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Discovery and Molecular Characterization of AGS-15/SLITRK6 as a Novel Target for Antibody-Mediated
Therapeutic Development in Bladder Cancer Yuriy Shostak, Suzanne Said, Deanna L. Russell, Michael D. Mattie, Mi Sook Chang, Ashley Christensen, Karen Morrison, Kendall Morrison, David Stover, Pia Challita-Eid. Agensys Inc., an affiliate of Astellas Pharma Inc, Santa Monica, CA.
Abstract
• AGS-15/SLITRK6 discovered at Agensys as a differentially expressed
gene in bladder cancer utilizing suppression subtractive hybridization
(SSH) methodology using:
- bladder cancer patient specimens pool vs. 9 vital normal tissue
mix and normal bladder
• SLITRK6 encodes a cell surface single-pass type I transmembrane
protein, with two N-terminal LRR domains with SLIT proteins homology
and C-terminal regions with neurotrophin receptors homology
• Member of the SLITRK family of factors with neuronal function activities
• Slitrk6 implicated in neurite outgrowth control, inner ear neural circuits
development, and auditory-vestibular functions
• SLITRK6 gene chromosomal location: 13q31.1
• SLITRK6 exhibits restricted tissue expression pattern, distinct from
other SLITRK family members
Discovery efforts utilizing suppression subtractive hybridization
identified AGS-15/SLITRK6 as a differentially expressed gene in bladder
cancer. SLITRK6 encodes a cell surface type I transmembrane protein, a
member of the SLITRK family of factors with neuronal function activities.
Comprehensive SLITRK6 RNA expression analysis in patient specimens
revealed limited restricted normal tissue expression and high frequent
expression in bladder cancers, as well expression in subsets of breast,
lung and several other cancer types. Tissue immunohistochemical
analysis validated SLITRK6 protein limited expression in normal tissues
and high expression levels in cancers. SLITRK6 expression profiling in
cancer cell lines and Agensys proprietary patient-derived xenografts
(PDXs) identified model systems in relevant cancer indications for further
target characterization and antibody therapeutics development. Genomic
analyses, including whole exome next generation sequencing,
chromosomal aberrations and gene copy number assessments, were
performed to evaluate SLITRK6 gene status in model cell lines and
PDXs. Thus, we discovered and characterized AGS-15/SLITRK6 as a
novel target with high levels and selective expression in tumors,
particularly in bladder cancer. This cell surface antigen’s attractive
expression profile is a major determinant of AGS-15/SLITRK6 as a
suitable and preferential candidate for antibody drug conjugate
therapeutic targeting in bladder cancer treatment and, potentially, in
several other cancer indications.
Copyright: Agensys, Inc. an affiliate of Astellas Pharma, Inc.
Printed by
Abstract # 2047
AGS-15/SLITRK6 Gene Limited Normal Tissue
Expression and High Expression in Cancers
