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. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancer
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Contents lists available at ScienceDirect
Seminars in Cancer Biology
j our nal home page: www. el sevi er . com/ l ocat e/ semcancer
Review
The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted
cancer therapy
Mohammad Hojjat-Farsangi
a
, Ali Moshfegh
a
, Amir Hossein Daneshmanesh
a
,
Abdul Salam Khan
a
, Eva Mikaelsson
a
, Anders sterborg
a,b,c
, Hkan Mellstedt
a,
a
Department of Oncology-Pathology, Immune and Gene Therapy Lab, Cancer Center Karolinska (CCK), Karolinska University Hospital Solna and Karolinska
Institutet, Stockholm, Sweden
b
Department of Hematology, Karolinska University Hospital Solna, Stockholm, Sweden
c
Department of Oncology, Karolinska University Hospital Solna, Stockholm, Sweden
a r t i c l e i n f o
Keywords:
ROR1
Tyrosine kinase inhibitors
Monoclonal antibodies
Cancer therapy
a b s t r a c t
Targeted cancer therapies have emerged as new treatment options for various cancer types. Among
targets, receptor tyrosine kinases (RTKs) are among the most promising. ROR1 is a transmembrane RTK
of importance during the normal embryogenesis for the central nervous system, heart, lung and skeletal
systems, but is not expressed in normal adult tissues. However, ROR1 is overexpressed in several human
malignancies and may act as a survival factor for tumor cells. Its unique expression by malignant cells
may provide a target for novel therapeutics including monoclonal antibodies (mAbs) and small molecule
inhibitors of tyrosine kinases (TKI) for the treatment of cancer. Promising preclinical results have been
reported in e.g. chronic lymphocytic leukemia, pancreatic carcinoma, lung and breast cancer. ROR1 might
alsobe an interesting oncofetal antigen for active immunotherapy. Inthis review, we provide an overview
of the ROR1 structure and functions in cancer and highlight emerging therapeutic options of interest for
targeting ROR1 in tumor therapy.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Cancer is a complex disorder of uncontrolled cell prolifera-
tion. Eight hallmarks have been suggested to explain the acquired
tumorigenic characteristics [1]. These properties include prolifer-
ation, evading growth suppression, resisting cell death, enabling
replicative immortality, activating invasion, metastasis, evading
from recognition of the immune system and reprogramming
energy metabolism[1,2].
The termoncogenic addiction by Weinstein [3] suggested that
tumor cells may exhibit dependence on an activated oncogenic
pathway to maintain survival and proliferation. Phosphorylation
of signaling proteins is central in the regulation of cellular activi-
ties and protein kinases play a key role in the normal development
as well as during tumorigenesis [2,4].
Protein kinases are enzymes that catalyze the transfer of a phos-
phate group fromadenosine three phosphates (ATP) to tyrosine or

Corresponding author at: Department of Oncology, Cancer Center Karolinska

(CCK), Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden.
Tel.: +46 8 51 77 43 08; fax: +46 8 31 83 27.
E-mail address: hakan.mellstedt@karolinska.se (H. Mellstedt).
serine/threonine residues. Receptor tyrosine kinases (RTKs) are a
family of kinases, consisting of a transmembrane receptor linked
to an intracellular kinase domain. Most RTKs are involved in the
normal cell development but might alsoact as oncogenes intumori-
genesis. Several tumors are addicted to oncogenic RTKs and their
signaling pathways are of importance for cell survival [2].
The human kinome includes about 90 tyrosine kinases (TKs)
and 43 TK-like functional genes. These proteins are regulators of
normal cellular processes as proliferation, differentiation, motility,
survival, metabolismand play critical roles in the normal develop-
ment and organogenesis [4] and are emerging as pharmacological
targets in oncology [5].
Current data indicate that deregulation of kinase activity is a
major mechanismby which tumor cells may escape normal phys-
iological controls for survival and growth [6]. Targeting TK activity
might be of importance to prevent tumor cell growth. Monoclonal
antibodies (mAbs) against the extracellular part of RTKs and small
molecules inhibiting the intracellular tyrosine kinase activity of
RTKs (TKI) respectively are drugs downregulating oncogenic activ-
ities through receptor or non-receptor tyrosine kinases.
In 1992, Masiakowski and Caroll described two new RTKs with
a high amino acid homology to the TK domain of growth factor
receptors and to the Trk family named ROR1 and ROR2 shown
http://dx.doi.org/10.1016/j.semcancer.2014.07.005
1044-579X/ 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hojjat-Farsangi M, et al. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancer
therapy. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.07.005
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to be involved in a network of regulatory interactions [7]. These
two members shared 58% amino acid homology and 98% homology
betweenthe mouse andhuman. Partial nucleotide sequences of the
rat genes revealed a strikingly evolutionary conservation between
the human and rat proteins. The level of expression of the rat genes
was high in the head and body of early embryos by decreased dra-
matically after embryonic day 16 and undetectable after birth [7].
ROR1 and ROR2 were considered to be a novel regulatory family of
cell surface receptors during development [7]. In 1993, the expres-
sion of ROR1 was described in Drosophila and to be of importance
during the embryonic development of the nervous system[8].
ROR1 has also been found to be overexpressed in several human
cancers. Here, we discuss the biology of ROR1 in health and malig-
nancies, and highlight a novel class of therapeutics targeting ROR1
which might be of interest for cancer therapy.
2. Receptor tyrosine kinases (RTKs)
RTKs are a large family of cell surface glycoproteins [9]. These
receptors with enzymatic activity regulate various cellular pro-
cesses [10,11]. RTKs are divided into 20 different receptor families
consisting of 58 members [12].
All RTKs have a similar molecular structure including an extra-
cellular region containing the ligand binding domains, a single
transmembrane part and an intracellular region with a conserved
TK domain. Ligand binding induces dimerization of the extracellu-
lar part and initiation of intracellular signaling cascades regulating
cellular functions [13].
Aberrant activation of RTK may be due to receptor overexpress-
ion, chromosomal translocation, gene amplication, mutations and
downregulationwhichmight contributetothedevelopment of var-
ious types of cancer (Table 1) [1416]. Deregulation of more than
30individual RTKs invarious malignancies has beendescribed[10].
Some important RTKs families in cancer are vascular endothelial
growth factor receptor (VEGFR), epidermal growth factor receptor
(ErbB/EGFR), platelet-derived growth factor receptor (PDGFR) and
broblast growth factor receptor (FGFR) families. Targeting these
RTKs by mAbs or TKI have shown signicant clinical effects [17].
ROR1 might be added of this group of oncogenic RTKs.
3. Receptor-tyrosine kinase ROR1
Receptor tyrosine kinase orphanreceptors 1 and2 (ROR1/ROR2)
belong to one of the twenty different RTK families and are
evolutionarily highly conserved [5]. ROR1 consists of 3 dis-
tinct extracellular regions including an immunoglobulin (Ig)-like
domain, a cysteine rich (CRD) domain and a kringle (KNG) domain
as well as an intracellular TK domain (Fig. 1). The cytoplasmic part
contains a TK domain with protein kinase activity, and further
downstreamserine, threonine- and proline-rich motifs.
