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Please cite this article in press as: Hojjat-Farsangi M, et al. The receptor tyrosine kinase ROR1 An oncofetal antigen for targeted cancertherapy. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.07.005

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Seminars in CancerBiology xxx (2014) xxxxxx

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Seminars in Cancer Biology

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Review

The receptor tyrosine kinase ROR1 An oncofetal antigen for targetedcancer therapy

Mohammad Hojjat-Farsangi a, Ali Moshfegh a, Amir Hossein Daneshmanesha,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, CancerCenterKarolinska (CCK), KarolinskaUniversityHospital Solnaand Karolinska

Institutet, Stockholm, Swedenb Department of Hematology, KarolinskaUniversityHospital Solna, Stockholm, Swedenc Department of Oncology,KarolinskaUniversity Hospital Solna, Stockholm, Sweden

a r t i c l e i n f o

Keywords:

ROR1Tyrosine kinase inhibitorsMonoclonal antibodiesCancer therapy

a b s t r a c t

Targeted cancer therapies have emerged as new treatment options for various cancer types. Amongtargets, receptor tyrosine kinases (RTKs) are among the most promising. ROR1 is a transmembrane RTKofimportance during the normal embryogenesis for the central nervous system, heart, lung and skeletalsystems, but is not expressed in normal adult tissues. However, ROR1 is overexpressed in several humanmalignancies and may act as a survival factor for tumor cells. Its unique expression by malignant cellsmay provide a target for novel therapeutics including monoclonal antibodies (mAbs) and small moleculeinhibitors oftyrosine kinases (TKI) for the treatment ofcancer. Promising preclinical results have beenreported in e.g. chronic lymphocytic leukemia, pancreatic carcinoma, lung and breast cancer. ROR1 mightalso bean interestingoncofetal antigen for active immunotherapy.Inthis review, we provide an overviewofthe ROR1 structure and functions in cancer and highlight emerging therapeutic options ofinterest fortargeting 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 acquiredtumorigenic characteristics [1]. These properties include prolifer-ation, evading growth suppression, resisting cell death, enablingreplicative immortality, activating invasion, metastasis, evadingfrom recognition of the immune system and reprogrammingenergy metabolism [1,2].

The term oncogenic addiction by Weinstein[3] suggested thattumor cells may exhibit dependence on an activated oncogenicpathway to maintain survival and proliferation. Phosphorylationof signaling proteins is central in the regulation of cellular activi-ties and protein kinases play a key role in the normal developmentas well as during tumorigenesis [2,4].

Protein kinases areenzymesthat catalyze thetransfer of a phos-phate group from adenosine 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 517743 08; fax:+46 8 3183 27.

E-mail address: [email protected](H. Mellstedt).

serine/threonine residues. Receptor tyrosine kinases (RTKs) are afamily of kinases, consisting of a transmembrane receptor linkedto an intracellular kinase domain. Most RTKs are involved in thenormal celldevelopmentbut might alsoact as oncogenesin tumori-genesis. Several tumors are addicted to oncogenic RTKs and theirsignaling 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 ofnormal cellular processes as proliferation, differentiation, motility,survival, metabolism and play critical roles in the normal develop-ment and organogenesis [4] and are emerging as pharmacologicaltargets in oncology [5].

Current data indicate that deregulation of kinase activity is amajor mechanism by which tumor cells may escape normal phys-iological controls for survival and growth [6]. Targeting TK activitymight be of importance to prevent tumor cell growth. Monoclonalantibodies (mAbs) against the extracellular part of RTKs and smallmolecules inhibiting the intracellular tyrosine kinase activity ofRTKs (TKI) respectively are drugs downregulating oncogenic activ-ities through receptor or non-receptor tyrosine kinases.

In 1992, Masiakowski and Caroll described two new RTKs witha high amino acid homology to the TK domain of growth factorreceptors and to the Trk family named ROR1 and ROR2 shown

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2 M. Hojjat-Farsangi et al. / Seminars in Cancer Biology xxx(2014) xxxxxx

to be involved in a network of regulatory interactions [7]. Thesetwo members shared58% amino acid homology and 98%homologybetween themouseand human. Partial nucleotide sequencesof therat genes revealed a strikingly evolutionary conservation betweenthe human and rat proteins.The level of expression of therat geneswas 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 ofcell surface receptors during development [7]. In 1993, the expres-sion of ROR1 was described in Drosophila and to be of importanceduring the embryonic development of the nervous system [8].