AGS-15/Slitrk6 Protein is Highly Expressed in
Cancer Tissues
Discovery of AGS-15/SLITRK6 as a Differentially
Expressed Gene in Bladder Cancer
Summary and Discussion
• AGS-15/SLITRK6 was identified and validated as a gene highly and
selectively expressed in tumors, particularly in bladder cancer, and
several other cancer indications
• Agensys proprietary patient-derived xenografts (PDXs) and cell lines
with SLITRK6 expression in relevant cancer indications were identified,
characterized, and utilized in antibody therapeutics development
- Immunohistochemical tissue expression analysis and therapeutic antibody
development: abstracts #1274 and #4332, 2013 AACR Annual Meeting
• DNA copy number analysis revealed a lack of SLITRK6 gene
amplification in a majority of samples examined, especially in bladder
cancer
• In addition to expression in bladder cancer, SLITRK6 expression was
detected in subsets of patient tumor specimens and cancer cell lines of
other cancer indications (e.g., lung and breast cancers, neuroblastoma,
HNSCC)
• AGS-15/SLITRK6 cell surface antigen attractive expression profile is a
major determinant of AGS-15/SLITRK6 as a suitable and preferential
candidate for antibody drug conjugate therapeutic targeting in bladder
cancer treatment and, potentially, in several other cancer indications
• Membranous and cytoplasmic immunoreactivity of anti-AGS-15 specific
mAb M15-68(2)22
Immunohistochemical Analysis of Slitrk6 Protein Expression
Bladder transitional cell carcinoma specimens:
AG-B7 patient-derived xenograft Patient tumor BL2082
Lung cancer specimens:
Patient tumor LC20810
Lung squamous cell carcinoma Bronchoalveolar carcinoma
NCI-H322M-XCL xenograft
Mouse IgG
Control IHC tissue staining
Anti-AGS-15 mAb M15-68(2)22
SLITRK6-positive AG-B7 SLITRK6-negative specimen
SLITRK6 mRNA Expression in Normal Tissues and Patient Cancer
Specimens: semi-quantitative RT-PCR analysis (normalized to GAPDH)
Normal Tissues
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
Bla
dd
er
Blo
od
ce
lls (
PB
MC
s)
Bo
ne
ma
rro
w
Bra
in
Bre
ast
Ce
rvix
Co
lon
He
art
Kid
ne
y
Liv
er
Lu
ng
Lym
ph
no
de
Ova
ry
Pitu
itary
gla
nd
Pa
ncr
ea
s
Pla
cen
ta
Pro
sta
te
Sa
liva
ry g
lan
d
Ske
leta
l mu
scle
Ski
n
Sm
all
inte
stin
e
Sp
lee
n
Sto
ma
ch
Te
stis
Th
ymu
s
To
nsi
l
Ute
rus
HU
VE
C
NP28-1
NP28-2
NP28-3
20.6
Bladder Cancer
141312 3 11 6 17 5 8 1815 19 4 16 1 9 2 10 7
Rela
tive A
bundance
Patient Specimen
SLITRK6 mRNA Expression in Normal Tissues, Patient Cancer
Specimens, and Patient-Derived Xenografts (PDX): whole genome
microarray analysis (SLITRK6 - 2 probes)
0 1000 2000 3000 4000 5000
N. Bladder 1
N. Bladder 2
AG-B10
AG-B11
AG-B4
AG-B7
AG-B8
AG-B1 (P0)
AG-B1(+) (S17)
AG-B10 (P10)
AG-B10 (P4)
AG-B11 (P3)
AG-B11 (P7)
AG-B12 (P3)
AG-B13 (P3)
AG-B4 (P17)
AG-B4 (P6)
AG-B7 (P17)
AG-B7 (P7)
AG-B8 (P13)
AG-B8 (P5)
N. PBMC 1
N. PBMC 2
N. Bone Marrow
N. Brain 1
N. Brain 2
N. Breast 1
N. Breast 2
N. Breast 3
AG-Br10
AG-Br12