3.1. ROR1 structure and function
ROR family members in human and rat similar to the Trk neu-
rotrophin receptors were isolated by a PCR-based search for RTK
genes in 1992 [7]. Later, ROR genes were described in Drosophila
[18], mouse [19] and C. elegans (cam-1) [20]. The ROR1 gene was
originally cloned from a neuroblastoma cell line [7]. A truncated
ROR1 gene was reported in the fetal and adult human central ner-
vous system, in human leukemia and lymphoma cell lines and in a
variety of human cancer derived fromthe neuroectoderm.
Human(h) ROR1 is located on chromosomal region 1p31.3 and
consists of 937 amino acids (908 after cleavage of the signal pep-
tide) with an estimated molecular weight of 105kDa. hROR1 and
hROR2 shared an overall 58% identity in amino acid sequence. The
Ig domain
Cysteine-rich
domain
-
Kringle domain
Tyrosine kinase
domain
Serine/threonine
rich domain
Proline rich
domain
Human ROR Mouse ROR
Drosophila ROR
Extracellular part
Cell membrane
Cytoplasmic part
Fig. 1. Structure of the ROR receptor tyrosine kinases in different species. Organiza-
tion of ROR proteins in human (ROR1/ROR2), mouse (ROR1/ROR2) and Drosophila.
amino acid sequence identity within the kinase domains was 68%.
The degree of sequence conservation was even higher within the
ROR1 and ROR2 subgroups. 97% amino acid identity was shared
between hROR1 and mice ROR1 (mROR1). The identity for ROR2
(hROR2 and mROR2) was 92% [21,22].
The immunoglobulin-like (Ig) domain of hROR1 consists of 106
amino acid residues and the corresponding number for ROR2 is 91.
The precise functions of the Ig domain are not known, but might
be involved in proteinprotein and/or proteinligand interactions
[23].
The extracellular CRD domain is dened by 10 conserved cys-
teines that form ve disulde bridges and consists of 135 amino
acids. The CRD domain is similar to the WNT binding domain of
Frizzled receptors and is one of the ligand binding motifs for RORs.
The KNG domains of ROR1 and ROR2, juxtaposed to the cell
membrane, consist of 80 and 79 amino acids, respectively. It is
highly conserved throughout species and might function as recog-
nition modules for binding of proteins including WNT regulatory
proteins and other ROR ligands [24]. The presence of a KNGdomain
is a hallmark of the ROR family members as ROR is the only RTK
family reported to contain the KNG domain with the exception for
Torpedo MuSK [22].
ROR1contains 40amino acids withinthe kinase domain[25,26].
Twenty-one amino acids of the forty consensus amino acids are
conservedinall RTKs, makingthis regionthe most highlyconserved
region not only of ROR, but also for all known RTKs [7,25].
ROR1, as well as the other member of this family has a conserved
sequence, YXXDYY (YSADYY in hROR1, position 641646) within
the activation site of the TK domain which is also present in Trk
and MuSK [27]. Phosphorylation of ROR1 at the rst and last tyro-
sine residues is critical. ROR1 is constitutively phosphorylated at
these tyrosine residues in chronic lymphocytic leukemia (CLL) cells
[28]. As will be discussed later, ROR1 is phosphorylated at these
tyrosine residues by SRC kinases [29]. Anti-ROR1 mAbs against the
CRD and KNG domains [30] induced dephosphorylation of ROR1 at
these tyrosine residues followed by apoptosis of the leukemic cells
[28]. Serineat position652was alsophosphorylatedinmROR1[31].
This specic serine is present at the same position in hROR1. ROR1
has also two other conserved sequence, YSLM (position 772775)
and YXXF (YGKF in hROR1, position 666669), which are potential
binding sites for the SRC homology 2 (SH2) region of intracellular
kinases, like the p85 subunit of PI3K [32]. All these sequences are
present in the TK domain of ROR1.
Please cite this article in press as: Hojjat-Farsangi M, et al. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancer
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Table 1
Expression of oncogenic RTKs in cancer.
RTK Chromosome
location
Normal function Malfunction leading to
overexpression
Malignancy (examples)
ROR1 1p31.3 ED, NSD Unknown CLL, ALL, AML, MCL, HCL, melanoma, prostate,
lung, breast, pancreas, colon, ovarian, uterus
cancers
ROR2 9q22.31 ED, NSD Mutations Melanoma, medulloblastoma, testicular cancer,
gastrointestinal stromal tumor, hepatocellular
carcinoma, colon cancer, renal cell carcinoma,
osteosarcoma, prostate carcinoma
ALK 2p23 NSD Translocation (t2:5)
overexpression
NSCLC, colorectal cancer, breast cancer,
oesophageal cancer (squamous cell), renal cell
cancer
ROS1 6q22 NSD Deletion, inversion,
translocation
NSCLC, cholangiocarcinoma, ovarian cancer,
gastric cancer, colorectal cancer
RET 10q11.2 NSD Mutations NSCLC, medullary thyroid carcinoma
NTRK1 (TrkA) 1q21-22 Development and maturation of central and
peripheral nervous system
Translocation Colorectal cancer, papillary thyroid cancer,
lung adenocarcinoma, breast cancer, oral
squamous cell carcinoma
NTRK2 (TrkB) Development and maturation of central and
peripheral nervous system
Translocation Neuroblastoma, astrocytoma, oral squamous
cell carcinoma
NTRK3 (TrkC) 15q25 Development and maturation of central and
peripheral nervous system
Translocation Neuroblastoma, breast cancer
PDGFRA 4q12 ED Mutations Lung adenocarcinoma, gastrointestinal stromal
tumors
PDGFRB 5q32 Regulation of embryonic development, cell
proliferation, survival, differentiation,
chemotaxis, migration and blood vessel
development
Mutations Gastrointestinal stromal tumors, glioblastoma
FGFR1 8p12 Regulation of embryonic development, cell
proliferation, differentiation and migration
Mutations Squamous cell lung cancer, breast cancer
FGFR2 10q26 ED Mutations,
overexpression
Squamous cell lung cancer, lung
adenocarcinoma, breast cancer, thyroid cancer,
prostate cancer, cholangiocarcinoma,
astrocytoma
FGFR3 4p16.3 Normal skeleton development Mutations Bladder cancer, squamous cell carcinoma
(lung, head and neck)
MET 7q31.2 Gastrulation, development and migration of
muscles and neuronal precursors, angiogenesis
and kidney formation
Mutations Hepatocellular carcinoma, CLL, breast cancer,
pancreatic cancer, lung cancer, gastric
adenocarcinomas
