Targeting Drug-Insensitive CML Stem/Progenitor Cells with

Combination Treatments of New ABL and PP2A Inhibitors

Damian LaiSRD 2014

CML (Chronic Myeloid Leukemia)

Chronic Phase(Accumulation of granulocytes)

Accelerated Phase

Blast Crisis (Adapted from Ren RB Nat Cancer Rev,2005)

HSC(BCR-ABL)

CMP CLP

GMP MEP

M RBCT cellB cellPlatelets

Sattler et al, Cytokine Growth Factor Rev 8:63, 1997

G

Chronic myeloid leukemia (CML) is a multistep, multilineage myeloproliferative disease.• Incidence 1/100.000, 20-30% of human leukemia in adults

DD Domain

Y177

S/T kinase

Rho-

GEF

Y1294

SH3 SH

2

BCR ABL

Tyrosine Kinase

NLS DNA BD

Actin BD

p210BCR/ABL

Ras

MAPK

PI3K

AKT

JAK2

STAT5

Increased proliferationDecreased apoptosis

Massive accumulation of myeloid cells in circulation

(Major characteristic of CML)

The First Line of CML Treatment: Tyrosine Kinase Inhibitors (TKIs)

Imatinib

BCR-ABL kinase domain

Current 1st line: Imatinib Alternatives: Dasatinib, Nilotinib

Druker et al, Blood 112:4808, 2008

Abelson Helper Integration Site 1 (AHI-1) and CML

lin- lin- lin+

34+38- 34+38+ 34-

Zhou et al, J Exp Med 205:2657, 2008

Jiang et al, Blood 102:2976, 2004

•AHI-1/Ahi-1 is down-regulated during normal hematopoietic cell differentiation.

•AHI-1 is highly deregulated in CML stem/progenitor cells, indicating a possible cooperative activity in leukemia development.

AHI-1

STAT5

Stem cell proliferation, survival

& maintenance

IL-3R

IL-3

Autocrine loop

Stem cell genomic instability

BCR-ABLJAK2P

SH3

WD40N

P

TKI -responseprotein

degradation

Chen et al, J Natl Cancer Inst 105:405, 2013

Drug Screen: Experimental Design

Hoechst YFP Merge

Prestwick compound library:

-1200 small molecules-100% marketed drugs-known bioavailability and safety in humans

AHI-1-YFP+ CML cells

48 H

K562-AHI-1

K562-AHI-1 + IM

24 H

Pres library (10M)

Pres library

FixationHCS protocol adjustmentHCS ScanData analysis

Min Chen & Kaiji Hu

K5 6 2 K5 6 2 Ah i-1 B5

0

1

2

3

4

5R

elat

ive

tran

scri

pt

leve

lsAHI-1

K562 K562 AHI-1

Cantharidin

control 0.5uM IM 5uM CAN 0.5uM IM+ 5uM

CAN

0%20%40%60%80%

100%120%

% V

iabl

e ce

lls

control 0.5uM IM 5uM CAN 0.5uM IM+ 5uM CAN

0%

20%

40%

60%

80%

100%

120%

% P

rolif

erati

ve c

ells

control 0.5uM IM 5uM CAN 0.5uM IM+ 5uM CAN

0%

20%

40%

60%

80%

100%

120%

control 0.5uM IM 5uM CAN 0.5uM IM+ 5uM CAN

0%

20%

40%

60%

80%

100%

120%K562 K562-IMR

P<0.001 P<0.001

P<0.001 P<0.001

un-treated

DMSO 5IM 2.5CAN 5IM + 2.5CAN

0%

20%

40%

60%

80%

100%

Normal Bone Marrow

• Not FDA approved• High toxicity in NBM• Need to find alternative drug with similar

mechanism of PP2A inhibition

Hypothesis and Objective

• Hypothesis• A new PP2A inhibitor, to selectively target and destabilize

multiple AHI-1-mediated complexes, including AHI-1-BCR-ABL-β-catenin-PP2A, in combination with new TKIs, is more effective in eliminating leukemic stem cells, a population highly resistant to current TKI monotherapy.

