strategies employing recombinant ROR1 protein and/or polynucleotide constructs combined with CD154 genetic or chemical adjuvants, or replication-defective adenovirus vectors encoding ROR1 and CD154.

28 ROR1 Expression in CLL % ROR1+ CD19+ Cells MFIR CLL samples N= Significant threshold

29 ROR isoform expression (RNA-seq) Progenitors (n=3) CD34+CD38+ CD34+ cells in culture (n=6) Stem CD34+CD38- Prog. CD34+CD38+ CLL primary patient samples (n=10)

30 Anti-ROR1 mabs Trigger Cell-Dependent Cytotoxicity

31 Anti-ROR1 Mab Blocks CLL Development Low Dose CD5 dull B220 + Leukemia Challenge Normal migg D10 4A5 1cm

32 Anti-ROR1 Antibodies Eliminate CLL LSC In Vivo niche-dependent, activity model

33 KinExA Antibody Affinity Determination Antibody Characterization Primary Ab Y Cy5 Secondary Ab Ligand Fluorescence emission is measured to give a Binding Signal

34 Anti-ROR1 Mabs Generated Across Extracellular Domain Binding sites of antibodies No. 5 -Ig-like Middle of Ig-like 3 -Ig-like CRD Kringle 4A5 G11 H11 2G3 D

35 11 of 21 AML samples showed a ROR1+ population Sample Subtype %ROR1+ % of ROR1+ that are CD AML-M5a AML-M AML-M IMF JAK AML-M AML-M AML 2ry MDS AML-M AML AML AML 7 0

36 In vitro studies: Determining Therapeutic Index Normal Cord blood in methylcellulose

37 Current Status of anti-ror1 Antibodies Humanized of ROR1 mab PK studies ongoing Bulk production in progress Pre-clinical pharm/tox studies planned for IND submission in

38 Summary (3) ROR1 is an embryonic antigen that is expressed by LSC in CLL, ALL and AML. ROR1 is not expressed by normal hematopoietic stem cells (HSC). ROR1 mab decreases niche dependent CLL LSC and does not inhibit normal HSC.

39 Overall Summary LSC express embryonic antigens related to the Wnt pathway that are dispensable for normal adult blood development. Monoclonal antibodies against ROR1 have potential for selective treatment of CLL and other refractory hematologic malignancies including AML. RNA Seq investigations may reveal biomarkers of response and resistance Stem cell targeted agents may need to be combined with drugs that block the pro-survival niche effects in bone marrow and other sites.

40 ACKNOWLEDGEMENTS Development of Highly Active Anti-Leukemia Stem Cell Therapy (HALT)

41 SHH INHIBITION ABROGATES LSC DORMANCY in vivo Shih et al submitted

42 LSC Signature Predicts Survival (Dick et al 2011)

43

44 SLM2