Il controllo della malattia minima residua Daniela Cilloni Università di Torino

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1 Milano, ottobre 2009 Il controllo della malattia minima residua Daniela Cilloni Università di Torino

2 Response to imatinib Two main aspects to be considered: Degree of response Time to achieve it

3 MONITORING RESPONSE IN CML: HIERARCHIC ORDER OF RESPONSES Hematologic remission CCyR PCR negativity Leukemic Burden log 2 logs 3/4 logs 10 5

4 Optimal Response (time schedule) CHR within 3 months PCyR at 6 months CCyR at 12 months MMR at 18 months ELN reccomandations 2009

5 Baccarani et al., Blood 2006

6 Progression-free Survival on First-line Imatinib by Molecular Response (MR) at 12 months p<0.001 Estimated rate (95% CI) at 42 months: n=138 75% (67-84) n= 94 90% (84-97) n=136 98% (96-100)

7 PROGNOSTIC IMPACT OF MOLECULAR RESPONSE IN CML Patients with residual disease reductions >3-logs have a very high protection from progression, especially when Imatinib treatment begins in early chronic phase In CCR patients, residual disease may progressively decrease for up to 4 years during Ima treatment

8 Molecular Response Rates Major molecular response (MMR) and the depth of molecular response increase over time 100 % of available samples BCR-ABL% (International Scale) 0.1% (MMR) 0.01% Sample Analysis Timepoints (months) Hughes et al. abstract 334 ASH 2008 IRIS 7 year update

9 CMR = cure? Pts IFN Sokal M0 M3 M6 M12 M18 M24 (months) 1 0 L 4 0 L 8 9 H I I I 3 32 I 6 33 L L 7 71 L I Rousselot et al. Blood 2007 PCR negativity Molecular relapse Imatinib retreatment Molecular response

10 Cortes et al Blood 2004

11 Merante et al. Haematologica 2005

12 Ph+ Stable disease??? Ph+ Ph+ Ph+ Ph+ Ph+ SC Ph+. Ph+ Additional genetic lesions? Point mutations? Decreased compliance? Ph+. Ph+. P Ph+. Ph+ Ph+ Different biology? Ph+. Ph+ Loss of Response Different condition at the stem cell Time levels?

13 Evidences of the role of the immune system for a complete eradication of the disease HSCT: donor derived T cells can exhibit a potent graft-vs-leukemia effect compared to syngeneic or T cell depleted grafts Allogeneic donor lymphocyte infusion (DLI) that demonstrates significant and durable responses in relapsed CML patients

14 Vaccine in CML: why? Vaccination could induce a graft versus leukemia without the graft versus host tipically of allogenic stem cell trasplantation or donor lymphocyte infusions Imatinib treatment leading to prolonged minimal residual disease allows vaccination strategies to be designed for patients with a very low tumor burner

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16 Leukemia-Associated Antigen: BCR-ABL, PR3, WT1, PRAME Leukemia Proteinase 3 Elastase Leukemia Cell Peptide CTL Selectively Kill CML cells PR1-Specific T Cell CTL

17 Do we really need to eradicate leukemic stem cells?

18 Primitive quiescent BCR-ABL+ leukemic stem cells are less sensitive to imatinib Holyoake TL, Blood 2002

19 imatinib dasatinib

20 Targeting the self renewal G0 SC G0 SC G0 SC G0 SC SC SC Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. Pr. What does self renewal means? The capacity to proliferate but not differentiate thus mantaining the ability to generate a stem cell progeny similar to the parental cell.

