Download GPRC5D Is a Cell Surface Plasma Cell Marker Whose Expression Is

GPRC5D is a cell surface plasma cell marker whose expression is high in myeloma cells and reduced
f ll i coculture
following
lt
with
ith osteoclasts
t
l t
Sathisha Upparahalli Venkateshaiah, Rakesh Bam, Xin Li, Sharmin Khan, Wen Ling, Randal S Shelton, and Shmuel Yaccoby
Myeloma Institute for Research and Therapy
Therapy, University of Arkansas for Medical Sciences
Sciences, Little Rock
Rock, AR
AR.
Results
Abstract
Figure 1. GPRC5D is expressed in plasma cells but not in B cells, osteoclasts (OC), MSC or donor
PBMC and its expression is higher in primary MM cells than normal plasma cells (NPC).
qRT-PCR
p<0.0001
GPRC5D is a cell surface maker detected in plasma cells but not in any other cell types in BM.
GPRC5D expression is higher in MM than normal plasma cells.
GPRC5D expression is downregulated in MM cells co-cultured with osteoclasts, along with other
plasma cell markers such as syndecan-1
syndecan 1 (SDC1) and CD38.
CD38
GRPC5D knockdown has no effect on short-term growth of CAG MM cell line.
Figure 5. GPRC5D cell surface expression in primary MM cells (Pt #11-14): Flow cytometry analysis
Pt#11
20000
15000
10000
GPRC5D
5000
MM Patients
MMCL
Figure 2. GPEC5D protein expression is detected in MM cell lines and primary MM cells (Pt #4-10)
but not MSC, osteoclasts or normal BM (NBM): Western blot Analysis
Positive
control
Pt#4
H929
CAG
ARPI
MSC
SC
NBM
Figure 6. Expression of GPRC5D, SDC1 and CD38 is reduced while CD45 is upregulated in MM cells
co-cultured with osteoclasts: Microarray analysis
30000
O t
Osteoclast
l t
GPRC5D
ß-Actin
Pt#6
Pt#7
Pt#8
Pt#9
25000
20000
15000
X20
GPRC5D
X20
X40
CD138-selected
primary MM cells
MM cell markers
GPRC5D
SDC1
Figure 7. Expression of GPRC5D is reduced in
primary MM cells co-cultured with osteoclasts
(Pt #15-17): qRT-PCR Analysis
1.2
MM
CD38
PTPRC (CD45)
GAPDH (house
keeping gene)
0.8
0.6
0.4
0.2
Pt #15
Figure 8. GPRC5D knockdown had no effect on
short-term MM cell growth.
CAG MM Line
Cocultured MM
1
0
H929 MM line
IRF4
•Primary MM cells from 8 patients were co-cultured with osteoclasts for 3-4 days.GEP was performed on freshly obtained (baseline)
and MM cells recovered from co-culture (co-cultured MM).
GPRC5D mRNA relativ
ve
to GAPDH
CONT AB
CONT AB
PC
AIM 1: Evaluate GPRC5D gene expression and cell surface localization in MM cells.
Baseline Co-culture Baseline Co-culture Baseline Co-culture Baseline Co-culture Baseline Co-culture Baseline Co-culture
GPRC5D
Figure
g
3. GPRC5D expression
p
is restricted to
MM plasma cells in whole myelomatous BM
aspirate.
p<0.02
p<0.007
0
Figure 4. Cell surface and nuclear localization
of GPRC5D in MM plasma cells.
GPRC5D gene expression and protein localization has yet not been demonstrated in MM.
AIMS of the study
p<0.001
5000
ß-Actin
The orphan receptor GPRC5D belongs to the group of five C GPCRs known as retinoic acidinducible genes (RAIGs) including RAIG1, RAIG2, GPRC5B, GPRC5C and GPRC5D.
p<0.004
10000
Pt#10
Pt #16
Pt #17
* Primary MM cells from 3 patients were co-cultured with
osteoclasts for 3-4 days. RT-PCR was performed in freshly
obtained (baseline) and MM cells recovered from co-culture
(co-cultured MM).