• Objective• To investigate role of PP2A in CML and evaluate the

therapeutic potential of two PP2A inhibitors, LB100 and LB102, alone and in combination with TKI to inhibit the growth of CML stem and progenitor cells in vitro and in vivo.

New Drugs Specifically Target PP2A and Efficacy is Affected by Expression of AHI-1

0 1 2 3 4 5 6 7 80%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Time Course Phosphatase Assay: 5uM Inhibitors

LB1.0LB1.2

Time (H)

Phos

phat

ase

Acti

vity

Rel

ative

to

Unt

reat

ed C

ells

(%)

p<0.05

0.5uM IM 2.5uM LB100 2.5uM LB1.20%

25%

50%

75%

K562 k562 SHRNA sorted

% V

iabi

lity

p<0.05 p<0.05

C 0.5uM IM 5uM LB100 5uM LB1.2 0.5uM IM + 5uM LB100

0.5uM IM + 5uM LB1.2

0%10%20%30%40%50%60%70%80%90%

100% K562 24h48h

% P

rolif

erati

ve C

ells

C 5uM IM 5uM LB100 5uM LB1.2 5uM IM + 5uM LB100

5uM IM + 5uM LB1.2

0%10%20%30%40%50%60%70%80%90%

100%

K562-IMR 24h

48h

% P

rolif

erati

ve C

ells

Control 0.5uM IM 2.5uM drug

5uM drug 0.5uM IM + 2.5uM

drug

0.5uM IM + 5uM drug

0%10%20%30%40%50%60%70%80%90%

100%K562

LB1.0

LB1.2

% V

iabi

lity

P<0.01

P<0.01

P<0.01

P<0.01

P<0.01

P<0.01

Control 5uM IM 2.5uM drug 5uM drug 5uM IM + 2.5uM drug

5uM IM + 5uM drug

0%10%20%30%40%50%60%70%80%90%

100%

K562-IMR LB1.0LB1.2

% V

iabi

lity

P<0.01

P<0.01

Inhibition of PP2A In Combination With IM Leads To Decreased Proliferation of K562 and K562 IM-Resistant Cells

Inhibition of PP2A in Combination With IM Leads To Apoptosis of K562 and K562 IM-Resistant Cells

LB1.0 5uMLB1.2 5uM

---

K562-IMR

IM 5uM ++-

+-+

+--

-+-

--+

---

++-

+-+

+--

-+-

--+

24H 48H

β-Actin

C. Caspase 8

C. Caspase 3

Control IM 5uM LB100 5uM LB1.2 IM+5uM LB100

IM+5uM LB1.2

0%

10%

20%

30%

40%

50%

60%

Apoptosis 48hk562

k562-IMR

% A

nnex

in V

pos

itve

cel

ls

P<0.05

P<0.05

Control 0.5 IM 1 LB100 2.5 LB100

5 LB100 1LB1.2 2.5 LB1.2

5LB1.2 0.5 IM + 1 LB100

0.5 IM + 2.5

LB100

0.5 IM + 5 LB100

0.5 IM + 1 LB1.2

0.5 IM + 2.5

LB1.2

0.5 IM + 5 LB1.2

0

5

10

15

20

25

30

35

40

45

50 K562 Cell Cycle AnalysisG1SG2/M

Treatment

Perc

enta

ge o

f Cel

ls (

%)

Control 5 IM 1 LB100 2.5 LB100

5 LB100 1LB1.2 2.5 LB1.2

5LB1.2 5 IM + 1 LB100

5 IM + 2.5

LB100

5 IM + 5 LB100

5 IM + 1 LB1.2

5 IM + 2.5

LB1.2

5 IM + 5 LB1.2

0

10

20

30

40

50

60 K562-IMR Cell Cycle AnalysisG1 S

G2/M

Treatment

Perc

enta

ge o

f Cel

ls (%

)

Inhibition of PP2A Leads to Mitotic Arrest (G2/M Phase)