AG-Br13
AG-Br2
AG-Br21
AG-Br26
AG-Br28
AG-Br29
AG-Br4
AG-Br7
AG-Br8
AG-Br21 (P2)
N. Colon 1
N. Colon 2
N. Colon 3
N. Heart 1
N. Heart 2
N. Intestine
N. Kidney 1
N. Kidney 2
AG-K12
AG-K18
AG-K24
AG-K9
AG-K18 (P10)
AG-K18 (P6)
N. Liver 1
N. Liver 2
N. Lung 1
N. Lung 2
N. Lung 3
AG-L11
AG-L12
AG-L13
AG-L14
AG-L15
AG-L16
AG-L18
AG-L19
AG-L21
AG-L25
AG-L29
AG-L4
AG-L8
AG-L9
AG-L11 (P1)
AG-L11 (P4)
AG-L13 (P14)
AG-L13 (P5)
AG-L14 (P10)
AG-L14 (P5)
AG-L15 (P5)
AG-L15 (P8)
AG-L4 (P14)
AG-L4 (P6)
AG-L8 (P7)
AGL8 (P17)
AG-L9 (P2)
AG-L9 (P5)
N. Lymph Node 1
N. Lymph Node 2
N. Lymph Node 3
N. Ovary 1
N. Ovary 2
N. Ovary 3
N. Pancreas 1
N. Pancreas 2
N. Placenta 1
N. Placenta 2
N. Prostate 1
N. Prostate 2
N. Prostate 3
AG-PC10
AG-PC10 (P3)
AG-PC10 (P7)
CWR22R
LAPC-9AD (PR16)
LAPC-9AD (PR2)
LAPC-9AI (PR10)
LAPC-9AI (PR3)
N. Sk. Muscle 1
N. Sk. Muscle 1
N. Skin 1
N. Skin 2
N. Skin 3
N. Spleen 1
N. Spleen 2
N. Spleen 3
N. Stomach
N. Testis
N. Tonsil
N. Uterus 1
N. Uterus 2
HUVEC
22RV-1
HPAC-LM-XCL
M14
H. Ref. RNA
H. Ref. RNA
H. Ref. RNA
NPa
tien
t Tum
orXe
nogr
aft
Nor
mal
Pati
ent T
umor
XN
Tum
orX
Nor
mal
Pati
ent T
umor
Xeno
graf
tN
orm
alT
Xeno
graf
t
Blad
der
Nor
mal
Ti
ssue
sBr
east
Nor
mal
Tis
sues
Kidn
eyN
Lung
Nor
mal
Tis
sues
Pros
tate
Nor
mal
Tis
sues
Cell
Line
sPo
oled
N
orm
al
Normalized Intensity
A_33_P3230189 Intensity
0 10000 20000 30000 40000 50000 60000
N. Bladder 1
N. Bladder 2
AG-B10
AG-B11
AG-B4
AG-B7
AG-B8
AG-B1 (P0)
AG-B1(+) (S17)
AG-B10 (P10)
AG-B10 (P4)
AG-B11 (P3)
AG-B11 (P7)
AG-B12 (P3)
AG-B13 (P3)
AG-B4 (P17)
AG-B4 (P6)
AG-B7 (P17)
AG-B7 (P7)
AG-B8 (P13)
AG-B8 (P5)
N. PBMC 1
N. PBMC 2
N. Bone Marrow
N. Brain 1
N. Brain 2
N. Breast 1
N. Breast 2
N. Breast 3
AG-Br10
AG-Br12
AG-Br13
AG-Br2
AG-Br21
AG-Br26
AG-Br28
AG-Br29
AG-Br4
AG-Br7
AG-Br8
AG-Br21 (P2)
N. Colon 1
N. Colon 2
N. Colon 3
N. Heart 1
N. Heart 2
N. Intestine
N. Kidney 1
N. Kidney 2
AG-K12
AG-K18
AG-K24
AG-K9
AG-K18 (P10)
AG-K18 (P6)
N. Liver 1
N. Liver 2
N. Lung 1
N. Lung 2
N. Lung 3
AG-L11
AG-L12
AG-L13
AG-L14
AG-L15
AG-L16
AG-L18
AG-L19
AG-L21
AG-L25
AG-L29
AG-L4
AG-L8
AG-L9
AG-L11 (P1)
AG-L11 (P4)
AG-L13 (P14)
AG-L13 (P5)
AG-L14 (P10)
AG-L14 (P5)
AG-L15 (P5)
AG-L15 (P8)
AG-L4 (P14)
AG-L4 (P6)
AG-L8 (P7)
AGL8 (P17)
AG-L9 (P2)
AG-L9 (P5)
N. Lymph Node 1
N. Lymph Node 2
N. Lymph Node 3
N. Ovary 1
N. Ovary 2
N. Ovary 3
N. Pancreas 1
N. Pancreas 2
N. Placenta 1
N. Placenta 2
N. Prostate 1
N. Prostate 2
N. Prostate 3
AG-PC10
AG-PC10 (P3)
AG-PC10 (P7)
CWR22R
LAPC-9AD (PR16)
LAPC-9AD (PR2)
LAPC-9AI (PR10)
LAPC-9AI (PR3)
N. Sk. Muscle 1
N. Sk. Muscle 1
N. Skin 1
N. Skin 2
N. Skin 3
N. Spleen 1
N. Spleen 2
N. Spleen 3
N. Stomach
N. Testis
N. Tonsil
N. Uterus 1
N. Uterus 2
HUVEC
22RV-1
HPAC-LM-XCL
M14
H. Ref. RNA
H. Ref. RNA
H. Ref. RNA
NPa
tien
t Tum
orXe
nogr
aft
Nor
mal
Pati
ent T
umor
XN
Tum
orX
Nor
mal
Pati
ent T
umor
Xeno
graf
tN
orm
alT
Xeno
graf
t
Blad
der
Nor
mal
Ti
ssue
sBr
east
Nor
mal
Tis
sues
Kidn
eyN
Lung
Nor
mal
Tis
