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MCL1-DependentRegulatoryNetworks
in the Context of B-Cell
Lymphomagenesis(Antonio Ruiz-Vela, PhD)
Theophilus Schweighart, 1604
CED9Bcl-2Bcl-xLMcl-1A1/Bfl-1Boo/DivaNr13
BH4 BH3 BH1 BH2 TM
Pro-Survival membersOncogenes
CED9 Family Members:(CED9 Family Members are Critical Regulators of Programmed Cell Death)
TMBaxBakBok/Mtd
BidBadBik/NbkBlkHrkBim/BodBnip3NixNoxa
BH3 BH1 BH2
Pro-Death membersTumour suppressors
BH3-only proteins
Multidomain proteins
BAX & BAK -dependent Remodeling of the MitochondrialCristae and Cyt-c Release
Scorrano L., Dev Cell. 2002 Jun;2(1):55-67.
IMboundary
Outermembrane Cristae
Class I
Class II
Class III
Acehan D., Mol Cell. 2002 Feb;9(2):423-32.Liu et al., Cell. 1996 Jul 12;86(1):147-57.
Pro-B cell
B220+IgM-CD23-ckit+CD43+CD24+CD23-
Schematic Representation of B-Cell Development
Pre-BII
V-DJ/IgHl5Pre-BCR
Early Pre-BI
CD25+D-J/IgH
Pre-BI
CD19+
ImmatureB cell
V-J/IgLBCR (IgM+)
Autoantigens
Programmed Cell Death‘Negative Selection Checkpoint & Tumour Suppressor Pathway”
(CED9 family members keep in check)
Bone Marrow
SpleenLymph nodes
TransitionalB cell
IgD+IgM+CD24+CD23-CD21-
MatureB cell
IgD++IgM+CD24-CD23+CD21+
HSC
Sca-1+Lin-ckit+
PAX5
IL7R/IL7
CED9BH4 BH3 BH1 BH2 TM
E2AE2A
Aberrant Release of Pro-Death Cyt-c from Mitocondrias inMurine B-Cell Lymphomas (WEHI-231 B-cells)
Ruiz-Vela et al., EMBO J. 1999 Sep 15;18(18):4988-98.
(BAX, BAK)
APAF-1/ CASP-9CAPN-1, -2
Cyt cCa2+
BCR
Autoantigen
Programmed Cell Death Pathway Initiated by B-CellReceptor (BCR) Regulatory Networks in B-Cells
(BAX, BAK)
APAF-1/ CASP-9CAPN-1, -2
Ruiz-Vela et al., Blood. 2008 Feb 1;111(3):1665-76.
Ruiz-Vela et al., EMBO J. 1999 Sep 15;18(18):4988-98.Ruiz-Vela et al., J Exp Med. 2001 Aug 6;194(3):247-54.
Takeuchi et al., Proc Natl Acad Sci U S A. 2005 Aug 9;102(32):11272-7.
Cyt cCa2+
BCR
Autoantigen
Combined Inactivation of Proapoptotic BAX and BAK Generates an Autoreactive B-cell Lymphomagenesis
Lindsten., Mol Cell. 2000 Dec;6(6):1389-99.Ruiz-Vela et. al EMBO Rep. 6 (4). 379-385. 2005
BAX-/-, BAK-/-
BAX-/-, BAK+/-
BAX+/-, BAK-/-
BAX+/-, BAK+/-
Lymph node Spleen
BAX, BAK
BAX, BAK
BAX, BAK
BAX, BAK
TMBaxBak
BH3 BH1 BH2
High risk-group Low risk-group
Somatic mutationLymphoid migration
BCR signalling
NFkB targets
Gene Expression Profiling of B-CLL:(BCR signaling pathway-associated signature of B-CLL cells derived from high-risk group patients)
It suggest a potential inhibition ofBCR-induced programmed
cell death in B cell transformation
(BAX, BAK)
APAF-1/ CASP-9CAPN-1, -2
Cyt cCa2+
BCR-Network ?
Experimental System to Dissect Key Molecules Linking BCR Signaling and Programmed Cell Death
Burkkit’s lymphoma (BL)hallmark
IgH enhancerMYC
t (8:14)ER membrane
Loss-of-functionstrategy (RNAi)
Lentiviral (HIV)-Based RNA Interference Screen:(>300 shRNAi Clones from BCR-Regulatory Network, HPRD bioinformatic tool)
EV#1#2#3
Methodology in Our RNA Interference Screen:(GFP-Negative B-Cells as Endogenous Negative Controls per Well)
96-well plate
RNA Interference Screen in the Context of BCR Signaling
Mcl-1BH4 BH3 BH1 BH2 TM
MCL1 Gain-of-Function
Wide Genome Gene Expression of MCL1-OverexpressingB-Cell Lymphomas: (Agilent, PantherDB and GEPAS Tools)
Gene Network Reconstruction Analysis:(Ingenuity, HiMAP and BioGrid Bioinformatic Tools)
Hallmarks of Many Types of B-Cell Lymphomas:(reciprocal translocation involving the non-productive rearranged IgH loci and a proto-oncogene)
Follicular lymphoma (FL)
IgH enhancerBCL2
t (18:14)
Burkkit’s lymphoma (BL)
IgH enhancerMYC
t (8:14) Mantle-cell lymphoma (MCL)
IgH enhancerCCND1
t (11:14)
Diffuse large B-celllymphoma (DLBCL)
IgH enhancerBCL6
t (3:14) Lymphoplasmacytoid lymphoma (LL)
IgH enhancerPAX5
t (9:14)
MALT lymphomas (ML)
IgH enhancerMALT1
t (1:14)t (18:14)
IgH enhancerBCL10
IgH enhancerFGFR3
Multiple myeloma (MM)
t (4:14)
IgH enhancerFGFR3
Chronic lymphocyticleukemia (B-CLL)
IgH enhancerBCL3
t (19:14)
Aberrant Expression of MCL1 in Follicular Lymphomas
At Least Two MCL1 Functions: (Pro-Survival & Cell Fate Reprogramming Oncoprotein)
Future approaches:
1) Cell Biology:MCL1 subcellular localization in B-cell leukaemic cancer stem cells models.(Ruiz-Vela et al., Oncogene 2005, 24(32), 5119-24)
2) Epigenetics:MCL1-induced cell fate reprogramming at the level of histone modifications.
3) Mouse Genetics:Generation & Characterization of MCL1 transgenic mice using stem-cell specificpromoters.
4) iPS Biology (KLF2/4, Sox2, Myc, Oct3/4):MCL1-dependent targets: CBX7, NOTCH2, HOXA4, KLF2.(Ruiz-Vela et al., Blood 2008, 11(3), 1665-76)
Acknowledgment:
Miguel Angel Piris Lab
Beatriz HerrerosDaniel MartinPierfrancesco VargiuAbel Sanchez-AguileraMohit AggarwalPaloma de la Cueva
Genomics Unit
Orlando Dominguez