Nuove potenziali strategie di sensibilizzazione a molecole di nuova generazione: analisi del ruolo delle serin-‐treonin chinasi CK2 e GSK3 Francesco Piazza Department of Medicine Hematology and Clinical Immunology University of Padova ITALY SIES – DISCUTIAMONE INSIEME “FARMACORESISTENZA” 20 MARZO 2014 FIRENZE, ITALY La farmacoresistenza • Meccanismi vari 1.Resistenza singola>>>polichemioterapia 2.FenoGpo MDR (resistenza mulGpla) Overespressione di P-‐gp 3.Mutazioni geneGche pathways bersaglio Overespressione molecole proliferaGve/anGapoptoGche (es. delezione PTEN, mutazioni PI3K) Esempio classico in ematologia mutazioni: BCR-‐ABL DifferenG meccanismi di farmacoresistenza possono impa[are in modo differente a seconda se il farmaco è “convenzionale” o di nuova generazione Nature Reviews Cancer 13, 714–726 (2013) DifferenG meccanismi di farmacoresistenza possono impa[are in modo differente a seconda se il farmaco è “convenzionale” o di nuova generazione Nature Reviews Cancer 13, 714–726 (2013) Farmacoreistenza: problemaGca pressante in ematologia e in oncologia JCO 2009 vol. 27 no. 3, 469-‐471. Inibitori del proteasoma • Bortezomib (Velcade) • Approvato per la terapia del mieloma mulGplo e del linfoma mantellare Nature 480, S40–S42 (15 December 2011) Il Bortezomib causa citotossicità inibendo NF-‐kB e aevando La via mitocondriale dell’apoptosi L’inibizione dle proteasoma sinergizza con l’inibizione della via aggresoma-‐autofagia • Resistenza a inibitori del proteasoma (bortezomib): Mutazioni di PSMB5 (subunità β5) Alterazioni vie di risposta allo stress Up regolazione di meccanismi anIapoptoIci Mechanisms of resistance and susceptibility to proteasome inhibition. Kumar S , and Rajkumar S V Blood 2008;112:2177-2178 ©2008 by American Society of Hematology Sites of action of proteasome inhibitors. Ruschak A M et al. JNCI J Natl Cancer Inst 2011;jnci.djr160 © The Author 2011. Published by Oxford University Press. COMPLEXITY OF MM GENESIS AND EVOLUTION EXTRA-‐BM MM BM (micro)environment Memory B Ly Long-‐lived PC Post-‐GC B Ly MGUS SMM CSR t G1-‐S passage Derangement (CyclinD) >>Myc EpigeneGcs NF-‐kB Non CSR t TP53 Ras EpigeneGcs MM Increased nutrients needs Increased metabolic acGvity Non-‐CSR t(Myc) Gains EpigeneGcs KNOWN GENETIC LESIONS IN MM • Inherited variaQons SNPs hyperdyploid D1+D2 • hyperdyploid D2 Primary geneQc events hyperdiploid D1 1. IGH@translocaQons partners t(4;14) FGFR3 and MMSET t(6;14): CCND3 t(11; 14): CCND1 t(14;16): MAF t(14; 20): MAFB 2. Hyperdiploidy Trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21 HYPERDIPLOID Trisomies of odd • 1. 