sues
Pros
tate
Nor
mal
Tis
sues
Cell
Line
sPo
oled
N
orm
al
Normalized Intensity
A_23_P65307 Intensity
Lung Cancer
4 9 3 19 6 18241321111620 5 2 232210 8 12 1 151417 7
Normal Tissues
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
Bla
dd
er
Blo
od
ce
lls (
PB
MC
s)
Bo
ne
ma
rro
w
Bra
in
Bre
ast
Ce
rvix
Co
lon
He
art
Kid
ne
y
Liv
er
Lu
ng
Lym
ph
no
de
Ova
ry
Pitu
itary
gla
nd
Pa
ncr
ea
s
Pla
cen
ta
Pro
sta
te
Sa
liva
ry g
lan
d
Ske
leta
l mu
scle
Ski
n
Sm
all
inte
stin
e
Sp
lee
n
Sto
ma
ch
Te
stis
Th
ymu
s
To
nsi
l
Ute
rus
HU
VE
C
NP28-1
NP28-2
NP28-3
20.6
Rela
tive A
bundance
Patient Specimen
• High levels of SLITRK6 mRNA expression in patient bladder cancer
AG-B7, AG-B8, and specimens from various cancer indications
• Corresponding proprietary PDXs exhibit retained and stable levels of
SLITRK6 expression, thus providing suitable representative models for
use in therapeutics development
AGS-15/SLITRK6 Gene is Highly and Frequently
Expressed in Bladder Cancer
AGS-15/SLITRK6 Expression in Cancer Cells –
Identification and Validation of Model Cell Lines
CL = Cell lines (listed in order):UM-UC-3, J82, SCaBER
P = PatientN = Normal Bladder
N AT = Normal adjacent tissue
T = Tumor
P1 - Transitional, grade 2P2 - Transitional, grade 3/2
P3 - Transitional,
P4 - Polypoid Cystitis
P5 - Papillary, grade 3/3
9.5-7.5-
4.4-
2.4-
1.4-
CL CL CL N NAT T NAT T NAT T T Tkb
P1 P2 P3 P4 P5
9.5-7.5-
4.4-
2.4-
1.4-
CL CL CL N NAT T NAT T NAT T T Tkb
P1 P2P2 P3P3 P4 P5
0 0 ND ND ND ND ND 3 ND ND ND 2
9.5-7.5-
4.4-
2.4-
1.4-
CL CL CL N NAT T NAT T T T T Tkb
P1 P2P2 P3P3 P4P4 P5P5 P6P6
RT-PCR
result
0 0 ND 0.5 ND ND ND 1 1 1 2 3 CL = Cell lines (from left to right): UM-UC-3, J82, SCaBER
P = PatientN = Normal Bladder
N AT = Normal adjacent tissueT = Tumor
P1 - Transitional, grade 4
P2 - Squamous inv.
P3 - Transitional, grade 3
P4 - Papillary non -inv, grade 1/3
P5 - Papillary, grade 3/3
P6 - Transitional, grade 3/2
AGS-15/SLITRK6 mRNA Expression and Transcript
Size Analysis by Northern Blot in Bladder Cancer
• SLITRK6 mRNA detected in bladder cancer specimens as a transcript
appearing to migrate predominantly as a single band around 4.4 kb
marker
SLITRK6 mRNA Expression in Normal Bladder and Bladder
Cancer Patient Specimens: real-time qPCR analysis
SLITRK6 mRNA Expression in Cell Lines: CCLE Bioinformatics Data
AGS-15/SLITRK6 RPL19 Actin
( .1/.2 primer set) .3/.4OriACTB
5'/3'
NCI-H1975 Lung Adenocarcinoma 2510 24370 37149
NCI-H1734 Lung Adenocarcinoma, NSCLC 1549 2689 24621
CHP-212 Brain Neuroblastoma 1160 5918 7562
SW 780 Bladder Transitional Cell Carcinoma 1089 8644 24188
HPAC-LM-XCL Pancreas Adenocarcinoma (mouse lung MET) 992 11293 12636
HCC1419 Breast Primary Ductal Carcinoma 787 1594 41333
NCI-H520 Lung Squamous Cell Carcinoma 736 13568 25287
NCI-H1930 Lung Carcinoma; Small Cell Lung Cancer - Lymph Node MET 531 4690 16234
A-375 Skin Malignant Melanoma 459 9294 2194
NCI-H322M-XCL Lung Bronchoalveolar carcinoma 449 5949 N/A