Axl 19q13.1 ND Unknown Lung cancer, colon cancer, breast cancer, AML,
CML, esophageal, thyroid cancer,
gastrointestinal stromal tumors,
astrocytomaglioblastoma
IGF1R 15q26.3 Embryonic and fetal development Mutations CLL, breast cancer, oral squamous cell
carcinoma cells. Gastrointestinal stromal,
squamous-cell laryngeal cancer tumors,
hepatocellular carcinoma, pancreatic cancer
IGF2R 6q25.3 Embryonic and fetal development Mutations Squamous cell carcinoma, breast cancer,
prostate cancer, hepatocellular carcinoma,
colorectal carcinoma, NSCLC, pancreatic cancer
EGFR1 (ERBB1) 7p11.2 ED Mutations Breast cancer, hepatocellular carcinoma, head
and neck squamous cell carcinoma
EGFR2 (ERBB2) 17q12 ED Mutations Breast cancer, gastric adenocarcinomas
EGFR3 (ERBB3) 12q13.2 ED Mutations Breast cancer
EGFR4 (ERBB4) 2q34 ED Mutations Breast cancer, melanoma
VEGFR1 (FLT1) 13q12.3 ED Mutations Ovarian cancer, NSCLC, colorectal carcinoma
VEGFR2 (KDR) 4q12 ED Mutations Renal cell carcinoma, hepatocellular carcinoma
VEGFR3 (FLT4) 5q35.3 ED Mutations
FLT3 13q12.2 Hematopoiesis Mutations AML, acute promyelocytic leukemia
KIT 4q12 Hematopoiesis, stemcell maintenance,
gametogenesis, mast cell development,
migration and function, and in melanogenesis
Mutations AML, melanoma, ovarian carcinoma,
gastrointestinal stromal tumors
RON (MST1R) 3p21.31 Regulates many physiological processes
including cell survival, migration and
differentiation
Mutations Pancreatic cancer, breast cancer, NSCLC,
laryngeal squamous cell carcinoma, head and
neck squamous cell carcinoma
INSR 19p13.2 Metabolic actions of insulin Mutations Colorectal cancer, prostate cancer
INSRR 1q23.1 Metabolic actions of insulin Mutations Neuroblastomas
CCK4 (PTK7) 6p21.1 ED Mutations Squamous cell carcinoma, small cell lung
cancer, breast cancer, gastric and colon cancer,
AML
EPHA1 7q35 NSD Mutations NSCLC, prostate cancer, esophageal squamous
cell carcinoma
EPHA2 1p36.13 NSD Mutations Hepatocellular carcinoma colorectal cancer,
osteosarcoma, breast cancer
EPHA3 3p11.1 NSD Mutations Glioblastoma, lung cancer, melanoma, ALL,
T-cell leukemia, Hodgkins lymphoma
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Table 1 (Continued)
RTK Chromosome
location
Normal function Malfunction leading to
overexpression
Malignancy (examples)
EPHA4 2q36.1 NSD Mutations NSCLC, gastric cancer
EPHA5 4q13.1 NSD Mutations Breast cancer, hepatocellular carcinoma, ALL
EPHA6 3q11.2 NSD Mutations
EPHB1 Xq13.1 NSD NSCLC, cervical cancer, ovarian Cancer
EPHB2 13q33.3 NSD Cervical cancer, breast cancer
EPHB3 3q27.1 NSD NSCLC, breast cancer, colorectal cancer
EPHB4 7q22.1 NSD Breast cancer, melanoma, glioma
MER 2q13 Survival, migration, differentiation, and
phagocytosis of apoptotic cells
Mutations Glioblastoma, hepatocellular carcinoma,
astrocytoma
TYRO3 15q15.1 Cell survival, migration and differentiation Mutations Colon cancer, melanoma, thyroid cancer,
breast cancer
TIE 1p34.2 Regulation of angiogenesis Mutations Glioblastoma
TEK 9p21.2 Regulates angiogenesis, endothelial cell
survival, proliferation, migration, adhesion and
cell spreading, reorganization of the actin
cytoskeleton, but also maintenance of vascular
quiescence
Mutations Bladder cancer, glioblastoma, AML
RYK 3q22.2 Neuron differentiation, axon guidance, corpus
callosumestablishment and neurite outgrowth
Translocation,
mutations
CML, ovarian cancer
DDR1 6p21.33 Regulates cell attachment to the extracellular
matrix, remodeling of the extracellular matrix,
cell migration, differentiation, survival and
proliferation
Mutations NSCLC, breast cancer, AML, ovarian Cancer,
hepatocellular carcinoma
DDR2 1q23.3 Regulates cell differentiation, remodeling of
the extracellular matrix, cell migration and cell
proliferation
Mutations Head and neck squamous cell carcinoma,
NSCLC, lung cancers, CML, breast cancer
LTK 15q15.1 ND Mutations Gastric cancer, lymphomas, leukemias
MUSK 9q31.3 NSD Mutations Ovarian cancer
ROR: receptor tyrosine kinase-like orphan receptor, ED: embryonic development, NSD: nervous system development, CLL: chronic lymphocytic leukemia, ALL: acute
lymphoblastic leukemia, AML: acute myeloid leukemia, HCL: hairy cell leukemia, NSCLC: non-small cells lung carcinoma, NTRK: Neurotrophic Tyrosine Kinase, PDGFR:
platelet-derived growth factor receptor, FGFR: broblast growth factor receptor, ND: normal development, CML: chronic myeloid leukemia, INSR: insulin receptor, EGFR:
epidermal growth factor receptor, VEGFR: vascular endothelial growth factor receptor, CCK: colon carcinoma kinase, RYK: receptor related to tyrosine kinases, DDR: discoidin
domain receptor, LTK: leukocyte tyrosine kinase, MuSK: muscle-specic kinase.
ROR1 expresses two serine/threonine-rich domains, (S/TRD1)
and (S/TRD2), on both sides of the proline-rich domain at the C-
terminal part of the TK domain. No domains similar to the S/TRDs
or proline-rich domains of the ROR family have been found in other
RTKs. The S/TRD1 domain of ROR1 and ROR2 showed a high degree
of homology (67%) but the S/TRD2s did not exhibit any appar-
ent homology. The proline-rich domains showed a lower degree
of homology comparing ROR1 and ROR2 (30%). These cytoplas-
mic domains participate in signaling by interacting with mediators
[24]. Within the serine/threonine-rich and proline-rich domains,
there are potential phosphorylation sites as well as SH2 and SH3
recognition motifs for protein interactions. These motifs might be
of importance in ROR1 mediated signaling by association with the
SH2 and SH3 domains of adaptor/signaling molecules [33].
ROR1 has also been shown to be phosphorylated at one or
more tyrosine residues at positions 786, 789, 822, 828 and 836
by the MET RTK. Such phosphorylation may facilitate the acces-
sibility of tyrosines in the kinase domain to be phosphorylated by
SRC kinases. The presence of consensus motifs of the SH3 domains,
promoted binding of SRC to the ROR1 proline-rich domain. Sub-
sequently the SRC kinase phosphorylated ROR1. Deletion of the
proline-rich domain prevented ROR1 phosphorylation within the
TKdomainby SRC. Impairment of the functionof SRCby saracatinib
(TKI) or silencing by siRNA prevented ROR1 phosphorylation [29].
