Humanized models to study myeloid malignancies. Prof. Dominique Bonnet Hematopoietic Stem Cell Lab Francis Crick Institute, London, UK

Transcription

1 Humanized models to study myeloid malignancies Prof. Dominique Bonnet Hematopoietic Stem Cell Lab Francis Crick Institute, London, UK

2 The true nature of stem cells can be learned only by discovering how they are regulated INTRINSIC and EXTRINSIC mechanisms BM NICHE AML/MDS-LSC? We demonstrate that AML-LSC could be maintained for 3 weeks ex vivo in co-culture with MSC Griessinger et al. Stem Cell Trans. Med,

3 Limitations of the xenotransplant models Not all AML at diagnosis engra1 (60-70%) Correla:on between engra1ment poten:al and clinical outcome (good risk group : NE) (Pearce et al., Blood 2006; Varga1ig et al., Leukemia 2011) Low level of engra1ment of MDS samples Could we improve engra1ment via humanizing the niche? Co-injec:on with human MSC 3D Scaffold seeded with hmsc 3

4 Engraftment of MDS samples in NSG versus NSG-S A MNCs or CD34+ BM MNCs NSG or NSG-S T cells W/O MSCs B 100 MNC Injected C 100 MNC Injected hcd45+ Cell Engraftment % NSG NSG-S hcd45+ Cell Engraftment % MNC MNC+MSCs MNC MNC+MSCs NSG NSG-S

5 Engraftment of MDS samples in NSG versus NSG-S A 100 CD34 + Injected B 8 CD34 + Injected hcd45+ Cell Engraftment % hcd45+ Cell Engraftment % RARS RCMD RAEB MDS/MPD CMML -2 CD34+ Alone nmscs pmscs C CD34 + Injected 100 hcd45+ Cell Engraftment % injected Tibia Non injected Bones

6 IBM injection of hmscs do not engraft long-term 6

7 Robust engraftment of human HSPC in 3D scaffold pre-seeded with human MSC

8 Human HSPC engraftment in human ossicles

9 Comparison of different procedures for seeding human HSPCs in 3D scaffold

10 Advantage of humanized stroma for engrafted human AML samples

11 3D scaffold showing good murine endothelial blood vessels inside the scaffold

12 3D scaffold showing humanized endothelial blood vessels 12

13 Summary of the new 3D humanized scaffold 3D scaffolds seeded with human MSC are an effective model to study human normal and malignant haematopoiesis (especially samples hard to engraft in NSG) Our data demonstrate that bone formation is not mandatory for human normal and leukemic engraftment, contrary to what was previously described Versatile model where different niche components can be changed or added depending on the context Model where the importance of particular signalling pathways within the hematopoietic niche could be study using genetically modified stroma Abarrategi et al., J Clin. Inv. 2017, Feb 13

14 Multiphoton intravital microscopy for 3D tracking of human leukaemia (HL60) in live bone marrow Day1 Day4 Day 8 NirV11D4-A-sk1det1z Bone TRITC-dextran GFP Endothelium Haematopoietic Stem Cell Lab

15 Vasculature in AML

16 Results: AML-induced toxicity on vessel permeability ns Bone ctrl KS2 hcd45 NT-Qdot kda Dextran *** ** **** *** Merge Merge M S C R PM ct rl hc B i.v. sac Qtracker kda dextran *** L + AML Patients uninjected 10 * K Engraftment Mean Sum Intensity OUT/IN TRITC Dextran kda K S2 SW 2 i.v.

17 Persistence of vessel leakiness after leukemia treatment with cytarabine (AraC)

18 Upregulation of nitric oxide (NO) pathway

19 Combination of Ara.C plus NOS inh have a synergistic effect on reducing AML engraftment

20 Conclusions We provide a global reliable picture of the bone marrow vasculature in AML using intravital two-photon confocal microscopy. We found several abnormalities in the vascular architecture and function in patient-derived xenografts (PDX), i.e. vascular leakiness. We identified an increase in nitric oxide (NO) as major mediator of this phenotype in PDX and in patient-derived BM biopsies. Moreover, induction chemotherapy failed to restore normal vascular permeability and NO levels. Strikingly, inhibition of NO production reduced vascular permeability, and significantly improved treatment response in PDX. Passaro D et al. Cancer Cell In Revision

21 Advancement of LSC therapeutic program Based on the heterogeneity of of LSC between pacents and within the same pacents Development of therapies against LSC will have to be combinatorial (targe:ng different pathways at the same :me) The role of the microenvironment and the cross-talk of LSCs and their niche should also be taking into account when targe:ng the LSCs

22 THE FRANCIS CRICK INSTITUTE, LONDON Discovery without Boundaries... Despite BREIXIT!! Current HSC lab members: Linda Ariza-McNaughton, Beatiz Montaner, Alexander Waclawiczek, Semiramis Popova, Kevin Rouault-Pierre, Syed Mian, Ander Abarrategi, Marion Piganeau, Diana Passaro, Antoniana Batvaris, William Grey 22

23 Fernando Anjos-Afonso Linda Ariza McNaughton Alexander Waclawiczek Amy Bradburn Beatriz Montaner Eamonn Morrison Alessandro Di Tullio Ashley Hamilton Kevin Rouault Pierre Ander Abarrategi Diana Passaro Thais Lavagnolli Syed Mian KaCe Foster LRI: Flow Cytometry Equipment Park Biological Resources Barts Hospital: Professor John Gribben King s College Hospital Professor Ghulam MuWi 23

24