RT4 Bladder Transitional Cell Papilloma 329 3971 6783
BxPC-3 Pancreas Adenocarcinoma 297 2780 8633
OVCAR-5 Ovary Carcinoma 220 11906 25530
TOV-112D Ovary Endomitrioid Adenocarcinoma 205 15695 12286
MDA-MB-453 Breast Metastatic Carcinoma - Pericardial effusion 160 8425 15973
SF-295 CNS Glioblastoma multiforme 155 2045 16509
A549 Lung Adenocarcinoma 143 9554 7607
CMK Peripheral Blood Acute Megakaryocytic Leukemia 125 6449 39246
NCI-H1581 Lung NSCLC, Large Cell 121 16101 19164
OVCA420 Ovary 103 4492 13244
HCC38 Breast Primary Ductal Carcinoma 89 5036 27562
PC3 Prostate Carcinoma 74 5173 16985
A-253 Submaxillary salivary gland Epidermoid Carcinoma 57 13416 43253
DMS-114 Lung Small Cell Carcinoma 47 15983 13358
HCC827 Lung Adenocarcinoma 42 5430 13239
G-292 Bone Osteosarcoma 38 4127 13832
NCI-H292 Lung Mucoepidermoid Pulmonary Carcinoma 33 4543 12274
Hep 3B2.1-7 Liver Hepatocellular Carcinoma (HBV integrated) 30 20436 24260
AG-OV1-XCL Ovary Adenosquamous Carcinoma 28 5576 12871
NCI-N87 Stomach Gastric Carcinoma 24 33554 13362
Capan-1 Pancreas Adenocarcinoma - Liver MET 23 8544 26073
SK-MEL-2 Skin Malignant Melanoma - Thigh MET 22 11136 5806
U-87 MG Brain Glioblastoma 18 3832 10327
CAL-120 Breast Adenocarcinoma 16 1506 7961
SNB-19 CNS Glioblastoma 15 1833 25637
UT-7 Bone Marrow Acute Myeloid Leukemia 15 7710 25286
HCC1806 Breast Primary Acantholytic Squamous Cell Carcinoma 14 1479 11190
NCI-H23 Lung Adenocarcinoma, NSCLC 12 3589 5585
T98G Brain Glioblastoma multiforme 12 11106 27477
UACC-62 Melanoma Malignant Melanoma 10 1666 6566
GRANTA-519 Peripheral Blood B Cell Lymphoma 0 61247 18975
OVCAR-3 Ovary Carcinoma - Ascites MET 0 1803 8493
AG-Mel2-PCL Skin Melanoma 0 5064 7044
M14 Melanoma Amelanotic Melanoma 0 2824 18102
JVM-3 Peripheral Blood Chronic B cell Leukemia (PLL) - EBV transformed 0 1915 12792
WM-266-4 Skin Melanoma 0 6772 16674
Capan-2 Pancreas Adenocarcinoma 0 2562 29484
Daudi Peripheral Blood Burkitt's lympohoma; B lymphoblast 0 11691 11742
HS-578T Breast Ductal Carcinoma 0 1092 20843
qPCR: Relative Abundance (10-4
GAPDH units)
Cell Line Name Cell Origin (Organ) Cell / Cancer Type
Copy number data for SLITRK6 extracted from CCLE_copynumber_2012-09-29.seg. http://www.broadinstitute.org/ccle
• SLITRK6 mRNA expression was detected in multiple cell lines of several cancer
indications, including neuronal, bladder, breast, lung, and other origins
AGS-15/SLITRK6 Gene DNA Copy Number Analysis in Cancer
Specimens with SLITRK6 mRNA Expression
• SLITRK6 gene, in general, is not amplified in cancer cell lines expressing SLITRK6 mRNA
• Lack of SLITRK6 gene amplification was observed in majority of samples examined, especially in
bladder cancer
• Similar observations as above were made from whole genome aCGH copy number analysis
conducted on select cancer specimens with SLITRK6 expression, as well as on additional
specimens with low/no SLITRK6 expression (data not shown)