ROR1 hasalso been found to be overexpressed in several humancancers. Here, we discuss the biology of ROR1 in health and malig-nancies, and highlight a novel class of therapeutics targeting ROR1which might be of interest for cancer therapy.

2. Receptor tyrosine kinases (RTKs)

RTKs are a large family of cell surface glycoproteins [9]. Thesereceptors with enzymatic activity regulate various cellular pro-cesses [10,11]. RTKs are divided into 20 different receptor familiesconsisting of 58 members [12].

All RTKs have a similar molecular structure including an extra-cellular region containing the ligand binding domains, a singletransmembrane part and an intracellular region with a conservedTK domain. Ligand binding induces dimerization of the extracellu-lar part and initiation of intracellular signaling cascades regulatingcellular functions [13].

Aberrant activation of RTK may be due to receptor overexpress-ion,chromosomal translocation, geneamplification, mutations anddownregulation which might contribute to thedevelopmentof var-ious types of cancer (Table 1) [1416]. Deregulation of more than30 individual RTKsin various malignancies hasbeendescribed[10].Some important RTKs families in cancer are vascular endothelialgrowth factor receptor (VEGFR), epidermal growth factor receptor(ErbB/EGFR), platelet-derived growth factor receptor (PDGFR) andfibroblast growth factor receptor (FGFR) families. Targeting theseRTKs by mAbs or TKI have shown significant clinical effects [17].ROR1 might be added of this group of oncogenic RTKs.

3. Receptor-tyrosine kinaseROR1

Receptor tyrosinekinase orphan receptors1 and 2 (ROR1/ROR2)belong to one of the twenty different RTK families and areevolutionarily highly conserved [5]. ROR1 consists of 3 dis-tinct extracellular regions including an immunoglobulin (Ig)-likedomain, a cysteine rich (CRD) domain and a kringle (KNG) domainas well as an intracellular TK domain (Fig. 1). The cytoplasmic partcontains a TK domain with protein kinase activity, and furtherdownstream serine, 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 RTKgenes in 1992 [7]. Later, ROR genes were described in Drosophila[18], mouse [19] and C. elegans (cam-1) [20]. The ROR1 gene wasoriginally cloned from a neuroblastoma cell line [7]. A truncatedROR1 gene was reported in the fetal and adult human central ner-vous system, in human leukemia and lymphoma cell lines and in avariety of human cancer derived from the neuroectoderm.

Human(h) ROR1 is located on chromosomal region 1p31.3 andconsists of 937 amino acids (908 after cleavage of the signal pep-tide) with an estimated molecular weight of 105 kDa. hROR1 and

hROR2 shared an overall 58% identity in amino acid sequence. The

Ig domain

Cysteine-richdomain-

Kringle domain

Tyrosine kinasedomain

Serine/threoninerich domain

Proline richdomain

Human ROR Mouse ROR

Drosophila ROR

Extracellular part

Cell membrane

Cytoplasmic part

Fig. 1. Structureof theROR receptor tyrosinekinasesin differentspecies.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 theROR1 and ROR2 subgroups. 97% amino acid identity was sharedbetween 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 106amino acid residues and the corresponding number for ROR2 is 91.The precise functions of the Ig domain are not known, but mightbe involved in proteinprotein and/or proteinligand interactions[23].

The extracellular CRD domain is defined by 10 conserved cys-teines that form five disulfide bridges and consists of 135 aminoacids. The CRD domain is similar to the WNT binding domain ofFrizzled receptors and is one of the ligand binding motifs for RORs.

The KNG domains of ROR1 and ROR2, juxtaposed to the cellmembrane, consist of 80 and 79 amino acids, respectively. It ishighly conserved throughout species and might function as recog-nition modules for binding of proteins including WNT regulatoryproteins and other ROR ligands [24]. The presence of a KNGdomainis a hallmark of the ROR family members as ROR is the only RTKfamily reported to contain the KNG domain with the exception forTorpedo MuSK [22].

ROR1 contains 40 aminoacids withinthe kinasedomain [25,26].Twenty-one amino acids of the forty consensus amino acids areconservedin all RTKs,makingthis region themosthighlyconservedregion not only of ROR, but also for all known RTKs [7,25].