The effects of ROR1 phosphorylation at different tyro-
sine/serine/threonine residues (within or outside the TK domain)
on tumor cell survival are, however, not well established as is the
case for e.g. members of the EGFR family [34].
As for other RTKs, ROR1 requires two steps for intracytoplasmic
activation: (a) increase in the intrinsic catalytic activity and (b) cre-
ation of docking sites for the recruitment of downstreamsignaling
proteins. These two processes are accomplished by autophospho-
rylation of tyrosine residues as a consequence of ligand-mediated
oligomerization. Autophosphorylation of tyrosine residues in the
activation loop, within the kinase domain, stimulates phos-
phorylation of tyrosine residues within the juxtamembrane and
carboxy-terminal regions. Phosphorylation induced generation of
binding sites for modular domains detecting phospho-tyrosine
residues in specic sequence contexts. SH2 and the phospho-
tyrosine-binding domains are two well-known phospho-tyrosine
binding modules of signaling proteins [35]. The activation loop of
the kinase domain contains one to three tyrosine residues, similar
for several RTKs [25].
There are studies indicating that RORs bind WNT proteins (lig-
ands) [24,36]. ROR1 and WNT5a have been shown to physically
interact with each other and activate NF-B when overexpressed
in HEK293 cells. Survival of immature cells during embryonic
development [37] and of leukemic cells was enhanced by sol-
uble WNT5a, which could be neutralized by anti-ROR1 antisera
in vitro [38]. These ndings may indicate that ROR1 might acti-
vate anNF-B-dependent survival signal inducedbyWNT5a during
embryogenesis and tumorigenesis [38]. ROR2 seems also to act as
a receptor for WNT5a. CRD of ROR2 was required for binding to
WNT5a and mediated signals to the cell interior [39,40].
3.2. RORs tyrosine kinase activity
Kinase activity has been observed for the two ROR family mem-
bers. Changes in conserved amino acids within the ROR kinase
region raised the question if kinase activity was lost. Kinase activity
has been shown for hROR2 [7]. Analyses of CAM-1 TK activity indi-
cated that kinase activity was not required for the function of ROR.
CAM-1encodes aCaenorhabditis elegans orphanRTKof theRORfam-
ily that was required for cell migration and cell polarity [41]. One
of the CAM-1 mutants (gm105) contained a stop codon, 74 amino
acids downstreamof thetransmembraneregionand25aminoacids
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upstreamof the TK domain. The encoded protein lacked the kinase
domain, but CAM-1 (gm105) was not a null mutant without func-
tion [9,41]. This observation indicated that some CAM-1 function
did not require kinase activity and CAM-1 mutants with lack of
kinase activity were involved in cell migration [20].
Biochemical assays have demonstrated that ROR1 might be a
pseudokinase devoided of catalytic activity or with low activity,
but silencing of ROR1 with specic siRNA suppressed cell growth
and subsequent apoptosis [42]. In lung adenocarcinoma, wild-type
ROR1 but not a kinase-dead ROR1 with silent mutations at the
siRNA binding site was able to neutralize siRNA ROR1-induced
growth inhibition. Enhancement of the wild-type ROR1 expression
but not the kinase-dead ROR1 in the ROR1-negative MSTO-211H
cell line to a level similar to that of the ROR1 expressing NCI-H1975
cell line increased in vivo growth of xenografts. These data suggest
that ROR1 kinase activity was required to fully confer a growth
advantage [43]. ROR1 kinase activity might differ between nor-
mal cells, cell lines and malignant cells and also between various
tumors, but overall, ROR1 seems to be an important RTK for cell
survival and migration.
3.3. Expression and function of ROR1 during organ development
ROR1 is expressedduring various embryonic stages of the skele-
tal, respiratory and cardiac systems as well as in neurite extension
of central neurons. The expression was tightly downregulated after
birth [7,19,4447]. hROR1 was expressed in fetal heart, lung and
kidney but to a lesser extent in placenta, pancreas and skeletal
muscles [21]. Analysis of ROR1 protein expression in 28 normal
adult tissues showed no expression in the majority of samples
with the exception for a very low level in testis, uterus, lung,
bladder, and colon [48]. In various normal hematopoietic and
non-hematopoietic tissues, the expression of ROR1 at the mRNA
level was not noted except for low levels in pancreas and adipose
tissues.
The ROR1 protein was also noted in an intermediate stage of
the normal B-cell maturation in the bone marrow (pre-BII stage)
whichcells proliferatedafter internalizationof the pre-Bcell recep-
tor complex. This process was necessary for the development to an
immature B-cell stage [49].
The functions of ROR1 and ROR2 have been studied in mice
lacking either of these genes. ROR1 mutant newborn mice were
similar in size as the wild-type mice and did not exhibit any gross
abnormalities. After birth ROR1 mutant mice showed respiratory
dysfunctions anddiedwithin24h[23,46]. Incontrast, ROR2mutant
mice exhibited dwarsm, short limbs and tail as well as facial
anomalies at birth. ROR2 mutant newborns showed forced respira-
tion and cyanosis and died within 6h after birth [50,51]. ROR1 and
ROR2 double decient newborn mice died shortly after birth due
to breathing difculties associated with incomplete expansion of
lung alveoli [23,46,51]. As ROR1 and ROR2 genes showed a similar
expression pattern in the developing face, limbs, heart and lung tis-
sues, the absence of morphological abnormalities in ROR1 mutant
mice could be explained by functional redundancy between ROR1
and ROR2 [44].
ROR1 and ROR2 modulated the rate of neurite elongation in cul-
ture of rat hippocampal neurons [52] and were highly expressed
in dendrites of central neurons [37]. ROR1ROR2 heterodimeriza-
tion was essential for neuron synapse formation [53]. Suppressed
expression of ROR1 and/or ROR2 led to the formation of fewer
synaptic contacts. Furthermore, ROR1ROR2 dimers interacted
withWNT5a andregulatedsynapse formationinhippocampal neu-
rons. WNT5a was also expressed by hippocampal neurons and
astrocytes and may act as an autocrine factor to stimulate ROR1
and ROR2 in synaptogenesis [53].
Table 2
Overexpression of ROR1 in malignancies.
Malignancy Constitutively
phosphorylated
ROR1
Association
to disease
progression
References
CLL + + [48,54]
B-ALL NI + [59,60,65,66,70]
CML NI NI [60,65,66]
AML NI NI [65,66]
Hairy cell leukemia NI NI [66]
Mantle cell lymphoma NI NI [66]
Pancreatic cancer NI + [67,79,94]
Prostate cancer NI NI [67]
Colon cancer NI NI [67]
Bladder carcinoma NI NI [67]
Ovarian cancer NI + [67]
Testicular cancer NI NI [67]
Uterus NI NI [67]
Adrenal carcinoma NI NI [67]
Breast cancer + + [62]
Lung cancer + + [43,67]
Melanoma + NI [63,64]
CLL: chronic lymphocytic leukemia, B-ALL: B-cell acute lymphoblastic leukemia, NI:
no information, CML: chronic myeloid leukemia, AML: acute myeloid leukemia.