SLITRK6 Gene DNA Copy Number in Cell Lines: CCLE Bioinformatics Data
• Various levels of SLITRK6 mRNA expression detected in majority
of bladder tumors evaluated
24915
23853
SLITRK6 gene DNA Copy Number
Evaluation: CNV qPCR assay
SLITRK6 mRNA Expression Analysis:
real-time qPCR
AGS-15/SLITRK6 SNP Analysis in Cancer Specimens
and Normal Individuals
aa position
aa
change Zygosity Model(s)
Variant
type Presence in genome panel (69) Comments Base Sequence used in the sequence comparability analysis:
25 L>F x1 Known 1 European, 1 Kenya Signal peptide NM_032229
414 Q>R x1 B1(+), L16 Known 6 Yoruba, 2 Kenya, 3 Afr-Amer ECD NP_115605
414 Q>R x2 - Known 1 Kenya, 2 Afr-Amer ECD >gi|40217825|ref|NP_115605.2| SLIT and NTRK-l ike protein 6 precursor [Homo sapiens]
451 L>Q x1 OV5 Novel - ECD MKLWIHLFYSSLLACISLHSQTPVLSSRGSCDSLCNCEEKDGTMLINCEAKGIKMVSEISVPPSRPFQLS
508 L>F x1 B7,B8,B9,C9 Known - ECD LLNNGLTMLHTNDFSGLTNAISIHLGFNNIADIEIGAFNGLGLLKQLHINHNSLEILKEDTFHGLENLEF
526 V>I x2 Br7 Novel - ECD LQADNNFITVIEPSAFSKLNRLKVLILNDNAIESLPPNIFRFVPLTHLDLRGNQLQTLPYVGFLEHIGRI
550 G>R x1 - Known 1 Kenya ECD LDLQLEDNKWACNCDLLQLKTWLENMPPQSIIGDVVCNSPPFFKGSILSRLKKESICPTPPVYEEHEDPS
635 R>C x1 Panc5 Novel - GSLHLAATSSINDSRMSTKTTSILKLPTKAPGLIPYITKPSTQLPGPYCPIPCNCKVLSPSGLLIHCQER
728 S>L x1 - Known 1 Kenya NIESLSDLRPPPQNPRKLILAGNIIHSLMKSDLVEYFTLEMLHLGNNRIEVLEEGSFMNLTRLQKLYLNG
732 G>E x1 B1(+) Known 1 Puerto Rico NHLTKLSKGMFLGLHNLEYLYLEYNAIKEILPGTFNPMPKLKVLYLNNNLLQVLPPHIFSGVPLTKVNLK
822 E>K x1 Mel9 Novel - TNQFTHLPVSNILDDLDLLTQIDLEDNPWDCSCDLVGLQQWIQKLSKNTVTDDILCTSPGHLDKKELKAL
832 D>N x1 C3 Novel - NSEILCPGLVNNPSMPTQTSYLMVTTPATTTNTADTILRSLTDAVPLSVLILGLLIMFITIVFCAAGIVV
549 Frameshi ft P>P x1 UG-B1, LAPC-9AD, LAPC-9AI Novel - LVLHRRRRYKKKQVDEQMRDNSPVHLQYSMYGHKTTHHTTERPSASLYEQHMVSPMVHVYRSPSFGPKHL
EEEEERNEKEGSDAKHLQRSLLEQENHSPLTGSNMKYKTTNQSTEFLSFQDASSLYRNILEKERELQQLG
ITEYLRKNIAQLQPDMEAHYPGAHEELKLMETLMYSRPRKVLVEQTKNEYFELKANLHAEPDYLEVLEQQ
T
158P01D07 ORF
841 aa
Putative Signal peptide
Putative TM Region (609-633)
pTag5 & psecFc (16-608)
ITIM motif
Putative Extracellular Domain (27-608)
Putative Intracellular Domain
SSH (282-357)
pTag5 (27-300) pTag5 (301-608)
SLITRK6 Gene Coding Region Mutational/SNP Analysis using Whole
Exome Next Generation Sequencing (NGS):
49 patient cancer specimens
69-sample normal specimens genome panel
• Several potential missense SNPs in SLITRK6 coding region have been identified,
including in AG-B7 and AG-B8 bladder cancer models with high SLITRK6 expression
• Discovery using suppression subtractive hybridization (SSH) was conducted as
previously described (Hubert, R.S., et al., PNAS 1999; 96(25):14523-8)
• Whole genome microarray RNA expression analysis: Agilent 8x60K arrays; Agilent
Feature Extraction software, data normalized using quantile method
• Real-time qPCR gene expression analysis: Bio-Rad CFX384 real time detection
system; modified deltaCq analysis method
• Copy number variation (CNV) analysis: SABiosciences qPCR assays, Mref norm.
