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