3.4. ROR1 expression in malignancies
The expressionof ROR1indifferent malignancies is summarized
in Table 2.
The main mechanisms leading to aberrant RTK activation in
human cancers are self-activation, chromosomal translocations,
overexpression, gain-of-functional mutations or loss-of-functionof
tumor suppressor genes. Mutations or chromosomal translocations
of ROR1 in several cancer types have not been shown. Mutation
analysis of the extracellular and cytoplasmic kinase domain of the
ROR1 gene in CLL cells indicated no major genomic aberrations
(mutation or truncation). FISH analysis showed no rearrangement
in the ROR1 locus [54].
Current evidence may suggest that ROR1 acts as a classical RTK
in cancer, but ROR2 might have a dual function, both as an onco-
gene[60,62] andas asuppressor genedependingonthemalignancy
[55,56]. This dual role of ROR2 is unusual and has not been shown
for any other RTKs.
In 2001, two independent, gene proling studies in CLL revealed
a 45-fold increase of the ROR1 expression compared to normal
mature B-lymphocytes [57,58]. Subsequently, ROR1 was shown to
be overexpressed not only in CLL [38,48,54], but also in acute lym-
phocytic leukemia (ALL) [59,60], renal cell carcinoma [61], breast
cancer [62], lung adenocarcinoma [43], melanoma [63,64], and
other lymphoid and myeloid malignancies [28,54,60,6266]. The
number of ROR1 receptors on the surface of CLL cells was esti-
matedto be 10,000 molecules per cell [38,54]. This number of ROR1
molecules should be sufcient to target ROR1 expressing tumor
cells by monoclonal antibodies [28,30,63].
WNT5a has been suggested to be a ROR1 ligand for tumor cells.
Coexpression of ROR1 and WNT5a in HEK293 cells activated NF-
B. CLL cells cocultured with WNT5a-expressing Chinese Hamster
Ovary (CHO) cells signicantly improved survival compared to
those cultured with CHO untransfected cells. The survival advan-
tage was abrogated by ROR1 antisera [38]. The data support the
notion that WNT5a activates ROR1 in CLL cells. Furthermore, ROR1
is constitutively phosphorylated at tyrosine and serine residues in
CLL as well as in other malignancies [28,42,63] and WNT5a seemed
to maintain phosphorylation of ROR1 [62]. A signicant correlation
between phosphorylation intensity of ROR1 and disease activity in
CLL was noted, i.e. a higher level of phosphorylation in progres-
sive than in non-progressive CLL as well as in breast, lung and
ovarian cancer cells from patients with aggressive disease [28].
Please cite this article in press as: Hojjat-Farsangi M, et al. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancer
therapy. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.07.005
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Moreover, the ROR1 protein was signicantly higher expressed in
more aggressive tumors [30,67]. Collectively the data suggest that
the expression pattern of ROR1 was related to the aggressiveness
of the disease.
The leukemic cells in ALL originate from different stages of B-
and T-cell maturation [68]. Overexpression of the ROR1 gene was
shown in fresh ALL cells as well as in cell lines [59,60]. High ROR1
expression was noted in ALL cells with the chromosomal translo-
cation t(1;19) exhibiting the oncogenic fusion protein E2A-PBX1
[69] which subtype has a poor prognosis [70]. ROR1 overexpress-
ion was related to a highly progressive disease and associated with
increased cell migratory capacity and an undifferentiated pheno-
type compared to non-ROR1 expressing ALL cells [69,70].
ROR1 has also been shown to be overexpressed both at the gene
andproteinlevels inmultiple myeloma [49,71], marginal zone lym-
phoma (MZL), mantle cell lymphoma (MCL), diffuse large B-cell
lymphoma (DLBCL), and follicular lymphoma (FL) [72].
Also in several solid tumors, ROR1 has been shown to be
overexpressed [42,61]. Surface expression of ROR1 was noted in
melanoma cell lines and phosphorylated at tyrosine and serine
residues [63]. Anti-ROR1 specic mAbs alone killed melanoma
cells [63]. ROR1 and ROR2 were inversely expressed in melanoma
cells suggesting that they may negatively regulate each other [64].
Hypoxia induced a shift of ROR1-positive melanoma cells to a more
aggressive ROR2-positive phenotype. The switch induced a 10-fold
decrease in sensitivity to BRAF inhibitors. In melanoma patients
treated with the BRAF inhibitor vemurafenib, WNT5a expression
in melanoma cells correlated with clinical response and resistance
to activation of the WNT-mediated canonical signaling pathway
(-catenin activation) [64].
In breast cancer, ROR1 was overexpressed and associated with
aggressive disease [62]. Breast cancer cell lines with a high ROR1
expression had a more aggressive and invasive behavior than those
with low ROR1 expression which were non-migrating cells. Spe-
cic ROR1 siRNAdown-regulated the expression of ROR1 in human
breast cancer cell lines and impaired the growth in immunode-
cient mice [62]. ROR1 could be shown to interact with casein
kinase 1epsilon(CK1e) activating PI3KmediatedAKTphosphoryla-
tion as well as the cAMP-response-element-binding protein (CREB)
Furthermore, WNT5a augmented the growth of breast cancer cells
expressingROR1inline withthe assumptionthat WNT5a is a ligand
for ROR1 [62,73].
ROR1 has also been suggested to be associated with
epithelialmesenchymal transition (EMT) during embryogenesis
and in cancer metastases [73]. ROR1 was highly expressed in breast
adenocarcinomas withhighlevels of a gene signature for EMT anda
high rate of relapse as well as the capacity to metastasize compared
to breast cancer patients with a better prognosis where the level
of ROR1 expression was low. Expression of ROR1 in breast cancer
cell lines witha highcapacity to metastasize include MDA-MB-231,
HS-578T and BT549 which had decreased expression of EMT asso-
ciated proteins, E-cadherin, epithelial cytokeratins, as well as tight
junction proteins, and a high migratory capacity in vitro as well as
in immunodecient mice. Similarly, MCF-7 cells transfected with
ROR1 showed a reduced expression of E-cadherin and CK-19, but
not of SNAIL-1/2 and vimentin [73], which molecules are of impor-
tance for homing of cells at proliferating sites. The data support the
notion that ROR1 is associated with less differentiated cells with a
high capacity to metastasize [70].
ROR1 has also been shown to localize to the nucleus (Fig. 2)
[28,74] suggesting that ROR1 might act as a transcription fac-
tor activating genes involved in tumorigenesis [74]. IL-6 induced
phosphorylation of signal transducer and activator of transcrip-
tion factor 3 (STAT3) and upregulated the ROR1 protein in multiple
myeloma cell lines indicating a role of STAT3 in the activation
and expression of ROR1 [75]. STAT3 was constitutively activated
in CLL and bound to gene promoters containing specic binding
elements. The ROR1 promoter contained -interferon activation
sequence-like elements whichwere activatedby STAT3[76]. STAT3
induced WNT5a expression [88,89]. Thus, STAT3 may activate both
ROR1 and WNT5a in tumor cells inducing activation of the WNT5a
signaling pathways through binding to the ROR1 promoter stimu-
lating cell survival, growth and migration.