21 Dormant and self-renewing HSCs Dormant HSCs Self-renewing activated HSCs Progenitors Mature self-renewal expansion What is their role in the body? Emergency, in response to injury? Injury Slide kindly provided by G. Martinelli (Wilson A., et al. Cell, 2008)

22 Acute stimulation with IFNα activates dormant HSCs Acute IFNα Dormant HSCs (repair) Self-renewing HSCs (tissue maintenance) expansion Progenitors Mature (M. Essers et al. (2009), Nature advanced online pub. 11. Feb. 09)

23 IFNα induces HSCs to rapidly exit G 0 Lin neg Sca1 + ckit + CD HSCs u IFNα h 8h 16h Ki67 <FL 1 Log>: Ki67 FITC G1 S/G2/M 10 S/G2/M 3 10 S/G2/M 3 <FL 1 Log>: Ki67 FITC G1 <FL 1 Log>: Ki67 FITC G G 0 G 0 G K 20K 30K FL 6 Area: hoechst 10 0 DNA (Hoechst) 0 10K 20K 30K FL 6 Area: hoechst K 20K 30K FL 6 Area: hoechst G 0 : 59% ± 2% 43% ± 4% 17% ± 3% (M. Essers et al. (2009) Nature, AOP 11.Feb. 09)

24 Acute stimulation with IFNα activates dormant HSCs Acute IFNα Dormant HSCs (repair) Self-renewing HSCs (tissue maintenance) expansion Progenitors Mature This effect of IFNα is only observed in vivo, but not in vitro! (M. Essers et al. (2009) Nature, AOP 11.Feb. 09)

25 Acute stimulation with IFNα activates dormant HSCs, while chronic stimulation leads to their exhaustion Acute IFNα Chronic IFNα Dormant HSCs (repair) Self-renewing HSCs (tissue maintenance) expansion Progenitors Mature This effect of IFNα is only observed in vivo, but not in vitro!

26 IFNα may also activate dormant CML stem cells making them sensitive to imatinib Imatinib CML- SC CML- SC CML- SC Expansion Expansion Dormant CML-SCs are resistant to imatinib Activated CML-SCs may be sensitive to imatinib More differentiated CML cells are effectively eliminated by imatinib

27 IFNα may also activate dormant CML stem cells making them sensitive to imatinib Imatinib CML- SC CML- SC CML- SC minimal residual disease Dormant CML-SCs are resistant to imatinib Activated CML-SCs may be sensitive to imatinib More differentiated CML cells are effectively eliminated by imatinib

28 IFNα may also activate dormant CML stem cells making them sensitive to imatinib IFNα CML- SC CML- SC CML- SC Expansion Expansion Dormant CML-SCs are resistant to imatinib Activated CML-SCs may be sensitive to imatinib More differentiated CML cells are effectively eliminated by imatinib

29 IFNα may also activate dormant CML stem cells making them sensitive to imatinib IFNα Imatinib CML- SC CML- CML- SC SC CML- SCCML- SCCML- CML- SC SC Expansion Expansion Dormant CML-SCs are resistant to imatinib Activated CML-SCs may be sensitive to imatinib More differentiated CML cells are effectively eliminated by imatinib

30 IFNα may also activate dormant CML stem cells making them sensitive to imatinib IFNα priming Imatinib Long term cure? Dormant CML-SCs are resistant to imatinib Activated CML-SCs may be sensitive to imatinib More differentiated CML cells are effectively eliminated by imatinib Case report: six patients who have been switched from IFNα to imatinib treatment did not relapse after stopping imatinib! (Rousselot et al., 2007) (M. Essers et al. (2009) Nature, advanced online pub. 11.Feb. 09) Similar results: Sato and Otheki (2009) Nature Medicine, June 15th)

31 Targeting the self renewal

32 Wnt/beta-catenin is critical for self-renewal and differentiation Several genes of Wnt Wnt or SMO signaling pathway are differen8ally expressed in CML CD34+ cells CBP/ p300

33 Hedgehog signalling and stem cell maintenance

34 Cancer cell 2008

35 SMO inhibitor treatment decreases the growth of imatinib resistant human CML Patient derived CD34+ imatinib resistant blast crisis CML cells xenotransplanted into immunocompromised mice: Smo inhibitor (100mg/kg PO qd x 15 days) treatment alone (n=8, p=.033) or in combination with Dasatinib (50mg/kg PO qd X 15 days) n=4, p=.004) reduces primary tumor burden (results verified in T315I mutants) Smo inhibitor treatment reduces tumor formation in secondary recipients (n=7, p=0.013). Communicated by C. Jamieson, UCSD Collaborator