2. 3. 4. 5. Secondary geneQc events Gains Seconday translocaQons EpigeneQc events Molecular hallmarks DeleQons None 11q13 CCND1 6p21CCND3 4p16 FGFR37MMSET 16q23 cMAF NON-‐HYPERDIPLOID 14q32 translocaitons chromosomes Cis and Trans-‐ Trans-‐dysregulaGon dysregulaGon of CCND1 of CCNDs Morgan GJ, Nature Rev Cancer, 2012 Chesi M, Bergsagel L, Int J Hematol, 2013 Whole exome/genome sequencing unravels pathway dysregulaGon in MM Frequently mutated genes • NRAS, KRAS, FAM46C, DIS3, TP53, CCND1, PNRC1, ALOX12B, HLA-‐A, MAGED-‐1 • Pathways RNA processing and protein homeostasis (DIS3, FAM46C, XBP1*, LRRK2) • IdenGcal mutaGons: gain of funcGon oncogenes IRF4, PRDM1 Clinically relevant mutaGons BRAF • Gene set mutaGons NF-‐κB pathway (BTRC, CARD11, CYLD, IKBIP, IKBKB, MAP3K1, MAP3K14, RIPK4, TLR4, TNFRSF1A, TRAF3) Histone modifying enzymes MLL, MLL2, MLL3, WHSC1, WHSC1L1>>> HOXA9 overexpression *not staGsGcally significant Chapman MA et al. Nature 2011 NF-κB mutations in MM Activation: intrinsic p50 IKBα IKKβ p65 CLASSICAL: Aberrations of: CYLD TACI NFκB1 TRAF2, 3 cIAP1,2 NIK NIK RelB NFKB2 p52 ALTERNATIVE: Truncation of NFκB2 TRAF3 NIK 20% ca. di patients & cell lines Keats J et al., Cancer Cell 12: 95-97 (2007) Annunziata C et al., Cancer Cell 12: 115-130 (2007) ER stress and Unfolded Protein Response Ischemia Endoplasmic reticulum BiP BiP PERK IRE1 BiP ATF6 P eIF2a >>>XBP1-short >>>ERAD >>>CHAPERONES High-rate protein synthesis Oxydative stress Gene mutations >>BiP >>GADD153 >>XBP1 PROTEIN SYNTHESIS >>>ATF4 >>BiP Adaptation=survival UPR “early” or “chronic” Overwhelming stress=apoptosis UPR “terminal” Antigen Terminal plasma cell differentiation Clonal expansion Resting B-lymphocytes (long lived) Stressed to work Increased Ig secretion Productive stress ? Stressed to kill Exuberant Ig secretion Proteasome insufficiency and Accumulation of poly-Ub proteins Increased metabolic, energetic and redox demand Fatal stress ? Adapted from: Cenci S, Sitia R FEBS Letters 2007, 581:3652. XBP1: what role in plasma cell malignancies? • ER stress UPR -‐ XBP1 overexpression and its inhibiGon -‐ XBP1 negaGve myeloma progenitor cells. XBP1s directs a compensatory UPR polyA XBP1u XBP1 mRNAu IRE1α polyA XBP1s XBP1 mRNAs XBP1s-‐dependent transcripGonal program COPING WITH ER STRESS Hetz C Nat Rev Mol Cell Biol 2013 ? The differenQaQon and stress response factor XBP-‐1 drives mulQple myeloma pathogenesis. - Transcription factor regulates th “unfolded protein response” - Indispensable