ROR1,as well as theother memberof this familyhas a conservedsequence, YXXDYY (YSADYY in hROR1, position 641646) within

the activation site of the TK domain which is also present in Trkand MuSK [27]. Phosphorylation of ROR1 at the first and last tyro-sine residues is critical. ROR1 is constitutively phosphorylated atthesetyrosine residues in chronic lymphocytic leukemia (CLL) cells[28]. As will be discussed later, ROR1 is phosphorylated at thesetyrosine residues by SRC kinases [29]. Anti-ROR1 mAbs against theCRD and KNG domains [30] induced dephosphorylation of ROR1 atthese tyrosine residues followed by apoptosis of the leukemic cells[28]. Serine at position652 wasalso phosphorylated in mROR1 [31].This specific serine is present at the same position in hROR1. ROR1has also two other conserved sequence, YSLM (position 772775)and YXXF (YGKF in hROR1, position 666669), which are potentialbinding sites for the SRC homology 2 (SH2) region of intracellularkinases, like the p85 subunit of PI3K [32]. All these sequences are

present in the TK domain of ROR1.
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Table 1

Expression of oncogenic RTKs in cancer.

RTK Chromosomelocation

Normal function Malfunction leading tooverexpression

Malignancy (examples)

ROR1 1p31.3 ED, NSD Unknown CLL, ALL, AML, MCL, HCL, melanoma, prostate,lung, breast, pancreas, colon, ovarian, uteruscancers

ROR2 9q22.31 ED, NSD Mutations Melanoma, medulloblastoma, testicular cancer,gastrointestinal stromal tumor, hepatocellular

carcinoma, colon cancer, renal cell carcinoma,osteosarcoma, prostate carcinomaALK 2p23 NSD Translocation (t2:5)

overexpressionNSCLC, colorectal cancer, breast cancer,oesophageal cancer (squamous cell), renal cellcancer

ROS1 6q22 NSD Deletion, inversion,translocation

NSCLC, cholangiocarcinoma, ovarian cancer,gastric cancer, colorectal cancer

RET 10q11.2 NSD Mutations NSCLC, medullary thyroid carcinomaNTRK1 (TrkA) 1q21-22 Development and maturation of central and

peripheral nervous systemTranslocation Colorectal c ancer, p apillary t hyroid c ancer,

lung adenocarcinoma, breast cancer, oralsquamous cell carcinoma

NTRK2 ( TrkB) Development a nd m aturation o f central a ndperipheral nervous system

Translocation Neuroblastoma, a strocytoma, o ral s quamouscell carcinoma

NTRK3 (TrkC) 15q25 Development and maturation of central andperipheral nervous system

Translocation Neuroblastoma, breast cancer

PDGFRA 4q12 ED Mutations Lung adenocarcinoma, gastrointestinal stromaltumors

PDGFRB 5q32 Regulation of embryonic development, cellproliferation, survival, differentiation,chemotaxis, migration and blood vesseldevelopment

Mutations Gastrointestinal stromal t umors, glioblastoma

FGFR1 8p12 Regulation o f embryonic d evelopment, c ellproliferation, differentiation and migration

Mutations Squamous cell lung cancer, breast cancer

FGFR2 10q26 ED Mutations,overexpression

Squamous cell lung cancer, lungadenocarcinoma, 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, d evelopment a nd m igration o f muscles and neuronal precursors, angiogenesisand kidney formation

Mutations Hepatocellular carcinoma, CLL, breast cancer,pancreatic cancer, lung cancer, gastricadenocarcinomas

Axl 19q13.1 ND Unknown Lung cancer, colon cancer, breast cancer, AML,CML, esophageal, thyroid cancer,gastrointestinal stromal tumors,

astrocytomaglioblastomaIGF1R 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, headand neck squamous cell carcinoma

EGFR2 (ERBB2) 17q12 ED Mutations Breast cancer, gastric adenocarcinomasEGFR3 (ERBB3) 12q13.2 ED Mutations Breast cancerEGFR4 (ERBB4) 2q34 ED Mutations Breast cancer, melanomaVEGFR1 (FLT1) 13q12.3 ED Mutations Ovarian cancer, NSCLC, colorectal carcinomaVEGFR2 (KDR) 4q12 ED Mutations Renal cell carcinoma, hepatocellular carcinomaVEGFR3 (FLT4) 5q35.3 ED MutationsFLT3 13q12.2 Hematopoiesis Mutations AML, acute promyelocytic leukemia