Relative GPRC5D
gene expression
NPC
GEP Sig
gnal
0
• Freshly obtained CD138-selected MM cells were stained with control antibody or GPRC5D antibody (R&D Systems,
Minneapolis,MN, USA. Cat no. FAB6300A) and analyzed using flow cytometry.
GPRC5D
In human tissues, GPRC5D expression was detected at high level in testis and much lower to
p y Acta
undetectable levels in other tissues ((Bra¨uner-Osborne et al., Biochim Biophys
2001;Atamaniuk et al, Eur J Clin Invest 2012).
Pt#14
25000
Pt#5
GPRC5D is highly expressed in myelomatous BM (Cohen et al, Hematology 2013) and its
expression
i iin MM patients
ti t bi
biopsies
i iis associated
i t d with
ith poor survival
i l (At
(Atamaniuk
i k ett al,
l Eur
E J
Clin Invest 2012).
Pt#13
Pt#12
30000
Background
AIM 2: Evaluate the changes in expression of GPRC5D and other
following coculture of primary MM cells with osteoclasts.
•
•
•
•
GPRC5D signal
Identification of unique cell surface markers on myeloma cells is important for development of targeted
therapies and detection of residual disease. GPRC5D is an orphan receptor reportedly expressed at high
level in bone marrow (BM) aspirates or biopsies from myeloma patients. Other studies detected
GPRC5D in skin and brain but not in other tissues. The aims of the study were to assess GPRC5D gene
expression and cell surface localization in myeloma cells,
cells the changes in expression of GPRC5D and
other typical myeloma cell markers following coculture of primary myeloma cells with osteoclasts and the
consequences of GPRC5D gene silencing on myeloma cell growth. Global gene expression profile
(GEP), qRT-PCR and immunohistochemistry revealed exclusive high expression of GPRC5D in normal
and myeloma plasma cells but not in B cells, MSCs, osteoclasts or BM mononucleated cells. GPRC5D
expression
p
was higher
g
in myeloma
y
plasma cells from newly
p
y diagnosed
g
patients ((n=698)) compared
p
p
to
normal plasma cells (n=26, p<0.0001)). Among molecularly classified groups GPRC5D expression is
higher in MS, MF and LB subgroups and lowest in CD2 and MM cell lines. Flow cytometry analysis and
immunohistochemistry detected cell surface expression of GPRC5D in myeloma plasma cells while
nuclear localization was also detected in certain myeloma cell lines (e.g. H929). Western blots analysis
confirmed GPRC5D expression in whole cell lysate and nuclear fraction. We and other demonstrated
phonotypical plasticity of myeloma plasma cells capable of altered expression of recognizable plasma
cell markers (e.g. CD138, CD45) following coculture with stromal cells (Dezorella et al., 2009) or
osteoclasts (Yaccoby, 2005). In coculture of primary CD138-selected myeloma cells with osteoclasts
(n=8), CD138 (p<0,004), CD38 (p<0.001) and GPRC5D (p<0.007) were commonly and significantly
downregulated, CD45 (p<0.02) was upregulated, and IRF4 expression was unaffected in cocultured
m eloma cells compared to the control freshly
myeloma
freshl obtained uncultured
nc lt red myeloma
m eloma cells assessed by
b GEP and
qRT-PCR. We also observed reduced expression of GPRC5D in MM cells purified from focal lesion
compared to interstitial marrow in paired clinical samples (n=176, p<1.21E-09), suggesting that high
activity of osteoclasts in osteolytic lesions mediates phenotypical alteration in myeloma cells. Stable
knockdown of GPRC5D by 70% in CAG myeloma line using lentiviral particles containing shRNA had no
effect on short-term growth of these cells assessed by MTT assay.
assay These data indicate that GPRC5D is a
novel cell surface marker for myeloma plasma cells and that its expression is reduced in dedifferentiated
myeloma cells.
Summary and Conclusions
Disclosure: All Authors have no relevant conflicts of interest to disclose
1.2
1
0.8
0.6
04
0.4
0.2
0
Control
Scr GPRC5D
shRNA shRNA