for PCs ontogenesis - 2 transcripts from alternative splicing - Overexpression of the spliced (XBP-1s) form in MGUS (50%) and MM 70% Carrasco et al Cancer Cell 2007 Animal Model of Multiple Myeloma: XBP1s Trangenic Mice Carrasco D et al. Cancer Cell 2007 InhibiGon of XBP1s generaGon as a therapeuGc strategy in MM polyA ? XBP1u XBP1 mRNAu IRE1α IRE1 inhibitors polyA XBP1 mRNAs XBP1s TERMINAL UPR APOPTOSIS XBP1s-‐dependent transcripGonal program COPING WITH ER STRESS Mimura N et al. Blood 2012 Hetz C et al. Nat Rev Drug Discov 2013 A role for XBP1 in the sensiGvity to proteasome inhibitors? PIs (bortezomib) Leung-‐Hagstejin C et al. Cancer Cell 2013 BiP BiP PERK ATF6 P eIF2a >>>XBP1-LONG Davenport et al 2007 Obeng 2006 BiP >>>XBP1-short >>BiP >>GADD153 >>XBP1 PROTEIN SYNTHESIS>>BiP >>>ATF4 >>>ERAD >>>CHAPERONES Adaptation=survival UPR “early” or “chronic” Overwhelming stress=apoptosis UPR “terminal” MM cell “tumor progenitors” PRE-‐PLASMABLASTS CD20+ XBP1 – De-‐commitment to secretory Ig producQon PIs RESISTANT BULK of MM cells XBP1 + Commitment to secretory Ig producQon PIs SENSITIVE Leung-‐Hagstejin C et al. Cancer Cell 2013 FARMACORESISTENZA (specialmente ai nuovi farmaci) • Meccanismi collegaG alla “ONCOGENE ADDICITON” (assuefazione ad un oncogene) • Meccanismi dipendenG dal fenoGpo “NON-‐ONCOGENE ADDICTED” (assuefazione a upregolazione di molecole/pathways di per sé non oncogeniche) Protein Kinase CK2 PKCK2 • Serine-threonine kinase ubiquitously expressed NON ONCOGENE ADDICITON • Is necessary and instrumental for cell survival • Has over 300 substrates Solimini N et al. Cell. 2007 130(6):986-‐8. Luo Jand et modified al. Cell. from: 2009 136(5):823-‐37. W. Litchfield Biochimica et Biophysica Acta 1784 (2008) 33–47 Adapted James S. Duncan, David PKCK2 is a Ser-‐Thr Kinases That Regulates oncogene and Non-‐oncogene addicGon related pathways • • • • NFκB MYC PI3K/AKT/MTOR ER stress/IRE1/XBP1 PKCK2 Piazza F et al Leukemia 2012 CK2 and NF-‐κB: a liason not enough explored CK2 Is a p65 Kinase Responsible for NF-‐kappaB AcQvaQon in physiological and pathological condiQons P CK2 p65 p50 NF-‐κB CK2 Is a C-‐Terminal IkappaB Kinase Responsible for NF-‐kappaB AcQvaQon during the UV Response. UV p50 p65 CK2 IrradiaQon NF-‐κB P P IκB TNFα Wang D et al. Journal Biol Chem 2000 Kato T et al Mol Cell 2003 Ub P IκB p50 Proteasome IκB p65 CK2 P Piazza et al. Blood,108: 1698. (2006) P P STAT3 P P STAT3 P SRC STAT3 STAT3 JAK P p50 p65 UPSTREAM SIGNALS (?) CK2 inhibitor CX-‐4945 CK2 Ser 529 Ser 536 NF-‐κB p-‐STAT3 S727 STAT3 