The Role, Relationship and Therapeutic Potential of AGL and HAS2 in Bladder Cancer

Sunny Guin, PhD Research Scientist (Principal Investigator) Kabara Cancer Research Institute Gundersen Medical Foundation La Crosse, WI

Adjunct Assistant Professor Department of Biology University of Wisconsin – La Crosse La Crosse, WI Identifying AGL as a Regulator of Bladder Cancer Growth

Constructs 400 AGL 350 GPR107

) OSR2 3 300 INMT ZBTB4 250 200 150

Read Count (10 CountRead 100 50 0 T1 T2 T3 T4 Guin et al, JNCI 2014 What is AGL?

. amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase ( glycogen debranching enzyme)

. Has 2 catalytic activities - oligo-1,4-1,4-glucanotransferase - amylo-1,6-glucosidase

Ritterson et al, Nat Rev Urol. 2015 AGL and Glycogen Storage Disease Type III

.Deficiency of GDE – incomplete glycogenolysis and accumulation of abnormal glycogen with shorter outer chains – Limit Dextrin

. Glycogen Storage Disease Type III (Cori disease or Forbes disease) results from deficiency of GDE

. Liver and Muscle affected

. Multiple mutations identified in AGL gene in Glycogen Storage Disease Type III patients

GDE - glycogen debranching enzyme

Demo et al, J Hepatol. 2007 AGL is a Prognostic Marker in Bladder Cancer

Guin et al, JNCI 2014 Loss of AGL Drives Bladder Cancer Growth

1200 shCTL n=2:10 1.2 shAGL n=3:10

shCTL ) 3 shAGL 900 AGL 0.9

* (mm GAPDH 600 0.6 P < 0.05 0.60 Limit Dextrin * 0.48 Fluorescence 0.3 300

0.36 volume Tumor

0.24 0 0 0 1 2 3 4 5 0 9 16 23 30 37 0.12 µmol per mg/cell per µmol Day Day 0.00 shCTL shAGL 0.45 * 0.36 Glycogen

0.27

0.18 µmol per mg/cell per µmol 0.09

0.00 shCTL shAGL

Guin et al, JNCI 2014 AGL Drives Bladder Cancer Growth Independent of its Enzymatic Activity

L620P R1147G

Transferase Glucosidase C Term

Cheng et al, Hum Mol Genet 2009

shAGL shAGL+wtAGL shAGL+AGL L620P shAGL+AGL R1147G

AGL

α-Tubulin 196±12.3 138±6.5* 112±15.6* 121±14.3* Guin et al, JNCI 2014 Identifying Drivers of Bladder Cancer Growth with AGL loss

shCTL* shAGL1* 16 AGL 14 UMUC3

α-tubulin shCTL 12 10 Association of Gene 8 Expression vs. 38 6 Clinicopathologic 4 Variables 2

Fold ChangemRNA vs 0 SEMA3A HAS2 RRAGD VCAN EREG TULP3 UCHL1 62 Tumor vs Normal (T) S, G, S, G, S, G, T, S, S, G, T, S, S, G, Tumor Stage (S) Expressed Genes Expressed Sr Sr Sr G, Sr G, Sr

Top 100 Differentially Differentially 100 Top Tumor Grade (G) Sr Sr Patient Survival (Sr) Clinicopathologic Variable Associations

Guin et al, CCR 2015 Loss of AGL Drives Bladder Cancer Growth via Hyaluronic Acid (HA) Synthase 2 (HAS2)

* siHAS2 siCTL ) 160 3 8 shCTL siCTL 140 7 shCTL siHAS2 120 6

) shAGL siCTL 100 5 shAGL siHAS2 * 80 siCTL 0.47 0.42 4

HAS2 HAS2 Expression 60 (vs (vs 0.28 * * 3 * 2 40 * 20

1 Avg. Colony Formation Relative Relative

Fluorescence Intensity (10 0 0 shCTL shAGL1 shAGL2 0 1 2 3 4 shCTL shCTL shAGL shAGL Days siCTL siHAS2 siCTL siHAS2 UMUC3

shHAS2 shCTL *

) 300

3 9 8 shCTL shCTL 250 7 shCTL shHAS2 * ) 6 shAGL shCTL 200 5 shAGL shHAS2 150 siCTL 4

0.33 HAS2 HAS2 Expression (vs (vs * 0.2 3 100 2 * 50

1 Avg. Colony Formation Relative

Fluorescence Intensity (10 0 0 shCTL shAGL 0 1 2 3 4 shCTL shCTL shCTL shAGL Days shCTL shHAS2 shAGL shHAS2

T24T Guin et al, CCR 2015 AGL and HAS2 Expression in Normal Bladder Tissues

hTERT TRT-HU1 WT AGL-KO 1.2

UMUC3 ) 1 shCTL AGL AGL

α-tubulin α-tubulin 0.8 shAGL

4 3 * shCTL 0.6 3.5 shAGL 2.5 * 3 WT AGL-KO Expression 2.5 2 0.4 *

HAS Expression Expression HAS (vs shCTL 2 1.5 HAS2 HAS2

1.5 (vs WT) 0.2 1

(vs shCTL for (vs each)shCTL 1 *

Relative 0.5

N.D Relative 0.5 * Relative Gene Gene Relative Expression 0 0 0 HAS1 HAS3 hTERT TRT-HU1 HAS1 HAS2 HAS3

Guin et al, CCR 2015 Loss of AGL Drives Bladder Cancer Growth by HAS2 Mediated HA Synthesis

A i) ii) B i) ii)