KIT 4q12 Hematopoiesis, s tem c ell m aintenance,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 p rocessesincluding cell survival, migration anddifferentiation

Mutations Pancreatic cancer, breast cancer, NSCLC,laryngeal squamous cell carcinoma, head andneck squamous cell carcinoma

INSR 19p13.2 Metabolic actions of insulin Mutations Colorectal cancer, prostate cancerINSRR 1q23.1 Metabolic actions of insulin Mutations NeuroblastomasCCK4 (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 squamouscell 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 Chromosomelocation

Normal function Malfunction leading tooverexpression

Malignancy (examples)

EPHA4 2q36.1 NSD Mutations NSCLC, gastric cancerEPHA5 4q13.1 NSD Mutations Breast cancer, hepatocellular carcinoma, ALL EPHA6 3q11.2 NSD Mutations EPHB1 Xq13.1 NSD NSCLC, cervical cancer, ovarian CancerEPHB2 13q33.3 NSD Cervical cancer, breast cancerEPHB3 3q27.1 NSD NSCLC, breast cancer, colorectal cancer

EPHB4 7q22.1 NSD Breast cancer, melanoma, gliomaMER 2q13 Survival, m igration, d ifferentiation, a ndphagocytosis 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 GlioblastomaTEK 9p21.2 Reg ulates ang iog enesis, endothelial cell

survival, proliferation, migration, adhesion andcell spreading, reorganization of the actincytoskeleton, but also maintenance of vascularquiescence

Mutations Bladder cancer, glioblastoma, AML

RYK 3q22.2 Neuron di fferentiation, axon gu ida nce, corpuscallosum establishment and neurite outgrowth

Translocation,mutations

CML, ovarian cancer

DDR1 6p21.33 Reg ulates cell attachment to the extracellularmatrix, remodeling of the extracellular matrix,cell migration, differentiation, survival andproliferation

Mutations NSCLC, breast cancer, AML, ovarian Cancer,hepatocellular carcinoma

DDR2 1q23.3 Regulates cell differentiation, remodeling of theextracellular matrix, cell migration and cellproliferation

Mutations Head and neck squamous cell carcinoma,NSCLC, lung cancers, CML, breastcancer

LTK 15q15.1 ND Mutations Gastric cancer, lymphomas, leukemiasMUSK 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: acutelymphoblastic 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: fibroblast growth factor receptor, ND: normal development, CML: chronic myeloid leukemia, INSR: insulin receptor, EGFR:epidermalgrowth factor receptor,VEGFR:vascular endothelial growth factor receptor,CCK: coloncarcinoma kinase, RYK:receptorrelatedto tyrosine kinases,DDR: discoidindomain receptor, LTK: leukocyte tyrosine kinase, MuSK: muscle-specific 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/TRDsor proline-richdomains of theROR familyhave been found in other

RTKs. The S/TRD1domain of ROR1 and ROR2 showeda high degreeof homology (67%) but the S/TRD2s did not exhibit any appar-ent homology. The proline-rich domains showed a lower degreeof 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 SH3recognition motifs for protein interactions. These motifs might beof importance in ROR1 mediated signaling by association with theSH2 and SH3 domains of adaptor/signaling molecules [33].

ROR1 has also been shown to be phosphorylated at one ormore tyrosine residues at positions 786, 789, 822, 828 and 836by 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 theproline-rich domain prevented ROR1 phosphorylation within theTK domainby SRC. Impairment of thefunction of 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 thecase for e.g. members of the EGFR family [34].

As for other RTKs, ROR1 requires two steps for intracytoplasmicactivation: (a)increase in theintrinsic catalytic activity and (b) cre-ation of docking sites for the recruitment of downstream signalingproteins. These two processes are accomplished by autophospho-

rylation of tyrosine residues as a consequence of ligand-mediated

oligomerization. Autophosphorylation of tyrosine residues in theactivation loop, within the kinase domain, stimulates phos-phorylation of tyrosine residues within the juxtamembrane andcarboxy-terminal regions. Phosphorylation induced generation of

binding sites for modular domains detecting phospho-tyrosineresidues in specific sequence contexts. SH2 and the phospho-tyrosine-binding domains are two well-known phospho-tyrosinebinding modules of signaling proteins [35]. The activation loop ofthe kinase domain contains one to three tyrosine residues, similarfor several RTKs [25].