β-‐acQn Tyr 705 Ser 727 STAT3 p-‐NF-‐κB p65 S529 NF-‐κB p65 β-‐acQn PROLIFERATION SURVIVAL RESISTANCE TO CHEMOTHERAPY INFLAMMATION ANGIOGENESIS Piazza FA et al Blood 2006 Manni S et al. PLOS One 2013 Unfolded client kinase Folded client kinase HSP90 CK2 HSP90 CK2 cdc37 P cdc37 cdc37 Gene amplificaGon In MM subsets Ischemia Endoplasmic reticulum Gene mutations BiP BiP PERK IRE1 ATF6 PROTEIN SYNTHESIS >>>ATF4 >>BiP Adaptation=survival UPR “early” or “chronic” CK2 BiP P eIF2a >>>XBP1-short >>>ERAD >>>CHAPERONES High-rate protein synthesis Oxydative stress >>BiP >>GADD153 >>XBP1 ? Overwhelming stress=apoptosis UPR “terminal” CK2 è in parte nel RE, è akva dop ER stress e la sua inibizione causa alterazioni della UPR C A B CK2α SERCA MERGE un TG p-‐Ser 13 CDC37 U-‐266 CDC37 β-acQn * 200 175 150 125 100 75 50 25 Kinase acQvity of CK2α (%) TG: INA-‐6 K27 5 μM: -‐ + IRE1α BIP/GRP78 p-‐Ser 51 EIF2α -‐ + -‐ + micro c EIF2α CHOP/GADD153 Cl PARP β-acQn CK2α rS6 kinase GAPDH un 3h 6h 24h PBMC IRE1α BIP/GRP78 CK2α β-acQn p-‐Ser 51 EIF2α EIF2α CHOP/GADD153 CK2α β-acQn p-‐Thr 981 PERK PERK CK2α Cl PARP β-acQn 1,2 1 0,8 0,6 0,4 0,2 0 * F 2,0 * 1,8 CK2α siRNA 1,6 XBP1u 500bp 400bp XBP1s 1,4 1,2 1 * * 0,8 0,6 0,4 0,2 0 CK2α scr CK2α siRNA scr mRNA levels XBP1s/XBP1u (arbitrary units) CK2α: E + -‐ -‐ + mRNA level scr: CK2α protein levels (arbitrary densitometric units) D TG 2.5 µM BIP/GRP78 1,2 1 0,8 * scr CK2α siRNA 0,6 0,4 0,2 0 EDEM CHOP/GADD153 Manni et al.,Clinical Cancer Res 2012 L’inibizione di CK2 sinergizza con inibitori di Hsp90 nell’arrestare la crescita di linee cellulari di MM in vitro e in vivo in un modello di xenotrapianto murino 3H-‐thymidine-‐incorporaQon (cpm) K27 50000 GA+K27 40000 30000 20000 10000 0 3H-‐thymidine-‐incorporaQon (cpm) 60000 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 µM GA 50000 GA+K27 40000 30000 Tumor volume mm3 CI=0.291 60000 350 300 250 200 150 100 50 0 0 VH tTBB COMBO 4 8 12 16 20 24 28 32 days 20000 10000 0 0 1 2 3 4 5 µM Manni S et al. Clin Cancer Res 2012 L’inibizione di CK2 distrugge il complesso tra Hsp90 IRE1a e Cdc37 Input K27: IP: IRE1α -‐ + -‐ + IRE1α HSP90 CDC37 CK2α β-acQn CK2 Cdc37 P Hsp90 Compensatory (oncogenic) UPR IRE1α Manni S et al. Clin Cancer Res 2012 Coopera con Hsp90 nella UPR nel mieloma? ER Bip IRE1 Bip Bip PERK ATF6 ? SURVIVAL CK2 CK2 HSP90 K27 GA CDC37 APOPTOSIS INHIBITION OF CK2 CAUSES APOPTOSIS OF MM and MCL CELLS and POTENTIATES THE EFFECT OF BORTEZOMIB Annexin V/CD19 posiQve cells (fold over control) MCL paGents (n=7) 2 * 1,5 1 0,5 0 CX4945 5 µM: -‐ + ? CK2 bortezomib INHIBITION OF CK2 AND BORTEZOMIB SYNERGIZE TO CAUSE CELL GROWTH ARREST OF MCL CELLS 80 60 40 20 0 0 2 4 6 8 100 CX-‐4945 80 60 40 20 0 0 2 4 6 8 10 µM 10 µM 100 BZ BZ+K27 80 60 CI=0.16 40 20 0 0 5 100 10 15 20 25 30 nM BZ BZ+CX-‐4945 80 60 CI=0.016 40 20 0 0 5 10 15 20 25 30 nM H 100 K27 80 60 40 20 0 0 2 4 6 8 100 80 CX-‐4945 60 40 20 0 0 2 4 6 8 3H-‐thymidine-‐incorporaQon (cpm % over untreated) K27 100 80 60 10 µM BZ BZ+K27 CI=0.5 40 20 0 10 µM 3H-‐thymidine-‐incorporaQon (cpm % over untreated) F 100 Rec-‐1 G 3H-‐thymidine-‐incorporaQon 3H-‐thymidine-‐incorporaQon (cpm % over untreated) (cpm % over untreated) 3H-‐thymidine-‐incorporaQon 3H-‐thymidine-‐incorporaQon (cpm % over untreated) (cpm % over untreated) E 3H-‐thymidine-‐incorporaQon 3H-‐thymidine-‐incorporaQon (cpm % over untreated) (cpm % over untreated) Jeko-‐1 0 5 10 15 20 25 30 35 40 nM 100 80 60 40 20 0 COMBINATION INDEX (CHOU and TALALAY METHOD): BX + K27= 0.16 Jeko1 BZ+ CX4945= 0.016 BX + K27= 0.5 Rec1 (bortezomib resistant) BZ+ CX4945= 0.69 BZ BZ+CX-‐4945 CI=0.69 0 5 10 15 20 25 30 35 40 nM A Siglo scrambled Bor 11.1% 27.2% M1 M1 siRNA CK2 α+β siRNA CK2 α+β + Bor 42.7% 21.2% M1 M1 Annexin V Manni S, PLOS One 2013 INA-‐6 * Annexin V posiQve cells (fold over control) 4 siRNA CK2 α+β: * ♦ 3.5 BZ 5 nM: -‐ -‐ + + -‐ + -‐ + CX-‐4945 5μM: Bcl2 Mcl1 β-‐acQn p-‐STAT3 Ser727 STAT3 β-‐acQn 3 2.5 2 p-‐NF-‐κB p65 Ser529 NF-‐κB p65 β-‐acQn 1.5 1 -‐ -‐ -‐ -‐ -‐ + -‐ -‐ + -‐ -‐ + CX-‐4945 2.5μM: -‐ -‐ -‐ -‐ + -‐ -‐ + -‐ -‐ + -‐ CX-‐4945 1μM: -‐ -‐ -‐ + -‐ -‐ + -‐ -‐ + -‐ -‐ BZ 1nM: -‐ + -‐ -‐ -‐ -‐ + + + -‐ -‐ -‐ BZ 5nM: -‐ -‐ + -‐ -‐ -‐ -‐ -‐ -‐ + + + Poly-‐ubiquiQn 0.5 0 siRNA CK2 α+β: BZ 5 nM: -‐ -‐ + + -‐ + -‐ + CK2α CK2β β-‐acQn Hsp70 β-‐acQn UPSTREAM SIGNALS ER stress Proteasome inhibiQon PROTEIN DEGRADATION TURN OVER CK2 Ser 529 Ser 536 NF-‐κB Tyr 705 Ser 727 STAT3 PROLIFERATION SURVIVAL RESISTANCE TO CHEMOTHERAPY INFLAMMATION ANGIOGENESIS CK2 inhibitors Collaborate With Proteasome And chaperone inhibitors INHIBITION OF CK2 SHUTS DOWN STAT3, NF-‐κB IN MCL/MM p<0.05 * 2 # 1,5 1 0,5 -‐ -‐ + -‐ -‐ + -‐ -‐ + p-‐NF-‐kB p65 Ser 536 * 2 1,5 1 0,5 0 2 1,5 * 1 0,5 # 2,5 IL-‐6 mRNA level NF-‐kB p65 p-‐STAT3 Ser727 Cyclin D1 p-‐NF-‐kB p65 Ser 529 3 0 2 1,5 1 * 0,5 # 0 1 2 # 0,5 1,5 0 CX4945 5 μM: BZ 2.5 nM: mRNA level 1,5 * 1 * 0,5 -‐ -‐ + + -‐ + -‐ + 0 2 mRNA level 2 Bcl2 β-‐acQn mRNA level STAT3 TNF-‐α BZ 25nM: 0 mRNA level -‐ + -‐ -‐ + -‐ -‐ + -‐ 3 2,5 mRNA level -‐ -‐ -‐ -‐ -‐ -‐ + + + NOS-‐2 BZ 15nM: * 3,5 IAP-‐2 K27 5µM: 4 mRNA level