• Whole genome DNA copy number profiling: Agilent SurePrint aCGH 2x400K
arrays, Partek Genomics Suite analysis
• Whole exome sequencing (Nimblegen EZ Exome V2.0, Illumina HiSeq 2000, DNA
Nexus)
• Immunohistochemical (IHC) analysis of SLITRK6: refer to poster, abstract #1274,
2013 AACR Annual Meeting
Methods
Real-time qPCR analysis
Gene Expression data for SLITRK6 extracted from CCLE_Expression_Entrez_2012-10-18.res :Gene-centric RMA-normalized mRNA expression data. http://www.broadinstitute.org/ccle
• SLITRK6 mRNA expression was detected at various levels in multiple cell
lines of several cancer indications; cell lines with high expression levels
were of lung, brain, bladder, breast, and other cancer indications
• Slitrk6 cell surface protein expression was evaluated and validated in
several high mRNA expressing lines using FACs analysis (data not shown)
AGS-15/SLITRK6 RPL19
(.1/.2 primer set) .3/.4
Transitional Cell Carcinoma MET AG-B8 (P13) X 3302 5471
Transitional Cell Carcinoma AG-B7 (P18) X 1297 7084
Papillary Urothelial Carcinoma AG-B10 (P11) X 779 2407
AG-B13 (P3) X 576 12507
Papillary Carcinoma AG-B1+ (P2) X 223 2303
Transitional Cell Carcinoma AG-B7 24915 39607
Transitional Cell Carcinoma MET AG-B8 24915 39607
Bld. Ca. 34 10503 TBD
Bld. Ca. 14 8423 19518
Bld. Ca. 15 5050 103344
Bld. Ca. 18 3798 33181
Bld. Ca. 11 2436 9070
Bld. Ca. 17 2353 36856
Bld. Ca. 3 2224 26067
Bld. Ca. 12 2217 24627
Bld. Ca. 6 1657 18965
Bld. Ca. 13 1497 9335
Bld. Ca. 19 1195 40170
Bld. Ca. 38 1122 TBD
Bld. Ca. 39 1047 TBD
Bld. Ca. 4 753 19247
Adenocarcinoma Bld. Ca. 8 623 14353
Transitional Cell Carcinoma Bld. Ca. 5 556 8108
Bld. Ca. 35 538 TBD
Bld. Ca. 37 317 TBD
Bld. Ca. 33 250 TBD
Transitional Cell Carcinoma Bld. Ca. 40 26 TBD
Br. Ca 2 7399 8844
Br. Ca 1 6339 9653
AG-Br21 3662 42052
Br. Ca 4 1167 4112
Br. Ca 5 328 5189
AG-Br40 316 18244
AG-Br26 310 8642
Br. Ca 3 302 5129
T AG-K18 2244 17315
X AG-K18 (P9) X 665 7408
Lrx. Ca 1 1314 5149
Lrx. Ca 2 692 8278
Lrx. Ca 3 228 4707
X Large Cell Carcinoma AG-L15 (P6) X 1993 76432
T AG-L36 219 7476
X Prostate Adenocarcinoma LAPC-9AI (PR11) X 320 13607
Adenocarcinoma NCI-H1975 2510 24370
Adenocarcinoma, NSCLC NCI-H1734 1549 2689
Brain Neuroblastoma CHP-212 1160 5918
Bladder Transitional Cell Carcinoma SW 780 1089 8644
Pancreas Adenocarcinoma (mouse lung MET) HPAC-LM-XCL 992 11293
Breast Primary Ductal Carcinoma HCC1419 787 1594
Squamous Cell Carcinoma NCI-H520 736 13568
Bronchoalveolar carcinoma NCI-H322M-XCL 449 5949
Bladder Transitional Cell Papilloma RT4 329 3971
CNS Glioblastoma multiforme SF-295 155 2045
Ce
ll L
ine
T
Xen
og
raft
(X
)
Transitional Cell Carcinoma
Transitional Cell Carcinoma