There are studies indicating that RORs bind WNT proteins (lig-ands) [24,36]. ROR1 and WNT5a have been shown to physicallyinteract with each other and activate NF-B when overexpressedin HEK293 cells. Survival of immature cells during embryonicdevelopment [37] and of leukemic cells was enhanced by sol-uble WNT5a, which could be neutralized by anti-ROR1 antiserain vitro [38]. These findings may indicate that ROR1 might acti-vateanNF-B-dependent survivalsignal induced by WNT5a during

embryogenesis and tumorigenesis [38]. ROR2 seems also to act asa receptor for WNT5a. CRD of ROR2 was required for binding toWNT5a 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 kinaseregionraised thequestionif kinaseactivitywas lost. Kinaseactivityhas 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-1encodesaCaenorhabditiselegans orphanRTKoftheRORfam-ily that was required for cell migration and cell polarity [41]. Oneof the CAM-1 mutants (gm105) contained a stop codon, 74 amino

acidsdownstreamofthetransmembraneregionand25aminoacids

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upstream of the TK domain. The encoded protein lacked the kinasedomain, but CAM-1 (gm105) was not a null mutant without func-tion [9,41]. This observation indicated that some CAM-1 functiondid not require kinase activity and CAM-1 mutants with lack ofkinase activity were involved in cell migration [20].

Biochemical assays have demonstrated that ROR1 might be apseudokinase devoided of catalytic activity or with low activity,but silencing of ROR1 with specific siRNA suppressed cell growthand subsequent apoptosis [42]. In lung adenocarcinoma, wild-typeROR1 but not a kinase-dead ROR1 with silent mutations at thesiRNA binding site was able to neutralize siRNA ROR1-inducedgrowth inhibition. Enhancement of the wild-type ROR1 expressionbut not the kinase-dead ROR1 in the ROR1-negative MSTO-211Hcell line to a level similar to that of theROR1 expressing NCI-H1975cell line increased in vivo growth of xenografts. These data suggestthat ROR1 kinase activity was required to fully confer a growthadvantage [43]. ROR1 kinase activity might differ between nor-mal cells, cell lines and malignant cells and also between varioustumors, but overall, ROR1 seems to be an important RTK for cellsurvival and migration.

3.3. Expression and function of ROR1 during organ development

ROR1 is expressedduring various embryonicstages of theskele-tal, respiratory and cardiac systems as well as in neurite extensionof central neurons. The expression was tightly downregulated afterbirth [7,19,4447]. hROR1 was expressed in fetal heart, lung andkidney but to a lesser extent in placenta, pancreas and skeletalmuscles [21]. Analysis of ROR1 protein expression in 28 normaladult tissues showed no expression in the majority of sampleswith the exception for a very low level in testis, uterus, lung,bladder, and colon [48]. In various normal hematopoietic andnon-hematopoietic tissues, the expression of ROR1 at the mRNAlevel was not noted except for low levels in pancreas and adiposetissues.

The ROR1 protein was also noted in an intermediate stage of

the normal B-cell maturation in the bone marrow (pre-BII stage)which cellsproliferated afterinternalizationof thepre-Bcell recep-tor complex. This process was necessary for the development to animmature B-cell stage [49].

The functions of ROR1 and ROR2 have been studied in micelacking either of these genes. ROR1 mutant newborn mice weresimilar in size as the wild-type mice and did not exhibit any grossabnormalities. After birth ROR1 mutant mice showed respiratorydysfunctionsanddiedwithin24h [23,46]. Incontrast,ROR2mutantmice exhibited dwarfism, short limbs and tail as well as facialanomalies at birth. ROR2 mutant newbornsshowed forced respira-tion and cyanosis and died within 6 h after birth [50,51]. ROR1 andROR2 double deficient newborn mice died shortly after birth dueto breathing difficulties associated with incomplete expansion of

lung alveoli [23,46,51]. As ROR1 and ROR2 genes showed a similarexpression pattern in the developing face, limbs,heartand lung tis-sues, the absence of morphological abnormalities in ROR1 mutantmice could be explained by functional redundancy between ROR1and ROR2 [44].