) ) 3 * 3 7 * 2 1 1.6 UMUC3 1.8 T24T UMUC3 T24T 6 1.4 1.6 0.8 1.2 5 1.4 shCTL shCTL

1.2 1 Thousands 4 0.6 Thousands shAGL shAGL 1 0.8 3 0.8 0.4 0.6 2 0.6 0.4 0.4 1 0.2 0.2 0.2 0

0 0 0

Relative HA Day perHA Media in Relative

Relative HA Day perHA Media in Relative HA in Media per Day Day perHA Media (ng/ml)(10 in shCTL shCTL shAGL shAGL shCTL shCTL shAGL shAGL Day perHA Media in (ng/ml)(10 siCTL siHAS2 siCTL siHAS2 shCTL shHAS2 shCTL shHAS2 0 200 400 600 800 1000 0 200 400 600 800 1000 4MU Conc (μM) 4MU Conc (μM)

C D )

3 12

) * 3 7

10 shCTL 6 * shCTL+4MU * 8 * 5 * shAGL Thousands 4 6 shAGL+4MU 3 4 2

2 1

Fluorescence Intensity (10 Fluorescence Intensity (10 0 0 0 1 2 3 4 shCTL shCTL shCTL shAGL shAGL shAGL Days 4MU 4MU+HA 4MU 4MU+HA

4MU – 4-methylumbelliferone Guin et al, CCR 2015 4-Methylumbelliferone Reduce Bladder Xenograft Growth Driven by AGL Loss

A B

)

) 3

1600 UMUC3 3 900 T24T 1400 800 shCTL siCTL shCTL shCTL 700 1200 shCTL siHAS2 shCTL shHAS2 600 1000 shAGL siCTL shAGL shCTL 500 * 800 shAGL siHAS2 shAGL shHAS2 400 600 * 300 400 200

200 100 Avg. Tumor Volume (mm Volume Tumor Avg.

0 (mm Volume Tumor Avg. 0 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 Days Days

C D )

1200 UMUC3 ) 700 T24T

3 3

1000 600 shAGL shAGL 500 shAGL+4MU 800 shAGL +4MU 400 600 * 300 * 400 200 4MU 4MU

200 100

Avg. Tumor Volume (mm Volume Tumor Avg. Avg. Tumor Volume (mm Volume Tumor Avg. 0 0 0 10 20 30 0 10 20 30 40 Days Days Guin et al, CCR 2015 HA Receptors CD44 and RHAMM are Essential for Bladder Tumor

Growth Driven by AGL Loss

) 3 16 )

3 14 14 shCTL siCTL 12 shCTL siCTL shAGL siCTL * 12 * shAGL siCTL shCTL siCD44 10 10 shCTL siRHAMM shAGL siCD44 8 8 shAGL siRHAMM 6 6 4 4 2 2 0

Fluorescence Intensity (10 0 0 1 2 3 4 5 Fluorescence Intensity (10 0 1 2 3 4 5 Days Days

* 250 * 200

150

100

Avg. Avg. Colony/ Well 50

0 shCTL shCTL shCTL shAGL shAGL shAGL siCTL siCD44 siRHAMM siCTL siCD44 siRHAMM

Guin et al, BMC Cancer 2016 (under review) Loss of CD44 or RHAMM Induce Apoptosis in Low AGL Expressing Bladder Cancer Cells

A C

60 * 50 *

shCTL 40 siCTL 20µm siHAS2 20µm siCD44 20µm siRHAMM 20µm 30

UMUC3 20

% Apoptosis % 10 shAGL siCTL 20µm siHAS2 20µm siCD44 20µm siRHAMM 20µm 0 siCTL siHAS2 siCD44 siRHAMM -10

B D

35 shCTL shAGL 30 *

25

shCTL 20 siCTL 20µm siHAS2 20µm siCD44 20µm siRHAMM 20µm

15 T24T

10 % Apoptosis %

5 shAGL siCTL 20µm siHAS2 20µm siCD44 20µm siRHAMM 20µm 0 siCTL siHAS2 siCD44 siRHAMM -5 Guin et al, BMC Cancer 2016 (under review) Summary

. AGL is a new regulator of bladder cancer growth

. AGL is a prognostic marker in bladder cancer

. Loss of AGL drives bladder cancer growth via Hyaluronic acid (HA) Synthase 2 (HAS2)- HA-CD44/RHAMM axis Ongoing Studies

. AGL KO transgenic mice studies

. Therapeutic inhibition of the HAS2-HA-CD44/RHAMM axis in AGL low bladder cancer model Acknowledgements

Dan Theodorescu Lab Center for Regulatory and Environmental Analytical Department of Surgery, University Metabolomics (CREAM), Department of Chemistry, of Colorado, Denver, CO, USA University of Louisville, Louisville, KY, USA

. Dan Theodorescu, MD, PhD . Pawel Lorkiewicz, PhD . All Lab members . Andrew N. Lane, PhD . Teresa W.-M. Fan, PhD Gundersen Medical Foundation Kabara Cancer Research Institute Sigma-Aldrich Research Biotech, La Crosse, WI, USA Saint Louis, MO, USA . Andrea Spencer, PhD . Darby Oldenburg, PhD . Scott Knight, PhD . Steve Cash, BS . Heather Holemon, PhD

Department of Pathology, Cleveland Funding Clinic, Cleveland, OH, USA . Cancer League of Colorado, Inc .Sounak Gupta, PhD . BCAN Young Investigator Award .Donna Hansel, MD . NIH Funding (RO1) to Dr. Theodorescu