ROR1 is extensively modied by N-linked glycosylation in CLL
cells [77]. Post-translational modications generated several ROR1
isoforms with different electrophoretic mobilities from 100 to
130kDa. Prevention of ROR1 glycosylation interfered with cell sur-
face expression of the fully mature ROR1 isoform(130kDa) and the
formation of lopodia in HEK293 cell line supporting the notion
that ROR1 has a role in cell migration and metastasis.
ROR1 signaling pathway(s) are not well established in cancer
cells. In breast cancer, the PI3K/AKT pathway was activated follow-
ing activation of ROR1 by WNT5a. ROR1 silencing or lack of WNT5a
inhibited the growth of breast cancer cells [62]. The gene expres-
sionproleof theMDA-MB-231breast cancer cell linesilencedwith
ROR1 siRNA showed lower expression of genes encoding proteins
induced by CREB. Treatment of MDA-MB-231 cells with recom-
binant WNT5a enhanced the viability of the tumor cells and the
expression of pAKT and pCREB, as well as a higher expression
of CREB target genes. Own preliminary data indicated activation
of SRC, PI3K, AKT, mTOR, and CREB in pROR1 positive pancreatic
carcinoma cells (Fig. 2). Treatment of ROR1 positive pancreatic car-
cinoma cells with anti-ROR1 mAbs or ROR1 TKIs induced specic
dephosphorylation of ROR1 as well as of SRC, PI3K, AKT, mTOR, and
CREB and subsequently tumor cell death [78,79].
ROR1 and the pre-B-cell receptor (pre-BCR) may interfere with
each other by modulating each other in a counterbalancing man-
ner of importance for tumor cell survival as shown for ALL cells
[70]. Downregulation of pre-BCR signaling by the kinase inhibitor
dasatinib (inhibiting the pre-BCR/SRC/AKT signaling pathway),
inhibition of AKT as well as of Ig and Ig (parts of BCR com-
plex) induced upregulation of ROR1 in ALL cells. Downregulation
of both ROR1 and pre-BCR induced permanent dephosphoryla-
tion of AKT and inhibited cell growth as well as increased tumor
cell killing. However, downregulation of either ROR1 or BCR alone
did not induce the same effects, suggesting complimentary effects
of ROR1 signaling on pre-BCR signaling [70]. The results indi-
cate a link between the pre-BCR and ROR1 receptor signaling
pathways.
ROR1 may also contribute to leukemogenesis of CLL cells
through binding to the T-cell leukemia antigen 1 (TCL1) as an acti-
vator of AKT (Fig. 2). Coexpression of ROR1 and TCL1 accelerated
leukemogenesis inducing increased phosphorylation of AKT, cell
proliferation and resistance to apoptosis. Targeting ROR1 with spe-
cic mAbs down-modulated ROR1 expression and decreased AKT
activity and subsequently the cells lost the tumorigenic character-
istics in a syngeneic mice model [80].
Our own unpublished and recent [29] data demonstrated the
involvement of the MET and SRC oncogenes in ROR1 phosphor-
ylation (Fig. 2). We could show constitutive phosphorylation of
ROR1 at tyrosine residues 641 and 646 in the ROR1 TK domain
which was of importance for cell survival [28]. Using COS-7 cell
line transfected with ROR1 mutants, Gentile et al. demonstrated
transphosphorylation of ROR1 by the SRC oncogene at tyrosines
641, 645 and 646 residues located within the TK domain. The
proline-rich domain was also transphosphorylated by MET and the
presence of this domain was required for SRC recruitment which
triggered transphosphorylation. The data may suggest that phos-
phorylation occurred in cells with overexpression of MET, but not
in cells with normal levels of MET expression. Moreover, phospho-
kinase arrays showedincreasedactivationof the JNKandp38MAPK
pathways and high phosphorylation of the JNK canonical substrate
Please cite this article in press as: Hojjat-Farsangi M, et al. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancer
therapy. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.07.005
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Fig. 2. A schematic proposed model for ROR1 signaling. The ROR1 receptor tyrosine kinase recruits canonical and non-canonical signaling pathways for cell survival and
invasion. A central pathway is the PI3K/AKT/mTOR pathway which activates the CREB transcription factor for nucleus translocation. ROR1 kinase-dependent SRC activation
is a key initiating event. Proteins like ROR1, STATs and CREB might act as transcription factors and bind to ROR1 promotor region to enhance the expression of the ROR1 gene.
Phosphate groups are denoted as green circles.
c-Jun in ROR1 knockdown HS746T cell lines. In contrast to the acti-
vation of JNK and p38 MAPK pathways, ROR1 knockdown showed
downregulation of -catenin, STAT5a and STAT4 but not of STAT3
and STAT6 proteins [29]. A high order function of SRC in promot-
ing activation of the STAT pathway in MET overexpressing cancer
cells may be suggested. ROR1-dependent MET transphosphoryla-
tionseemedtobe necessary for preventing apoptosis andinduction
of proliferation. SRC-dependent phosphorylation of ROR1 was of
importance for cell invasion. Specic inhibition of SRC activity by
saracatinib impaired cell invasion without affecting proliferation
(Fig. 2) [29].
Current data suggest that ROR1 might recruit different signaling
proteins and transcription factors and activate various signaling
pathways in different cancers, but the nal outcome is increased
tumor survival, proliferation and metastasis.
Most studies in primary tumor cells have suggested activation
of the PI3K/AKT/mTOR axis following ROR1 activation. The initial
activation of ROR1 may differ in various malignancies, but ROR1
signaling through the AKT/PI3K/mTOR axis might be important
irrespective of the initiation event. This pathway may be turned
on by small GTPases followed by SRC phosphorylation and conse-
quent binding of pSRC to ROR1. Axl [81], MET [42] and probably
other TKs may also be involved. Furthermore, ROR2 may dimer-
ize with ROR1 phosphorylating ROR1 to provide a docking site for
kinases with the SH2 domain. EGFR might be another partner for
ROR1 [43]. These signaling processes may accelerate the develop-
ment and progression of tumor cells activating genes involved in
growth, migration and metastasis [73]. Further studies are needed
to explore the activation of signaling pathways following ROR1
activation.
Schematic suggested ROR1 signaling pathways are depicted in
Fig. 2.