ROR1 andROR2 modulatedthe rate of neurite elongation in cul-ture of rat hippocampal neurons [52] and were highly expressedin dendrites of central neurons [37]. ROR1ROR2 heterodimeriza-tion was essential for neuron synapse formation [53]. Suppressedexpression of ROR1 and/or ROR2 led to the formation of fewersynaptic contacts. Furthermore, ROR1ROR2 dimers interactedwithWNT5aand regulatedsynapseformation in hippocampal neu-rons. WNT5a was also expressed by hippocampal neurons andastrocytes and may act as an autocrine factor to stimulate ROR1

and ROR2 in synaptogenesis [53].

Table 2

Overexpression of ROR1 in malignancies.

Malignancy ConstitutivelyphosphorylatedROR1

Associationto diseaseprogression

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 acutelymphoblasticleukemia,NI:no information, CML: chronic myeloid leukemia, AML: acute myeloid leukemia.

3.4. ROR1 expression in malignancies

Theexpressionof ROR1in differentmalignanciesis summarizedin Table 2.

The main mechanisms leading to aberrant RTK activation inhuman cancers are self-activation, chromosomal translocations,overexpression,gain-of-functional mutationsor loss-of-functionoftumor suppressorgenes. Mutations or chromosomal translocationsof ROR1 in several cancer types have not been shown. Mutationanalysis of the extracellular and cytoplasmic kinase domain of theROR1 gene in CLL cells indicated no major genomic aberrations(mutation or truncation). FISH analysis showed no rearrangementin 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]andasasuppressorgenedependingonthemalignancy[55,56]. This dual role of ROR2 is unusual and has not been shownfor any other RTKs.

In 2001, two independent, gene profiling studies in CLLrevealeda 45-fold increase of the ROR1 expression compared to normalmature B-lymphocytes [57,58]. Subsequently, ROR1 was shown tobe overexpressed not only in CLL[38,48,54], but also in acute lym-phocytic leukemia (ALL) [59,60], renal cell carcinoma [61], breastcancer [62], lung adenocarcinoma [43], melanoma [63,64], andother lymphoid and myeloid malignancies [28,54,60,6266] . Thenumber of ROR1 receptors on the surface of CLL cells was esti-mated to be 10,000 molecules percell [38,54]. This numberof ROR1molecules should be sufficient 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 HamsterOvary (CHO) cells significantly improved survival compared tothose cultured with CHO untransfected cells. The survival advan-tage was abrogated by ROR1 antisera [38]. The data support thenotion that WNT5a activates ROR1 in CLL cells. Furthermore, ROR1is constitutively phosphorylated at tyrosine and serine residues inCLLas well as in other malignancies [28,42,63] and WNT5a seemedto maintain phosphorylation of ROR1 [62]. A significant correlationbetween phosphorylation intensity of ROR1 and disease activity inCLL 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].

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Moreover, the ROR1 protein was significantly higher expressed inmore aggressive tumors [30,67]. Collectively the data suggest thatthe expression pattern of ROR1 was related to the aggressivenessof the disease.

The leukemic cells in ALL originate from different stages of B-and T-cell maturation [68]. Overexpression of the ROR1 gene wasshown in fresh ALL cells as well as in cell lines [59,60]. High ROR1expression 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 withincreased cell migratory capacity and an undifferentiated pheno-type compared to non-ROR1 expressing ALL cells [69,70].

ROR1 hasalso been shown to be overexpressed both at thegeneandproteinlevels in multiplemyeloma [49,71], marginalzone lym-phoma (MZL), mantle cell lymphoma (MCL), diffuse large B-celllymphoma (DLBCL), and follicular lymphoma (FL) [72].

Also in several solid tumors, ROR1 has been shown to beoverexpressed [42,61]. Surface expression of ROR1 was noted inmelanoma cell lines and phosphorylated at tyrosine and serineresidues [63]. Anti-ROR1 specific mAbs alone killed melanomacells [63]. ROR1 and ROR2 were inversely expressed in melanomacells suggesting that they may negatively regulate each other [64].

Hypoxia induced a shift of ROR1-positive melanomacells to a moreaggressive ROR2-positive phenotype. The switch induced a 10-folddecrease in sensitivity to BRAF inhibitors. In melanoma patientstreated with the BRAF inhibitor vemurafenib, WNT5a expressionin melanoma cells correlated with clinical response and resistanceto activation of the WNT-mediated canonical signaling pathway(-catenin activation) [64].

