Advancing Our Understanding of Leukemogenesis and Therapeutics Through Animal Models

Advancing our understanding of leukemogenesis and therapeutics through animal models

Levi J. Beverly, Ph.D. Assistant Professor Department of Medicine Division of Hematology and Oncology James Graham Brown Cancer Center Department of Pharmacology and Toxicology University of Louisville School of Medicine

Monday April 18th 3:00pm to 5:00pm

Beverly Lab Lavona Casson Parag Shah, Ph.D. *Kumar Saurabh, Ph.D. Zimple Kurlawala *Aditya Barve Past members *Michael Scherzer Sahiba Chandel Danial Malik Sean Shannon Amy Song Sanjay Yadav, Ph.D.

Siskind Lab Sharon Willer Tess Dupre Cierra Sharp Cameron Conway Doug Saforo Mark Doll

U OF L COLLABORATORS: John Trent Geoff Clark Jun Yan Jill Steinbach Gavin Arteel Jesse Roman

‘OUTSIDE” COLLABORATORS: John C. Reed Sanford-Burnham Institute William Lockwood; NHGRI/NIH (now BCCC) Daniel Starczynowski/ Jim Mulloy; Cincinnati Children’s

Andrew Kraft MUSC (now U. of Arizona)

Old Lab mantra:

Use mouse models to guide biochemical and molecular experimentation on the processes affecting tumorigenesis. New Lab mantra: Develop new models that better reflect clinical scenarios and use these models to find more effective and safer therapeutics for the treatment of cancer patients.

2 Quotes : “If you have cancer and you are a mouse, we can take good care of you.” – Judah Folkman

“We (JGBCC) are focused on curing cancer in humans, not mice” – Don Miller (Jason Chesney)

“Mice lie, monkeys exaggerate and ferrets are weasels” - Geoff Clark

Today’s talk: 1. Using mouse models to understand biochemistry of leukemogenesis. 2. Developing clinically relevant models of human leukemia. 2. Identifying novel drug regimens for the treatment of human leukemia.

Can we use in vivo mouse models to understand biochemical properties of leukemogenic proteins???

Bone marrow reconstitution assay for tumor development Tet-O-MYC 1. Harvest Tet-O-MYC bone marrow

X

MYC

hypothesis

2. Infect BM, ex vivo

Mit retrovirus LTR MCS IRES tTA LTR

-Dox

tTA

TRE

MYC

-Doxycycline

3. Transplant into lethally irradiated Syngeneic recip.

Empty virus Survival

TRE

MYC MYC + X Days

4. Expression of MYC and Protein X Beverly LJ, Bone Marrow Transplantation, 21012

6 Anti-apoptotic BCL2 members have similar domain structure BH4

BCL2

240

Gene BCL2

BCLxl

234

BCL2L1

BCLw

194

BCL2L2

MCL1

351

MCL1

BFL1

176

BCL2A1

BCLb

205

BCL2L10

BH3 BH1BH2 TM protein aa

43.8% 36.5% 22.5%

23.7%

22.1%

-Each has slightly different sub-cellular localizations, and different BH3 binding partners. -All BCL family members are presumed to be oncogenic, but only data existed for BCL2, BCLxl and MCL1

-At least 11 anti-BCL compounds in pre-clinical/clinical trials ABT-737; inhibits BCL2/xl/w

Anti-apoptotic BCL2 family members cooperate with MYC in leukemogenesis relative survival

1

MYC (n=10)

p=0.0048

0.8

MYC+BFL1 (n=9) MYC+BCLb (n=7) MYC+MCL1 (n=9)

0.6 0.4

MYC+BCL2 (n=8) MYC+BCLxl (n=11) MYC+BCLw (n=9)

0.2 0 0

Migrx

20

40

Days

60

80

100

MYC+ BCLxl

Beverly and Varmus, 2009

MIT-BCLxl

Spleen

Thymus

BM

Spleen

CD8

BM

FVB/n Thymus

CD11b

CD4

GR1 Blood

Liver

Spleen

FVB/n

FVB/n

MYC+ BCLxl

10X

10X

10X

10X

10X

10x

MYC+ BCLxl Beverly and Varmus, 2009

Conclusions: -anti-apoptotic BCL2 proteins are expressed in human tumor cell lines and patient samples.

-All six BCL2 genes can cooperate with MYC to accelerate leukemogenesis

Why is BCLb so much less potent than BCLxl??

CH

MG

MIG-BCLb

-

CH

MG

-

MIG-BCLxl 100 75 50 37 25 20

wb: BCL

15

37 25

wb: GFP

Are Lysine residues responsible for the lack of BCLb stability? KK K BH4

BH3

BH1

K

BH2

TM

BCLb

BCLbK0 is much more stable than BCLbwt CH 20hr

CH 10hr

BCLbK0

-

CH 20hr

CH 10hr

-

BCLbwt

37

wb: BCLb

25 20

37

wb: GFP

25 20

Does increased stability = increased oncogenic potential????

Bone marrow reconstitution assay for tumor development Tet-O-MYC 1. Harvest Tet-O-MYC bone marrow TRE

X

MYC 2. Infection ex vivo

Mit-BCLbwt LTR Bwt

IRES tTA LTR

VS. LTR BK0 IRES tTA LTR

-Dox

Mit-BCLbK0

tTA

TRE

MYC

-Doxycycline

3. Transplant into lethally irradiated mice

4. Expression of MYC and BCL

BCLbK0 is a more potent oncogene than BCLbwt

1

Relative survival

0.8

0.6

Mit-BCLxl; n=9 p=