3.5. ROR1 isoforms in cancer
Expression of the full length ROR1 (105130kDa) has been
noted in all studied malignancies [28,30,42,62,63]. Other isoforms
might also be present. A truncated ROR1 isoform(t-ROR1) lacking
both the extracellular and transmembrane parts was reported in
1996 [21]. Northern blot experiments revealed that mRNA encod-
ing the t-ROR1 was abundantly expressed in fetal and adult human
CNS, in human leukemia, lymphoma cell lines, and in a variety of
human cancers derived fromthe neuroectoderm. In normal human
heart, lung and kidney cells a 6kb mRNA encoding ROR1 has been
described [21]. NTera2, a neuronogenic teratocarcinoma cell line,
expressed a 2373 nucleotide transcript encoding a 388 amino acid
protein identical to the cytosolic C-terminal region of ROR1 (ROR1-
201, ENSP00000441637). A 50kDa ROR1 isoform was identied
originating from a splice variant [48]. The presence of a 260kDa
ROR1 isoform which might represent dimerized ROR1 (homo- or
hetero-dimerization) was also recently reported [28]. This isoform
might represent ROR1 dimerization either with ROR2 or physical
association with EGFR as described in lung adenocarcinoma [43].
In lung adenocarcinoma, ROR1 and EGFR were physically attached
to each other. We also found the presence of a 64kDa ROR1 in CLL
cells that was detectable only in the nucleus [28] in line with the
suggestion that ROR1 may act as a transcription factor [74].
Theassociationof different ROR1isoforms tothemalignant phe-
notype of tumor cells needs further evaluation. However, the fully
mature and glycosylated ROR1 isoform (130kDa) was reported to
Please cite this article in press as: Hojjat-Farsangi M, et al. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancer
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Fig. 3. Strategies totarget thereceptor tyrosinekinaseROR1. Monoclonal antibodies
(mAbs) and small molecules (tyrosine kinase inhibitor TKI) (red circles) may inter-
fere with cell proliferation, differentiation, migration, metastasis and invasion as
well as induction of cell death by apoptosis or necrosis. Dual blockage by mAbs and
TKI may further augment the anti-tumor effects as has been shown for anti-EGFR
mAb/getinib and anti-HER2 mAb/lapatinib combinations [8890].
be more frequently seeninCLL patients withprogressive compared
to non-progressive disease [28]. It should be noted that also HER2
isoforms were related to clinical characteristics in breast cancer
[82].
3.6. RTKs and targeted cancer therapies
Oncogenic RTKs are multi-target proteins. Strategies to inhibit
RTK signaling include mAbs and TKI (Fig. 3). MAbs targeting the
extracellular part of theRTKmay disrupt TKsignalingbyneutraliza-
tionof theligand, hinderingligandbinding, receptor internalization
and degradation but may also activate the immune system to kill
the tumor cells. MAbs have been developed against different RTKs
and their ligands for a variety of cancers as HER-2, EGFR, VEGFRand
VEGF. Trastuzumab was the rst anti-HER2 mAb approved for the
treatment of HER2positive breast cancer patients [83]. Pertuzumab
is another approved anti-HER2 mAb preventing dimerization of
HER2 with other members of the EGFR family.
TKIs targeting the intracellular TKdomainof the RTKs have been
developed against EGFR/HER2, VEGFR and PDGFR family members
and approved for clinical use. Getinib [84] and erlotinib [85] are
potent TKIs of EGFR and lapatinib of HER2 [86].
Dual blockade of extra- and intracellular parts of RTKs by mAbs
and TKIs is likely to represent an advancement in the treatment.
Combination of trastuzumab and lapatinib in xenografted mice
with HER2 overexpressing cell lines showed signicant inhibition
of tumor growth [87]. Dual targeting of EGFR using cetuximab and
getinib showed synergistic effects on prevention of proliferation
and induction of apoptosis in colon cancer cell lines [88]. Combi-
nation of trastuzumab and lapatinib has been examined in several
clinical trials [89,90]. Dual HER2-targeting with trastuzumab and
lapatinib had better clinical activity than either agent alone in vivo.
3.6.1. Targeting ROR1 using mAbs
MAbs targeting ROR1 in a therapeutic intent have been devel-
oped by different groups [30,48,71]. MAbs against the Ig, CRD and
KNGdomains of the extracellular part of ROR1have beenproduced.
The mAbs could induce direct apoptosis or kill tumor cells through
activation of complement or immune effector cells in vitro [28,30].
Most effective anti-ROR1 mAbs were those against the CRD and
KNG domains [30]. Treatment of pancreatic cancer cell lines with
anti-ROR1 CRD mAb induced programmed cell death. Apoptosis
was preceded by dephosphorylation of ROR1, the PI3K isoform
(p110), AKT and mTOR proteins indicating inactivation of these
signaling proteins by the anti-ROR1 mAb. However, no change in
the phosphorylation levels of ERK and PKC proteins was observed.
The ndings might suggest that ERK and PKC signaling pathways
are not involved in ROR1 signaling in pancreatic carcinoma cell
lines, but the PI3K/AKT/mTOR signaling axis through ROR1 acti-
vation [79].
Anti-ROR1 mAbs could also kill melanoma cell lines [63]. Three
of four anti-ROR1mAbs induceda signicant direct apoptosis of the
ESTDAB049, ESTDAB112, DFW and A375 melanoma cells as well
as by activating CDC and ADCC. Two cell lines (ESTDAB081 and
094) were resistant to direct apoptosis by the mAb but not to mAb
mediatedCDCor ADCC. Transfectionof ESTDAB081 cells withROR1
siRNA downregulated ROR1 at the mRNA and protein levels and
induced apoptosis.
Anti-ROR1 antibodies might also prevent metastasis by down-
regulation of proteins involved in cell motility. ROR1 expression
was associated with epithelialmesenchymal transition of tumor
cells [73]. Treatment of breast cancer cells with anti-ROR1 mAbs
down-regulated proteins involved in metastasis. These cells lost
their ability to migrate and invade in vitro and to form metastasis
in vivo [73].
Antibodies labeled with cytotoxic agents including radioiso-
topes or toxins [antibody drug conjugates (ADC)] have shown
clinical benets in cancer treatment [91]. Anti-ROR1 antibodies
may be used as a carrier of toxic compounds. AROR1-immunotoxin
conjugate (BT-1) consisting of a toxin fromPseudomonas exotoxin
(PE38) andthe variable fragments of ananti-ROR1 mAb (clone 2A2)
showed a dose-dependent and selective binding to leukemic cells
fromCLL and MCL patients. The immunotoxin was internalized and
induced a strong apoptosis in vitro [92]. The apoptotic effects were
due to exposure of inner-membrane phosphatidylserine to the cell
surface, changes in mitochondrial membranes and activation of
caspase 3.
Invivo effects of anunconjugatedanti-ROR1mAbwere analyzed
in a ROR1xTCL1 transgenic mice model crossing ROR1
+
and TCL1
transgenic mice [93]. These mice developed ROR1
+
/CD5
+
/B220
low
leukemic B-cells with high levels of phosphorylated AKT and seem
to be a relevant model for in vivo studies. Anti-ROR1 mAbs against
distinct epitopes of ROR1 had different effects in vivo. The activity
of two anti-ROR1 mAbs, D10 and 4A5 was evaluated. Treatment of
ROR1xTCL1 leukemia cells with the D10 anti-ROR1 mAb reduced
the expression of phosphorylated AKT, but not by the 4A5 mAb.