In breast cancer, ROR1 was overexpressed and associated withaggressive disease [62]. Breast cancer cell lines with a high ROR1expression had a more aggressive andinvasive behavior than thosewith low ROR1 expression which were non-migrating cells. Spe-cific ROR1 siRNA down-regulated theexpression of ROR1 in humanbreast cancer cell lines and impaired the growth in immunode-ficient mice [62]. ROR1 could be shown to interact with casein

kinase 1 epsilon (CK1e) activating PI3Kmediated AKT phosphoryla-tionas well as the cAMP-response-element-bindingprotein (CREB)Furthermore, WNT5a augmented the growth of breast cancer cellsexpressing ROR1 inline with theassumptionthatWNT5ais a ligandfor ROR1 [62,73].

ROR1 has also been suggested to be associated withepithelialmesenchymal transition (EMT) during embryogenesisand in cancer metastases [73]. ROR1 was highlyexpressed in breastadenocarcinomas with high levelsof a gene signaturefor EMTandahigh rate of relapse as well as thecapacityto metastasize comparedto breast cancer patients with a better prognosis where the levelof ROR1 expression was low. Expression of ROR1 in breast cancercell lines witha highcapacityto 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 tightjunction proteins, and a high migratory capacity in vitro as well asin immunodeficient mice. Similarly, MCF-7 cells transfected withROR1 showed a reduced expression of E-cadherin and CK-19, butnot of SNAIL-1/2 andvimentin [73], which molecules are of impor-tance for homing of cells at proliferating sites. The data support thenotion that ROR1 is associated with less differentiated cells with ahigh 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 inducedphosphorylation of signal transducer and activator of transcrip-tion factor 3 (STAT3) and upregulated the ROR1 protein in multiplemyeloma 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 specific bindingelements. The ROR1 promoter contained -interferon activationsequence-like elementswhich wereactivatedby STAT3 [76]. STAT3induced WNT5a expression [88,89]. Thus, STAT3 may activate bothROR1 and WNT5a in tumor cells inducing activation of the WNT5asignaling pathways through binding to the ROR1 promoter stimu-lating cell survival, growth and migration.

ROR1 is extensively modified by N-linked glycosylation in CLLcells [77]. Post-translational modifications generated several ROR1isoforms with different electrophoretic mobilities from 100 to130kDa. Prevention of ROR1 glycosylation interfered with cell sur-face expression of the fully mature ROR1 isoform (130 kDa) andtheformation of filopodia in HEK293 cell line supporting the notionthat ROR1 has a role in cell migration and metastasis.

ROR1 signaling pathway(s) are not well established in cancercells. In breastcancer, the PI3K/AKTpathway was activated follow-ingactivation of ROR1 by WNT5a. ROR1 silencingor lack of WNT5ainhibited the growth of breast cancer cells [62]. The gene expres-sionprofileoftheMDA-MB-231breastcancercelllinesilencedwithROR1 siRNA showed lower expression of genes encoding proteinsinduced by CREB. Treatment of MDA-MB-231 cells with recom-binant WNT5a enhanced the viability of the tumor cells and theexpression of pAKT and pCREB, as well as a higher expression

of CREB target genes. Own preliminary data indicated activationof SRC, PI3K, AKT, mTOR, and CREB in pROR1 positive pancreaticcarcinomacells (Fig. 2). Treatment of ROR1 positive pancreatic car-cinoma cells with anti-ROR1 mAbs or ROR1 TKIs induced specificdephosphorylation of ROR1 as well as of SRC, PI3K, AKT, mTOR,andCREB and subsequently tumor cell death [78,79].

ROR1 and the pre-B-cell receptor (pre-BCR) may interfere witheach 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 inhibitordasatinib (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. Downregulationof both ROR1 and pre-BCR induced permanent dephosphoryla-

tion of AKT and inhibited cell growth as well as increased tumorcell killing. However, downregulation of either ROR1 or BCR alonedid not induce the same effects, suggesting complimentary effectsof ROR1 signaling on pre-BCR signaling [70]. The results indi-cate a link between the pre-BCR and ROR1 receptor signalingpathways.

ROR1 may also contribute to leukemogenesis of CLL cellsthrough binding to the T-cell leukemia antigen 1 (TCL1) as an acti-vator of AKT (Fig. 2). Coexpression of ROR1 and TCL1 acceleratedleukemogenesis inducing increased phosphorylation of AKT, cellproliferation and resistance to apoptosis. Targeting ROR1 with spe-cific mAbs down-modulated ROR1 expression and decreased AKTactivity and subsequently the cells lost the tumorigenic character-istics in a syngeneic mice model [80].