CX4945 2.5μM: 4,5 COX-‐2 Rec-‐1 -‐ -‐ -‐ + + + -‐ -‐ -‐ 1,5 1 0,5 * # 0 CX4945 5 μM: BZ 2.5 nM: -‐ -‐ + + -‐ + -‐ + Schematic representation of mammalian GSK-3α and GSK-3β. Doble B W , and Woodgett J R J Cell Sci 2003;116:1175-1186 ©2003 by The Company of Biologists Ltd (A) Regulation of GSK-3β activity by serine phosphorylation. Doble B W , and Woodgett J R J Cell Sci 2003;116:1175-1186 ©2003 by The Company of Biologists Ltd Journal of Cell Science 2003 116, 1175-‐1186 GSK3 Espressione di forme fosforilate inibitorie/aevatorie di GSK3 nel MM PBMC 1 2 3 4 nPC PATIENTS CELL LINES 1 2 3 4 5 6 7 8 9 U-266 OPM-2 RPMI 8226 INA-6 GSK-3α GSK-3β pSer 21 GSK-3α pSer 9 GSK-3β β-actin PATIENTS 7 3 1 GSK-3α GSK-3β pTyr 279 GSK-3α pTyr 216 GSK-3β β-actin C D GSK-3 total DAPI (nuclei) MERGE GSK-3 total OPM2 MM1 INA-6 MM9 U-266 MM10 RPMI 8226 E DAPI (nuclei) MERGE GSK-3 total BMMC DAPI (nuclei) MERGE 100 90 80 70 60 50 40 30 20 10 0 SB415286: (µM) * - * ** * 2 4 8 12 16 ** 2 4 8 12 16 SB415286 4 mM: * 100 90 80 70 60 50 40 30 20 10 0 INA-6 * * - * - + * * - + - + U-266 RPMI 8226 INA-6 Annexin V negative cells (% of total cell number) U-266 100 90 80 70 60 50 40 30 20 10 0 Annexin V negative cells (% of total cell number) 3H-thymidine incorporation (arbitrary units as % of untreated) Inibitori di GSK3 causano arresto della crescita e apoptosi di cellule di MM 100 90 80 70 60 50 40 30 20 10 0 - + normal PBMC Inibitori di GSK3 sono efficaci nell’inibire la forma fosforilata in Grosina e aevatoria e inducono apoptosi nonostante auemnG di βcatenina e P-‐ERK U-266 RPMI-8226 SB216763: - - 5mM 10mM pTyr 279 GSK-3a pTyr 216 GSK-3b GSK-3a GSK-3b PARP Cleaved-PARP Smac/DIABLO n SB216763 (5mM) c n c GSK-3a GSK-3b pTyr 279 GSK-3a pTyr 216 GSK-3b b-catenin pThr 202/Tyr 204 ERK1,2 ERK 1,2 INA-6 SB415286 4 mM: - 8h 24 h PARP Cleaved-PARP b-actin PARP Cleaved-PARP nucleophosmin GAPDH GSK3 Inibitori di GSK3 cooperano con bortezomib nell’indurre citotossicità di cellule di MM INIBITORE GSK3 Annexin V positive cells (arbitrary units) B U-266 A INIBITORE GSK3 + bortezomib * 7 U-266 * 6 SB415286 (4µM): BZ (5nM): 5 * 4 -‐ -‐ + -‐ -‐ + + + n c n c n c n c 3 PARP Cleaved PARP 2 1 pSer 473 AKT 0 AKT MCL-1 Nucleophosmin RPMI-8226 GAPDH Annexin V positive cells (arbitrary units) * 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 SB415286 (4µM): BZ (5nM): * - + - + + + GSK-3α GSK-3β GSK-3β 25% 15% R1 R1 dead cells (fold increase arbitrary units) scr GSK-3α * 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 F * scr - + GSK-3α + * - dead cells (fold increase arbitrary units) GSK-3β scr GSK-‐3α E * scr scr R1 GSK-3α GSK-3β uncleaved PARP β-actin * dead cells (fold increase arbitrary units) 2.5 2 1.5 1 0.5 0 siRNA: BZ (5nM) : 21% 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 scr GSK-‐3β dead cells (fold increase arbitrary units) scr 2.5 2 1.5 1 0.5 0 siRNA: BZ (5nM) : R1 GSK-3β D BZ (5nM) GSK-3α 22% scr GSK-3β + + 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 siRNA: BZ (5nM) : * * BZ (5nM): siRNA: dead cells (fold increase arbitrary units) GSK3β scr GSK3α scr GSK-3α GSK-3β uncleaved PARP β-actin scr scr GSK-3α GSK-3β β-actin GSK-3α GSK-3β β-actin C B 72h scr scr un GSK-3β 48h 72h scr GSK-3α un scr 48h GSK-3α A - + - - - - + + + + - - scr α/β α β scr α/β α β 1 2 3 4 5 6 7 8 9 10 GSK-3α GSK-3β pSer 473-AKT AKT MCL-1 scr - α/β - scr + α/β + β-actin Bortezomib causa aevazione di GSK3 e shi| nel nucleo in cellule di MM untreated BZ (5nM) U-266 BZ (5nM): INA-6 n + c n c pSer 21/9 GSK-3α/β GSK-3α GSK-3β PARP cleaved-PARP β-actin % of cells with cytolsolic-only GSK3 U-266 untreated BZ 18h (5 nM) 100 80 60 40 20 0 * * * RPMI 8226 INA-6 C U-266 U-266 SB216763: -‐ 5µM 10µM -‐ BZ (5nM): -‐ -‐ + n pTyr 279 GSK-3α pTyr 216 GSK-3β GSK-3α GSK-3β GAPDH nucleophosmin c n -‐ c n c n 5µM 10µM + c n c + n c Fbxw7α-‐ and GSK3-‐mediated degradaGon of p100 is a pro-‐ survival mechanism in mulGple myeloma (2012) Luca Busino, Sco[ E. Millman, Luigi Sco[o, Christos A. Kyratsous, Venkatesha Basrur, Owen O’Connor, Alexander Hoffmann, Kojo S. Elenitoba-‐Johnson & Michele Pagano GSK3 GSK3 Unstressed Bortezomib ? GSK3α ? GSK3β SURVIVAL NF-‐kB STAT3 GSK3α SURVIVAL P-‐AKT P100 NF-‐kB GSK3β ? GSK3 MAF AKT NF-‐κB β-‐catenin Mcl-1 osteoblast PROLIFERATION BONE LOSS SURVIVAL PROLIFERATION PROLIFERATION MIGRATION SURVIVAL RESISTANCE TO CHEMOTHERAPY ANGIOGENESIS plasmacells CONCLUSIONS UpregulaGon of prosurvival signaling can be mediated by ONCOGENE and NON ONCOGENE ADDICTION MECHANISMS in blood tumors The idenGficaGon of the underlying mechanisms can be helpful To idenGfy Achillee’s heels exploitable for “syntheGc lethality” CK2 and GSK3 are two PKs with a potenGal applicaGon as therapeuGc targets in MM and other blood cancers. Hematology Division University of Padova Laboratory of Myeloma and Lymphoma Pathobiology VeneQan InsQtute of Molecular Medicine A. Brancalion A. Cabrelle A. Colpo C. Gurrieri E. Mandato S. Manni L. Quoe Tubi F. Zaffino Prof. G. Semenzato Biochemistry University of Padova L.A. Pinna M. Ruzzene
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