36 Potential pathways to target Wnt/catenin CBP inhibitors (ICG-001) Hedgehog: SMO inhibitors PF (phase 1/2) Cyclophamine LDE225

37 p21 is critical in maintaining self renewal of LSC p21 p21 G0 SC G0 SC SC SC SC SC Pr. Pr. Pr. Pr. The cell cycle inhibitor p21 activates DNA repair system Reduced self-renewal potential of HSCs in p21-/- mice Viale et al. Nature 2009

38 New drugs working on DNA repair system MDM2 inhibitors PARP inhibitors CHk1 and CHk2 inhibitors MS as a Chk1 inhibitor

39 Targeting the self renewal PML is required for HSC maintenance PML is indispensable for LIC maintenance As 2 O 3 decreases PML expression in HSCs Nature 2008

40 Ito et al: Nature 2008

41 Targeting quiescent CML stem cell FoxOs (FOXO1, FOXO3a, FOXO4) code for trascription factors belonging to the Forkhead family of transcription factors Regulated by PI3K/AKT pathway FoxO transcription factors: regulate cell cycle mediate apoptosis protect HSCs from oxidative stress (ROS mediated)

42 FOXO is inactive in BCR-ABL CD34+ cells CML healthy subject Pellicano and Cilloni. Blood in press

43 TKIs restores FOXO localization and activity Antiproliferative effect Pellicano and Cilloni. Blood in press

44 FOXO1, 3a and 4 phosphorylation is lower in primitive stem cells compared to more mature progenitor cells Quiescence Pellicano and Cilloni. Blood in press

45 Targeting CML stem cells The anti-proliferative activity of TKIs against primary CML CD34+ cells is mediated, at least in part, by the reactivation of FOXO1, 3a and 4. BCR-ABL appears to play a different role in more mature progenitor cells compared to primitive quiescent stem cells. Incomplete activity of BCR-ABL at the stem cell level? FOXO activity at this level provides the dominant signal responsible for intrinsic quiescence? Foxo as a target for blocking self renewal? ATO ( Cilloni et al. Preliminary data)

46 Targeting CML stem cell: : the microenvironment as target? Endothelial Cells Stromal Cells Shh AMD3100 AMD SDF-1 VEGFC CXCR4 FLT4 CML LSC Cell cycling, 27 Kip1 PI3K/Akt & MAPK signaling mtor (S6 kinase) Bcl-2 expression FLT3 KIT Notch CD44 VLA-4 CD62L VCAM-1 Extracellular matrix Osteopontin Fibronectin Collagen Hyaluronin Nataluzimab Bio5192 GMI-1070 Cytokine & Chemokine reservoirs Osteoblasts Tight junctions Slide kindly provided by G. Martinelli

47 Berger and Kipps. Blood 2006:107;

48 Jin et al. Mol Cancer Ther 2008

49 Targeting quiescent CML stem cell

50 Take home message To eradicate leukemic stem cells is probably better than control MRD Combined therapy targeting the skewed proliferation, survival, differentiation and self renewal of CML progenitors may rapresent a future approach for those patients who obtain suboptimal response or who are diagnosed in advanced phase disease Both innate and adoptive immune response against CML progenitors may accelerate disease eradication

51 University of Turin Giuseppe Saglio Emanuela Messa Paolo Nicoli Antonella Rotolo Elisabetta Greco Francesca Messa Francesca Arruga Sonia Carturan Monica Pradotto Chiara Maffè Enrico Bracco University of Bologna Michele Baccarani Giovanni Martinelli Ilaria Iacobucci Simona Soverini Roberto Bernardoni University of Naples Fabrizio Pane