4°WORKSHOP NAZIONALE della Società di Ematologia Sperimentale Ematologia traslazionale Bologna, 14-­‐15 novembre 2013 Aemilia Hotel Borsa di studio SIES-­‐AIL Analysis of signaling pathways PI3K/AKT/mTOR and JAK/
STAT in chronic myeloprolifera?ve neoplasms in vitro and in vivo Costanza Bogani Dipar/mento di Ematologia Università di Firenze Gruppo AGIMM Ac1va1on of the JAK/STAT pathway plays a central role in MPN pathogenesis Survival Differen/a/on Prolifera/on Oncogenesis Vannucchi AM et al., CA Cancer J Clin. 2009; 59:171-­‐91 PI3K/mTOR pathway PI3K-­‐AKT-­‐mTOR pathway regulates cellular metabolism, prolifera>on and survival Dysregulated Ac1va1on of PI3K/Akt in JAK2VF Cells •  PI3K/Akt cytokine-­‐independent ac/va/on in JAK2 V617F cells •  JAK2 V617F allele burden-­‐
dependent PI3K/Akt ac/va/on FDCP Epo-­‐R WT Epo -­‐ + V617F -­‐ + p-­‐JAK2 JAK2 p-­‐STAT5 STAT5 p-­‐AKT AKT James C et al., Nature 2005; 434:1144-­‐8, Akada H et al. , 2010; 115:3589 BM SPLEEN Erythr. Increased Phospho-­‐Akt and –mTOR in MPN Cells P-­‐STAT5 Megakaryocytes Hematopoie1c cells Control ET P-­‐Akt PMF P-­‐mTOR Grimwade LF et al., BJH, 2009; 147:495 Vicari L et al., BJH, 2012 PI3K and/or mTOR Inhibitors TKR PI3K KI • BKM120 PI3K – mTOR KI PI3K
•  BEZ235 •  XL765 •  GDC0980 •  GSK2126458 •  PF04691502 ……….. mTORC2 mTOR
mTOR KI • PP242 • OSI-­‐027 •  AZD8055 •  INK128 (not exhaus/ve) RAPALOGS •  Everolimus •  Temsirolimus •  Ridaforolimus Akt
mTOR
mTORC1 Aim of the Study I •  To explore in-­‐vitro and in-­‐vivo the poten?al relevance of targe?ng PI3K/mTOR pathway with a specific inhibitor BEZ235 alone or in combina?on with JAK1/2 inhibitor Ruxoli?nib. BaF3 JAK2 WT BaF3 JAK2 V617F BaF3 EpoR WT BaF3 EpoR V617F 100
80
60
40
20
Viable cells (%) Viable cells (%) BEZ235 Inhibits MPN Cell Lines Prolifera1on K562 HEL SET2 100
80
60
40
20
0
0
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
10
0
2
BEZ235 (µM) Cell Line BEZ235 IC50 (nM) BaF3 EpoR JAK2 WT 676 ± 200 BaF3 EpoR JAK2 V671F 87 ± 50 * BaF3 JAK2 WT BaF3 JAK2 V671F 10000 ± 500 64 ± 10 * 4
6
8
10
BEZ235 (µM) Cell Line BEZ235 IC50 (nM) SET2 334 ± 40 HEL 387 ± 90 K562 5000 ± 1000 * P<0 .01 BEZ235 Induces Cell Cycle Block and Apoptosis in MPN Cell BaF3 EpoR JAK2 V617F 200
180
* * 180
160
140
120
100
80
60
* * 40
* * 140
120
100
80
* 60
* 40
20
20
0
0
G 2/M
G2/M G 0/G 1
S
G0/G1 G 2/M
S G2/M * 80
70
* 60
* 50
40
30
S
S 90
80
70
60
40
30
20
10
10
0
0
0.5 1 5 BEZ235 10 * 50
20
µM 0.1 G 0/G 1
G0/G1 100
90
Annexin V+ (%) 100
Annexin V+ (%) 0 .3 3 4 uM
3 .3 4 uM
8 .4 uM
200
% of Control % of Control 160
SET2 0 .1 uM
1 uM
2 uM
µM 0.06 0.33 1.67 3.33 10 BEZ235 * P< 0.01 Inhibi1on of mTOR, 4E-­‐BP1 and STAT5 phosphoryla1on by BEZ235 in wild-­‐type and JAK2V617F Ba/F3-­‐EPOR cells Ba/F3 EPOR wt
1
0.81 0.57 0.44
Ba/F3 EPOR VF
1
0.77 0.43 0.23
P-mTOR
mTOR
1
0.2
0.1
0.05
1
0.3
0.23 0.19
P-4eBP1
4eBP1
1
0.6
0.27 0.18
P-STAT5
STAT5
Tubulina
BEZ235, nM
-
87
435
870
-
87
435
870
BEZ235 Preferen1ally Inhibits MPN Progenitors 80
C F U -­‐G M (P e rc e nt of c ontrol) B F U -­‐E 100
60
40
20
0
BFU-­‐E 0
100
