Inhibiting Glucosylceramide Synthase Sensitizes CML T315I Mutant to Bcr-Abl Inhibitor and Cooperatively Induces Glycogen Synthase Kinase-3-regulated Apoptosis

(1)

FIG.5.

FIG.5. GNFGNF--2/PDMP combination2/PDMP combination overcame T315I

overcame T315I--mutationmutation--mediatedmediated GNF

GNF--2 resistance 2 resistance in vitroin vitro and and in vivoin vivo..

FIG. 3.

FIG. 3. Exogenous Exogenous ceramide ceramideor endogenousor endogenous ceramide

ceramide accumulation triggered GSKaccumulation triggered GSK--3 activation.3 activation.

Inactivation of glycogen synthase kinase-3 (GSK-3) has been implicated in cancer progression. We previously showed abundance of inactive GSK-3 in human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl-inhibition- induced apoptosis. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr- Abl activity is therefore confirmed. To re-activate GSK-3, we inhibited glucosylceramide synthase (GCS) to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found silence of GCS and GCS inhibitor increased Bcr-Abl-inhibition-induced apoptosis. Furthermore, GCS inhibitor sensitized the most clinical problematic drug-resistant CML T315I mutants to Bcr-Abl inhibitor GNF-2- or imatinib-induced apoptosis. Combining GCS and Bcr-Abl inhibitors eliminated transplated-CML-T315I-mutants in vivo and dose-dependently sensitized primary cells from CML T315I patients to Bcr-Abl inhibitor-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl-restricted and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to re-activate GSK-3 and abrogate drug resistance.

A

BSTRACT

FIG. 7. FIG. 7. GNFGNF--2/PDMP2/PDMP combination overcame combination overcame T315I

T315I--mutationmutation--mediatedmediated GNF

GNF--2 resistance in 2 resistance in primary cells from primary cells from CML

CML--T315I patients.T315I patients.

NCKU

Inhibiting

Glucosylceramide

Synthase

Sensitizes

Chronic

Myeloid

Leukemia

T315I

Mutant

to

Bcr

-

Abl

Inhibitor

and

Cooperatively

Induces

Glycogen

Synthase

Kinase

-

3

3 -

-

regulated

Apoptosis

Wei-Ching Huang,1-3 _{Cheng-Chieh Tsai,}1,2,4 _{Chia-Ling Chen,}3 _{Tsai-Yun Chen,}5 _{Ya-Ping Chen,}1,5

Yee-Shin Lin,2,3 _{Pei-Jung Lu,}1-3 _{Chiung-Wen Tsao,}4 _{Chi-Yun Wang,}1,2 _{Yi-Lin Cheng,}1,6 _Chia-Yuan

Hsieh,1 _{and Chiou-Feng Lin}1-3

1_{Institute of Clinical Medicine,}2_{Institute of Basic Medical Sciences,}3_{Department of Microbiology and Immunology,}5_{Internal Medicine,}

and 6_{Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701,}

Taiwan; 4_{Department of Nursing, Chung Hwa University of Medical Technology, Tainan 717, Taiwan}

R

ESULTS

FIG. 4.

FIG. 4. Inhibiting GCS by either PDMP or Inhibiting GCS by either PDMP or shRNAshRNA increasedincreased GNF

GNF--22--induced GSKinduced GSK--3 activation and apoptosis.3 activation and apoptosis.

FIG. 2.

FIG. 2. Inhibiting Inhibiting BcrBcr--AblAbl activated GSKactivated GSK--3 and induced apoptosis in a 3 and induced apoptosis in a GSK

GSK--33--dependent manner.dependent manner. FIG. 1.

FIG. 1. Activating GSKActivating GSK--3 3 downregulateddownregulated cellcell proliferation and

proliferation and upregulatedupregulated BcrBcr--AblAbl--inhibitorinhibitor GNF

GNF--22--induced apoptosis.induced apoptosis.

FIG. 8.

FIG. 8. GNFGNF--2/PDMP combination restored the inhibitory2/PDMP combination restored the inhibitory effect of GNF

effect of GNF--2 against T315I2 against T315I--mutated mutated BcrBcr--AblAbl and inducedand induced GSK

GSK--33--dependent apoptosis in Ba/F3dependent apoptosis in Ba/F3--p210p210T315IT315I _cells._cells.

Acknowledgement



GSK

-

3 negatively regulates the

oncogenic

activity of

Bcr

-

Abl

in chronic myeloid leukemia.



Combining

glucosylceramide

synthase

inhibitor PDMP

with

Bcr

-

Abl

inhibitor GNF

-

2 or

imatinib

abroagates

gatekeeper mutation T315I

-

directed drug resistance.

C

ONCLUSIONS

GNF

-

2

2 Proliferation / Apoptosis resistance

or

GSK

-

3

3 Bcr

Bcr

-

Abl

imatinib

PDMP

Ceramide

?

T315I mutation

Summary

FIG.6.

FIG.6. Combining PDMP restoredCombining PDMP restored Bcr Bcr--AblAbl inhibitor inhibitor imatinib imatinib- -induced induced proliferation proliferation Inhibition Inhibition and and apoptosis. apoptosis.

I

NTRODUCTION

A

IMS

Cell culture / Colony forming assay / Proliferation test (WST

Cell culture / Colony forming assay / Proliferation test (WST--8)8)

DAPI,

DAPI, AnnexinAnnexin V and PI staining / Western blotting / Flow V and PI staining / Western blotting / Flow cytometrycytometry Transfection

Transfection / RNA silencing / / RNA silencing / in vivoin vivo study / Patient study (study / Patient study (ex vivoex vivo))

M

aterials

M

ethods

&

 Oncogenic Tyrosine Kinase Bcr-Abl  Chronic Myeloid Leukemia (CML)  Glycogen Synthase Kinase-3 (GSK-3)  Tumor Suppressor Lipid Ceramide  Glucosylceramide Synthase (GCS)  Anti-Cancer Drug Resistance