Papillary Transitional Cell Carcinoma
Infiltrating Ductal Carcinoma
Infiltrating Ductal Carcinoma
Invasive Urothelial Carcinoma
HNSSC
Pa
tie
nt
Tu
mo
r (T
)P
ati
en
t T
um
or
Type
Bladder
Breast
Kidney
Larynx
Cancer TypeCancer
Origin
(Organ)
Specimen Name
qPCR: Relative
Abundance (10-4
GAPDH
units)
Lung
Lung
Lung
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Calibrator
AG-B8 (P13) X
AG-B7 (P18) X
AG-B10 (P11) X
AG-B13 (P3) X
AG-B1+ (P2) X
AG-B7
AG-B8
Bld. Ca. 34
Bld. Ca. 14
Bld. Ca. 15
Bld. Ca. 18
Bld. Ca. 11
Bld. Ca. 17
Bld. Ca. 3
Bld. Ca. 12
Bld. Ca. 6
Bld. Ca. 13
Bld. Ca. 19
Bld. Ca. 38
Bld. Ca. 39
Bld. Ca. 4
Bld. Ca. 8
Bld. Ca. 5
Bld. Ca. 35
Bld. Ca. 37
Bld. Ca. 33
Bld. Ca. 40
Br. Ca 2
Br. Ca 1
AG-Br21
Br. Ca 4
Br. Ca 5
AG-Br40
AG-Br26
Br. Ca 3
AG-K18
AG-K18 (P9) X
Lrx. Ca 1
Lrx. Ca 2
Lrx. Ca 3
AG-L15 (P6) X
AG-L36
LAPC-9AI (PR11) X
NCI-H1975
NCI-H1734
CHP-212
SW 780
HPAC-LM-XCL
HCC1419
NCI-H520
NCI-H322M-XCL
RT4
SF-295
Bla
dd
er C
ance
rB
reas
t Can
cer
Kd
Ca
HN
SSC
Lu C
aP
r C
aC
ance
r Ce
ll L
ine
s
SLITRK6 Gene DNA Copy Number
Ave. of triplicates, standard deviation shown
Normal Tissues
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
Bla
dd
er
Blo
od
ce
lls (
PB
MC
s)
Bo
ne
ma
rro
w
Bra
in
Bre
ast
Ce
rvix
Co
lon
He
art
Kid
ne
y
Liv
er
Lu
ng
Lym
ph
no
de
Ova
ry
Pitu
itary
gla
nd
Pa
ncr
ea
s
Pla
cen
ta
Pro
sta
te
Sa
liva
ry g
lan
d
Ske
leta
l mu
scle
Ski
n
Sm
all
inte
stin
e
Sp
lee
n
Sto
ma
ch
Te
stis
Th
ymu
s
To
nsi
l
Ute
rus
HU
VE
C
NP28-1
NP28-2
NP28-3
20.6
Rela
tive A
bundance
NP28-1,2,3 – 3 normal specimens panels containing 3 distinct samples for each normal tissue
AGS-15/SLITRK6 (.1/.2) 0 2000 4000 6000 8000 10000 12000
NAT - AG-B6
N. Bladder 1
N. Bladder 3
N. Bladder 2
AG-B7
AG-B8
Bld. Ca. 34
Bld. Ca. 32
Bld. Ca. 14
Bld. Ca. 28
Bld. Ca. 24
Bld. Ca. 15
Bld. Ca. 22
Bld. Ca. 27
Bld. Ca. 18
AG-B10
Bld. Ca. 20
Bld. Ca. 11
Bld. Ca. 17
Bld. Ca. 1
Bld. Ca. 3
Bld. Ca. 12
Bld. Ca. 9
Bld. Ca. 6
Bld. Ca. 25
Bld. Ca. 13
Bld. Ca. 19
Bld. Ca. 38
Bld. Ca. 23
Bld. Ca. 39
Bld. Ca. 26
Bld. Ca. 4
Bld. Ca. 8
AG-B9
Bld. Ca. 5
Bld. Ca. 35
AG-B5
Bld. Ca. 37
Bld. Ca. 21
AG-B6
Bld. Ca. 30
Bld. Ca. 33
AG-B11
AG-B4
Bld. Ca. 31
Bld. Ca. 29
Bld. Ca. 10
Bld. Ca. 40
No
rmal
B
lad
de
r /
NA
T
Tr
C
Ca
Tr
C
Ca M t
Tr C
C
aTr
C C
a
Sq
Ca
Tr C
Ca
P.
U.
Ca
Tr C
Ca
P.
Tr
C
Ca
Tran
siti
on
al C
ell
Car
cin
om
aTr
ansi
tio
nal
Ce
ll C
arci
no
ma
Ad
C
aTr
. C
Ca
P.
Tr
C
Ca
Tr
C
Ca
P.
Tr
C
Ca
Tr C
C
a
Tr
C
Ca
P.
Tr
C
Ca
Sq
Ca
U.
Ca
M tTr
C C
a
Bla
dd
er
Can
cer
Pat
ien
Tu
mo
rs
Relative Abundance (10-4 GAPDH units)
AGS-15/SLITRK6 (.1/.2)
Ave. of triplicates, standard deviation shown
24915
23853
2013 AACR Annual Meeting – Washington, DC