Intravenous injections of D10 or 4A5 to ROR1 transgenic mice
engrafted with CD5
+
/B220
low
/ROR1
+
leukemia cells showed that
only the D10 mAb was able to clear leukemic cells fromthe blood
and spleen [93].
A humanized anti-ROR1 mAb, cirmtuzumab (UC-961) devel-
oped from the D10 anti-ROR1 mAb had a high specicity and
afnity (Kd=4nM) for ROR1. Intravenous injection of cirm-
tuzumab followed by infusion of human ROR1
+
CD5
+
B220
low
murine leukemia cells from ROR1xTCL1 transgenic mice, as well
as of human ROR1
+
CLL cells into the peritoneal cavity of Rag-
2//c/immunedecient mice, inducedclearanceof leukemic
cells in the spleen and peritoneal cavity. Cirmtuzumab had not
only a direct killing effect of tumor cells, but was also internalized
Please cite this article in press as: Hojjat-Farsangi M, et al. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancer
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by malignant B cells. An ADC using this mAb showed promising
results with enhanced cytotoxic activity against ROR1 expressing
cells. Cirmtuzumab-ADC cleared ROR1 expressing CLL cells in vivo
in xenografted mice and in vitro using adenocarcinoma cell lines of
the breast and pancreas [94].
3.6.2. Small-molecule tyrosine kinase inhibitors (TKI) targeting
ROR1
TKI targeting ROR1 are under development (www.kancera.
com). ROR1 TKI could efciently kill CLL cells with a high speci-
city. The best compounds killed 50 times more CLL cells than
normal blood lymphocytes. ROR1 was specically dephosphory-
lated as well as inactivated PI3K/AKT/mTOR proteins [95].
Interestingly, ROR1-TKIs killed not only treatment naive CLL
cells (EC50<2M), but also efciently udarabine-resistant CLL
cells [96].
Similar effects were seen on pancreatic adenocarcinoma cells.
Preliminary data showed that incubation of PaCa2 and PANC1 pan-
creatic carcinoma cells with ROR1 TKI induced a signicant cell
death including dephosphorylation of ROR1 as well as of SRC, PI3K,
AKT, mTOR and CREB. Gemcitabine killed the tumor cells as well
but did not dephosphorylate ROR1 [78].
3.6.3. Immunotherapeutic strategies targeting ROR1
ROR1 is expressed during embryogenesis but downregulated
in normal adult tissues. ROR1, as an onco-fetal antigen, might
be recognized by the immune system. In 2008, Fukuda et al.
described the induction of a humoral immune response against
ROR1 in CLL patients vaccinated with Ad-CD154 transduced CLL
cells. A subset of patients developed anti-ROR1 antibodies capable
of blocking the ROR1-WNT5a interaction [38]. Induction of anti-
ROR1 antibodies was also shown in CLL patients treated with the
immune-modulating drug lenalidomide [9799]. The results may
indicate that ROR1 is an immuno-dominant epitope.
Patients with CLL may also spontaneously mount a type 1 T cell
responseagainst ROR1as well as anIgGantibodyresponse. Ina sub-
set of patients the response was directed against the KNG domain
[100,101].
Antibodies against ROR1might haveclinical effects invivo. ROR1
transgenic mice immunized with a ROR1 peptide developed high
titers of anti-ROR1 antibodies inhibiting engraftment of human
ROR1 positive CLL cells [102]. These data suggest that ROR1 may
be a tumor vaccine candidate, similar to results presented for a
HER2 derived vaccine in breast cancer patients [103].
T-cells can be genetically engineered to express specic tumor
antigens, representing a promising technique for targetedcell ther-
apy [104,105]. Chimeric antigen receptor T cells (CARTs) combine
in a chimeric protein the recognition part of an antibody against
a tumor antigen with intracellular domains of the T cell receptor
stimulatory molecules as the CD8-CD3 zeta chain protein [106].
CART mediated responses might be further improved by inclusion
of another co-stimulatory domain as the CD137 (4-1BB) signaling
domain[107,108]. The specicity of recognitionof CARTs is dened
bythe antibodydomainandis independent of HLAantigens andcan
be extended to any target for which an antibody is available.
T cells genetically modied to express ROR1-CARTs targeting
ROR1 positive tumor cells have been developed [49,109]. Hudecek
et al. reportedthe constructionof the rst ROR1-CART fromhealthy
donors or CLL patients [49]. The scFv fragment of ananti-ROR1mAb
(clone 2A2) targeting the ROR1 Ig-like domain was linked to IgG4-
Fc domain, theCD28costimulatorydomainandtheTCRCD3 chain.
CD8
+
T cells engineered to express a ROR1-specic CART lysed CLL
andMCL cells, but not normal mature Bcells in vitro suggesting that
ROR1-CARTs might be a treatment option for patients with ROR1
positive tumors [49]. The group optimized the ROR1-CART con-
struct by modifying the extracellular spacer domains and produced
a panel of ROR1-CARTs with the same specicity but with differ-
ent scFv afnities. The modications of the extracellular spacer
region showed a better recognition of ROR1 expressing tumor cells
in vitro [109]. In vivo studies indicated that those ROR1-CARTs were
as effective as a CD19-CART in an immunodecient mice model of
humanMCL [109]. Clinical studies using ROR1-CARTs are just about
to be started.
4. Concluding remarks
RTKs play an important role in the normal cell development
and in cancer. ROR1 has been shown to be overexpressed in var-
ious malignancies but not in normal tissues. The role of ROR1 in
cancer is not fully clear. WNT5a seems to be a ligand for receptor
mediating growth stimulatory signals at least through the well-
recognized signaling pathway PI3K/AKT/mTOR. Aggressive tumors,
irrespective of type, had a higher expression of activated ROR1.
Silencing of ROR1 induced specic tumor cell death. These char-
acteristics make ROR1 an attractive target for therapy, using mAbs
as well as small molecules (TKIs). Preclinical data support the fur-
ther development of a new class of anti-cancer drugs. A deeper
understanding of the function of ROR1 in malignancies is required
to optimally develop ROR1 targeted therapy for hematological as
well as non-hematological tumors.
Conict of interest statement
HMis founder of Kancera AB. MHF, AM, AHD, ASK, A and HM
are shareholders of Kancera AB.
Acknowledgments
This study was supported by grants from the CLL Global
Research Foundation, The Torsten and Regnar Sderberg Foun-
dation (MT9/12 and MT4/13), the Cancer and Allergy Foundation
(149351, 149746, 150288), the Swedish Research Council (K2013-
64X-21464-04-3), the Swedish Cancer Society (CAN2009/852), the
Cancer Society in Stockholm(121332), the King Gustaf Vth Jubilee
Fund (124272), Felix Mindus Foundation (2013mind39290), Vin-
nova (348-2005-6481), the Karolinska Institute Foundations, AFA
Insurance (130054) andthe StockholmCounty Council (20120051).
The excellent secretarial help fromMs. Leila Relander and Gunilla
Buren is highly appreciated.
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