(P e rc e nt of c ontrol) C trl
P V
MF
80
60
40
20
0
50
100
150
200
CFU-­‐GM 0
50
B E Z 235 (nM)
200
80
60
40
20
CFU-­‐Mk 0
5
10
B E Z 235 (nM)
15
(P e rc e nt of c ontrol) 100
E E C (P e rc e nt of c ontrol) C F U -­‐Mk 150
B E Z 235 (nM)
100
0
100
80
60
40
20
0
BFU-­‐E IC50 (nM) Ctrl 177 ± 50 PV 98 ± 20 * MF 99 ± 8 * CFU-­‐GM IC50 (nM) Ctrl 143 ± 20 PV 44 ± 10 * MF 108 ± 7 CFU-­‐Mk IC50 (nM) Ctrl 11 ± 3 PV 2 ± 1 ** MF 0.7 ± 0.1 ** EEC IC50 (nM) PV 20 ± 10 EEC 0
10
20
30
B E Z 235 (nM)
40
50
* p< 0.01 Combo-­‐Therapy: PI3K/mTOR and JAK2 Inhibitor TKR PI3K
P VF JAK2
BEZ235 Ruxoli1nib mTOR
STAT5
P Akt
P STAT5
mTOR
mTORC1 JAK2
P P P mTORC2 VF BEZ235 Synergizes in vitro with Ruxoli1nib -­‐ -­‐ Ba/F3 EpoR V617F Combina/on Index 1 .2
31 -­‐ -­‐ 78 31 78 BEZ235 (nM) Ruxoli/nib P-­‐mTOR 1 .0
mTOR 0 .8
0 .6
P-­‐4eBP1 0 .4
4eBP1 0 .2
0 .0
0 .2
0 .4
0 .6
0 .8
Frac/onal Effect Chou -­‐Talalay P method Calcusyn Sooware CI <1 SYNERGISM 1 .0
P-­‐STAT5 STAT5 Tubulin C om bina tion Inde x
BEZ235 and Ruxoli1nib have synergis1c ac1vity against EEC colonies 1 .2
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
F ra c tiona l E ffe c t
Fa
CI
BEZ235
Ruxolitinib
(nM)
(nM)
0.300
0.13
1
0.09
0.400
0.16
2.2
0.2
0.500
0.21
4.4
0.4
0.600
0.27
9
0.700
0.39
19,3
1.7
0.800
0.65
49.5
4.4
0.8
Ba/F3 JAK2 V617F-­‐Luc mouse model NVP-­‐BEZ 235: PI3K – mTOR inhibitor Ruxoli1nib: JAK1/JAK2 inhibitor Strain: SCID beige daily administra/on oral gavage Every 7 days evalua/on •  Vehicle •  NVP-­‐BEZ235 45 mpk dose • Ruxoli1nib 60 mpk dose • COMBO 45+60 mpk dose D-­‐luciferin i.v. Ba/F3 VF-­‐Luc injec1on Start treatment (daily administra1on) Leukemic burden monitoring (7-­‐14-­‐21-­‐28 days) Endpoints Combined BEZ235 and Ruxoli1nib Treatment Prolongs Survival in Ba/F3VF-­‐luc Mouse Model Vehicle 60mpk Ruxo 45mpk BEZ235 60mpk Ruxo + 45mpk BEZ235 Baseline Day 7 Day 14 Day 21 Day 28 Counts per Minute 7
1,0x10
6
8,0x10
6
6,0x10
6
4,0x10
* 6
2,0x10
0,0
* Veh a
c
d
Ruxo b BEZ235 BEZ235 + Ruxo **, P<0.01 Overall Survival BEZ235 + Ruxo ** Vehicle Ruxo BEZ235 Time (Days) Conclusion I •  These in vitro data indicate that BEZ235 is ac/ve against JAK2V617F cell lines and MPN cells; •  Effects on apoptosis and cell cycle; •  Combina/on with Ruxoli/nib resulted in synergism. •  Studies in murine model confirm these results in vivo. Journal of Cellular and Molecular Medicine “Co-­‐targe?ng the PI3K/mTOR and JAK2 signaling pathways synergis?c ac?vity against myeloprolifera?ve neoplasms” Aim of the Study II •  To explore in-­‐vitro and in-­‐vivo the poten?al relevance of targe?ng PI3K pathway with a specific inhibitor BKM120 alone or in combina?on with different drugs PI3K and/or mTOR Inhibitors TKR PI3K KI • BKM120 PI3K – mTOR KI PI3K
•  BEZ235 •  XL765 •  GDC0980 •  GSK2126458 •  PF04691502 ……….. mTORC2 mTOR
mTOR KI • PP242 • OSI-­‐027 •  AZD8055 •  INK128 (not exhaus/ve) RAPALOGS •  Everolimus •  Temsirolimus •  Ridaforolimus Akt
mTOR
mTORC1 BaF3 JAK2 WT BaF3 JAK2 V617F BaF3 EpoR WT BaF3 EpoR V617F 100
80
60
40
20
Viable cells (%) Viable cells (%) BKM120 Inhibits MPN Cell Lines Prolifera1on K562 HEL SET2 100
