Meccanismi di resistenza all inibizione della via di segnale PI3K/Akt/mTOR nella LAM

Transcription

1 Meccanismi di resistenza all inibizione della via di segnale PI3K/Akt/mTOR nella LAM University of Modena and Reggio Emilia Cell Signaling Unit, ChiMOMo Sandra Marmiroli Jessika Bertacchini Laura Mediani Hematology Department Mario Luppi Fabio Forghieri Patrizia Barozzi University of Padova Oncohematology Laboratory Giuseppe Basso Benedetta Accordi George Mason University Manassass Virginia USA Center for Applied Proteomics and Molecular Medicine Lance Liotta Emmanuel Petricoin

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3 RTKs! GPCR Ras! IRS! p85! p110α/β/δ PIP 2! PTEN! PTEN! PIP 3! PIP 2! Gβγ p87/101! p85! p110γ p110β PDK-1! P Akt1/2/3! P mtorc2! S6K! mtorc1! Bad! IKK! MDM2! FOXO! GSK3! TSC2! PFK2! ACL! Cell! Survival! Cell! Cycle! Cell! Growth! Glycolysis! Metabolism!

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5 Signalling of the PI3K/AKT/mTOR pathway and relevant drugs that target each of the components of the pathway Rodon, J. et al. (2013) Development of PI3K inhibitors: lessons learned from early clinical trials Nat. Rev. Clin. Oncol. doi: /nrclinonc

6 Growth factor Receptor Tyr kinase PI3K Ras NF1 Energy stress PTEN PI-3,4,5P 3 Raf AMP AKT PDK1 MEK Tuberin Erk LKB1 AMPK Rheb Rsk A.A. RagB/RagD Raptor/mTOR 4EBP1 S6-kinase S6 Protein synthesis

7 Bertacchini et al. Leukemia 2014

8 Bertacchini et al. Leukemia 2014

9 Bertacchini et al. Leukemia 2014 Bertacchini et al. Leukemia 2014

10 mtor inhibitors AKT inhibitors

11 Perifosine, Akti 1/2 TCN Torin 1 Rapamycin

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13 Bertacchini et al. Leukemia 2014

14 Bertacchini et al. Leukemia 2014

15 Activation of PI3K/Akt pathway in AML is modulated by feedback Akt inhibitors reactivate PI3K/Akt through stabilization of IRS-1 and FOXO, which in turn increases IR or other RTKs The results highlight the limitations of these drugs if used as monotherapy Association of Akt and RTKs inhibitors prevents re-induction of Akt

16 Vitalità %/CTRL THP1 (PF ) μm Dose (μm) CEM (PF ) μm Vitalità %/CTRL Dose (μm) IC50 1,1 µm in cellule THP1 1,3 nm in cellule CEM.

17 Etoposide + Citarabina + GSK Etoposide + Citarabina + GDC-0068 Vitalità %/CTRL DMSO GSK E+C E+C+GSK Vitalità %/CTRL DMSO GDC E+C E+C+GDC Vitalità %/CTRL Etoposide + Citarabina + PF DMSO PF E+C E+C+PF INDICI DI COMBINAZIONE Trattamenti ED50 ED75 ED90 ETO + AraC + GSK 0,047 0,013 0,045 ETO + + AraC + GDC 0,051 0,011 0,0037 ETO + + AraC + PF 0,0062 0,052 0,055.

18 Bertacchini et al. Leukemia 2014

19 Our data support the development of targeted treatment paradigms for PI3K/Akt/mTOR-altered adult leukemias and also demonstrate that therapies must be tailored to the specific RTKs and phosphorylome context.

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21 3 fold change upon treatment Akt T308 Akt S473 PRAS40 T246 GSK3 S 21/9 P70 S6K T389 4EBP1 S65 IRS-1 rapamycin Torin1-3

22 Phosphorylation profile of receptor tyrosine kinases in human primary blast cells

23 RTKs! GPCR Ras! IRS! p85! p110α/β/δ PIP 2! PTEN! PTEN! PIP 3! PIP 2! Gβγ p87/101! p85! p110γ p110β PDK-1! P Akt1/2/3! P mtorc2! S6K! mtorc1! Bad! IKK! MDM2! FOXO! GSK3! TSC2! PFK2! ACL! Cell! Survival! Cell! Cycle! Cell! Growth! Glycolysis! Metabolism!

24 sample collection 84 samples with >80% blast cells analyzed by RPPA, then validated by western blotting Fresh peripheral blood and bone marrow specimens with newly diagnosed AML. Patients are diagnosed according to blast content, FAB classification and cytogenetic analysis.

25 Overlapping profiles of peripheral blood (PB) or bone marrow (BM) derived blast cells from the same patients. 18 blood and bone marrow specimens from the same patient were available. Overlapping profiles of fresh and cryopreserved samples from the same patient blast samples derived from the same patient before and after cryopreservation in 10% DMSO were analyzed, with similar results

26 Reverse Phase Protein Array Automated sample printing on nitrocellulose-coated glass Sypro Ruby staining for protein quantification Statistical analysis Detection and quantification of specific endpoints

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28 Through RPPA technology 90 endpoints were analyzed, involved in different signaling pathways: survival, apoptosis, oxidative-stress and metabolism. Areas with pathways hyper-activated in limited clusters of patients are highlighted.

29 Correlation of protein profiles with FAB groups : p-akt, p-pka, p-pkc, p-mtor, p-gsk, p-p70s6k, p-p90rsk and p-p38 cluster preferentially in M4/M5 FAB subtype; pro-apoptotic proteins cluster preferentially in M1/M2 FAB subtype. M1 M2 M4

30 C-Kit receptor expression correlates with hyperphosphorylation of the PI3K/Akt pathaway

31 % SURVIVAL AKT INHIBITORS LY PERIFOSINE VIII TRICIRIBINE DMSO % SURVIVAL % SURVIVAL DM RAPAMYCIN [µm] TORIN RAPAMYCI N TORIN [µm] AKT INHIBITORS + TORIN 1 % SURVIVAL

32 pakt S473 2,5 2 fold change upon treatment 1,5 1 0,5 0-0,5-1 -1,5-2 -2,5

33 Akti VIII 20 hr 3 2,5 2 1,5 1 0,5 0 Akt S473 FOXO3a IRS1 S612 PRAS40 Akt T308 4EBP1 p70s6k CC9 3 2,5 2 1,5 Perifosine 20 hr 1 0,5 0

34 The Akt inhibitor paradox: sustained apoptosis sustained Akt activation

35 0 Fold change upon treatment -0,5-1 -1,5-2 -2,5

36 pakt S473 4 fold change upon treatment Perifosine Akti VIII - 2 hr

37 Bertacchini et al. Leukemia 2014

38 Etoposide + Citarabina Vitalità %/CTRL , Dose (μm)