G2/M phase arrest is dosage dependent

*

**

*

*

*

*

*

N=3, *, P < 0.05

Control 0.5 IM 1 LB100 2.5 LB100

5 LB100 1LB1.2 2.5 LB1.2

5LB1.2 0.5 IM + 1 LB100

0.5 IM + 2.5

LB100

0.5 IM + 5 LB100

0.5 IM + 1 LB1.2

0.5 IM + 2.5

LB1.2

0.5 IM + 5 LB1.2

0

5

10

15

20

25

30

35

40

45

50 K562 Cell Cycle Analysis G1SG2/M

Treatment

Perc

enta

ge o

f Cel

ls (

%)

Control 5 IM 1 LB100 2.5 LB100

5 LB100 1LB1.2 2.5 LB1.2

5LB1.2 5 IM + 1 LB100

5 IM + 2.5

LB100

5 IM + 5 LB100

5 IM + 1 LB1.2

5 IM + 2.5

LB1.2

5 IM + 5 LB1.2

0

10

20

30

40

50

60 K562-IMR Cell Cycle Analysis G1 S

G2/M

Treatment

Perc

enta

ge o

f Cel

ls (%

)

Inhibition of PP2A Leads to Mitotic Arrest (G2/M Phase)

*

**

** *

**

*

*

*

*

*

*

*

*

N=3, *, P < 0.05

G2/M Arrest Leads to Mitotic Catastrophe

K562: 20uM LB100 – 40X

Multipolar Spindles

Multipolar Cell Division

Multipolar Cell Division with Lagging Chromosomes

G2/M Arrest Leads to Mitotic Catastrophe

Untreated – 40X

5uM LB1.2 – 40X

DAPIAnti-α-tubulin Merge

5LB100 5LB1.20%

5%

10%

15%

20%

25%

30%

35%

40%

Cells Undergoing Mitotic Catastrophe

% ca

tast

roph

ic ce

lls

• BCR-ABL has been shown to stabilize β-Catenin in CML through tyrosine phosphorylation at y86 and y654 residues• PP2A activity supports Wnt signalling pathway leading to stabilization of β-Catenin

• Effect of dual inhibition of BCR-ABL and PP2A may converge on β-Catenin

PP2A Inhibition in Combination with IM Affects AHI-1-BCR-ABL-JAK2-STAT and AKT Pathways

Phosho-β-Catenin (Y86)

β-Catenin (total)

K562 IM Resistant Cells

1: Control2: 5uM IM3: 5uM LB1004: 5uM LB1.25: 5uM IM + 5uM LB1006: 5uM IM + 5uM LB1.2

1 2 3 4 5 6

24H

Phosho-β-Catenin (T41,S45)

GAPDH

48H

1 2 3 4 5 6

Imatinib

BCR-ABL

β-catenin transcriptional activation repressed

β-catenindegradation

T41S45 Y654

β-catenin

p pY86

PP2A

PP2A inhibitorsBCR-ABL

T41S45 Y654

β-catenin

Y86

PP2A

p p

β-catenin transcriptional activation

β-cateninstability

IP: β-Catenin (total)WB: B-Catenin (total)

IP: β-Catenin (total)WB: PP2A-C

35kDa

40kDa

+C IP IgG

IgGIP

100kDa

70kDa

+C

IP: β-Catenin (total)WB: BCR-ABL

100kDa130kDa

+C IP IgG

170kDa

Potential mechanism β-Catenin Phosphorylation

Conclusion

• Combination of LB100 and LB102 with IM significantly inhibits proliferation and induces apoptosis of IM-resistant cells.

• LB100 and LB102 induces G2-M arrest leading to mitotic catastrophe.

• Combination of LB100 and LB102 with IM affects the AHI-1-BCR-ABL-JAK2-STAT5 pathway and potentially targets β-catenin for protein degradation.

• Inhibition of PP2A leads to mitotic catastrophe. This could occur via the activation of CyclinB/CDK1 complex.

Acknowledgments

• Dr Xiaoyan Jiang

• Will Liu

• Min Chen

• Katharina Rothe

• Kevin Lin

• Leon Lin

• Rachel Huang

• Sharmin Esmailzadeh

• Kyi Min Saw

• Helena Wang

• Josephine Leung

• Elianne Abramovich

•Dr. Catherine Pallen•Dr. Sandra Dunn

Committee members