80
60
40
20
0
0
0
1
2
3
4
5
6
0
1
BKM120 (µM) Cell Line 2
3
4
5
6
BKM120 (µM) IC50 (nM) Cell Line IC50 (nM) BaF3 EpoR JAK2 WT 3,122 ± 1,000 SET2 1,000 ± 300 BaF3 EpoR JAK2 V671F 1,100 ±207* HEL 2,000 ± 500 BaF3 JAK2 WT 5,300± 800 K562 4,500 ± 800 BaF3 JAK2 V671F 364 ± 200* * P<0 .05 BKM120 increases G2/M phase and induces Apoptosis in MPN Cell BaF3 EpoR JAK2 V617F * 500
0 .3 µM
1 µM
5 µM
300
* 100
* 300
200
* 100
0
0
GG2/M 2/M GG0/G1 0/G 1 GG2/M 2/M SS 100
GG0/G1 0/G 1 SS * * * * 40
20
0
µM 0.1 0.5 1 2 BKM120 Annexin V+ (%) 100
80
60
1 µM
3 µM
1 0 µM
* 400
% of Control 400
Annexin V+ (%) % of Control 500
SET2 80
60
* * * 3 6 10 BKM120 * 40
20
0
5 10 µM 1 20 * P< 0.01 BKM120 synergizes in vitro with different drugs IC50(nM)
IC50 (nM)
Drug combination
CI index
BKM120
1,100 ± 207
Ruxolitinib
220 ± 20
BKM120
507±230
Ruxolitinib
105±45
0.88±0.3
BKM120
1,100 ± 207
RAD001
651 ± 50
BKM120
272±142
RAD001
174± 96
0.2± 0.05
BKM120
1,100 ± 207
PP242
500 ± 100
BKM120
453±177
PP242
215±82
0.93±0.33
BKM120
1,100 ± 207
SGI1776
3,000±1,000
BKM120
419±243
SGI1776
1,258±729
0.78±0.08
BKM120
1,100 ± 207
AS703026
1,000±100
BKM120
459±256
AS703026
459±256
0.67±0.18
Drug combination
IC50 (nM)
IC50(nM)
BKM120
1100 ± 207
PP242
500 ± 100
Ruxolitinib
220 ± 20
BKM120
1100 ± 207
RAD001
651 ± 50
Ruxolitinib
220 ± 20
BKM120
171±87
BKM120
224±150
PP242
81±40
Ruxolitinib
35±18
RAD001
Ruxolitinib
143±100
48±34
CI index
0.52 ± 0.3
0.27 ± 0.16
CI<1 Sinergism BKM120 Preferen1ally Inhibits MPN Progenitors BKM120 IC50 (nM) BFU-­‐E CFU-­‐GM CFU-­‐MK Ctrl 900 ± 370 760 ± 300 >100 PV 187 ±76** 77±50** 14±1 MF 148±63** 312±200* 16±8 EEC 9±5 **P < .001 * P < .005 BKM120 effects on Ba/F3 JAK2 V617F-­‐Luc mouse model Methods D-­‐luciferin 3x106 cells Ba/F3 VF-­‐Luc i.v. injec1on Athymic Nude Female Mice Treatment Start day 5 post-­‐inj. in-­‐vivo imaging Endpoints Results BKM120 administered by oral gavage once daily Treatment Cohort Mean Lifespan post-­‐Inj. Mean Lifespan post-­‐Treat. Mean Lifespan increment (%) vs Veh BKM120 60mpk 27.8±4.9 20.8±4.9 + 31.1 % BKM120 45mpk 27.3±7.8 20.3±7.8 + 28.8 % Vehicle 21.2±6.6 14.2±6.6 \ BKM120 treatment OS – NUDI Vehicle Overall Survival 45 mpk BKM120 60 mpk BKM120 Conclusion II •  These in vitro data indicate that BKM120 is ac/ve against JAK2V617F cell lines and MPN cells; •  Effects on apoptosis and cell cycle; •  Combina/on with different drugs resulted in synergism. •  Studies in murine model confirm an increased lifespan. Two ABSTRACTs accepted like posters: SIE 2013 “The PI3K inhibitor BKM120 alone and in mul?ple combina?ons inhibits growth of JAK2V617F cells” ASH 2013 “The PI3K specific inhibitor BKM120 results effec?ve and synergizes with Ruxoli?nib in preclinical models of Myeloprolifera?ve Neoplasms” Thus, concurrent targe>ng of PI3K/mTOR and JAK/STAT pathway might represent a new therapeu>c strategy to op>mize efficacy and reduce toxicity in pa>ents with MPN. Thanks to Bartalucci N. Mar/nelli S. Tozzi L. Calabresi L. Mannarelli C. Bosi A. Vannucchi A.M. Gruppo AGIMM