Our own unpublished and recent [29] data demonstrated theinvolvement of the MET and SRC oncogenes in ROR1 phosphor-ylation (Fig. 2). We could show constitutive phosphorylation ofROR1 at tyrosine residues 641 and 646 in the ROR1 TK domainwhich was of importance for cell survival [28]. Using COS-7 cellline transfected with ROR1 mutants, Gentile et al. demonstratedtransphosphorylation of ROR1 by the SRC oncogene at tyrosines641, 645 and 646 residues located within the TK domain. Theproline-rich domain was also transphosphorylated by MET and thepresence of this domain was required for SRC recruitment whichtriggered transphosphorylation. The data may suggest that phos-phorylation occurred in cells with overexpression of MET, but notin cells with normal levels of MET expression. Moreover, phospho-kinase arrays showed increasedactivationof theJNK andp38 MAPK

pathways and high phosphorylation of the JNK canonical substrate

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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 andinvasion.A central pathway is thePI3K/AKT/mTOR pathway which activatesthe CREB transcription factorfor nucleus translocation. ROR1 kinase-dependent SRC activationis a keyinitiating event.Proteins like ROR1, STATs andCREB might actas transcription factors andbind to ROR1 promotor regionto enhance theexpression of theROR1 gene.Phosphate groups are denoted as green circles.

c-Jun in ROR1 knockdownHS746T cell lines. In contrast to the acti-

vation of JNK and p38 MAPK pathways, ROR1 knockdown showeddownregulation of-catenin, STAT5a and STAT4 but not of STAT3and STAT6 proteins [29]. A high order function of SRC in promot-ing activation of the STAT pathway in MET overexpressing cancercells may be suggested. ROR1-dependent MET transphosphoryla-tionseemed to be necessaryfor preventing apoptosisand inductionof proliferation. SRC-dependent phosphorylation of ROR1 was ofimportance for cell invasion. Specific inhibition of SRC activity bysaracatinib impaired cell invasion without affecting proliferation(Fig. 2) [29].

Current data suggest thatROR1 might recruit different signalingproteins and transcription factors and activate various signalingpathways in different cancers, but the final outcome is increasedtumor survival, proliferation and metastasis.

Most studies in primary tumor cells have suggested activationof the PI3K/AKT/mTOR axis following ROR1 activation. The initialactivation of ROR1 may differ in various malignancies, but ROR1signaling through the AKT/PI3K/mTOR axis might be importantirrespective of the initiation event. This pathway may be turnedon by small GTPases followed by SRC phosphorylation and conse-quent binding of pSRC to ROR1. Axl [81], MET [42] and probablyother TKs may also be involved. Furthermore, ROR2 may dimer-ize with ROR1 phosphorylating ROR1 to provide a docking site forkinases with the SH2 domain. EGFR might be another partner forROR1 [43]. These signaling processes may accelerate the develop-ment and progression of tumor cells activating genes involved ingrowth, migration and metastasis [73]. Further studies are neededto 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 beennoted in all studied malignancies [28,30,42,62,63]. Other isoformsmight also be present. A truncated ROR1 isoform (t-ROR1) lackingboth the extracellular and transmembrane parts was reported in1996 [21]. Northern blot experiments revealed that mRNA encod-ingthe t-ROR1was abundantly expressed in fetal and adult humanCNS, in human leukemia, lymphoma cell lines, and in a variety ofhuman cancers derived from the neuroectoderm. In normal humanheart, lung and kidney cells a 6 kb mRNA encoding ROR1 has beendescribed [21]. NTera2, a neuronogenic teratocarcinoma cell line,

expressed a 2373 nucleotide transcript encoding a 388 amino acidprotein identical to thecytosolic C-terminal regionof ROR1 (ROR1-201, ENSP00000441637). A 50kDa ROR1 isoform was identifiedoriginating from a splice variant [48]. The presence of a 260kDaROR1 isoform which might represent dimerized ROR1 (homo- orhetero-dimerization) was also recently reported [28]. This isoformmight represent ROR1 dimerization either with ROR2 or physicalassociation with EGFR as described in lung adenocarcinoma [43].In lung adenocarcinoma, ROR1 and EGFR were physically attachedto each other. We also found the presence of a 64kDa ROR1 in CLLcells that was detectable only in the nucleus [28] in line with thesuggestion that ROR1 may act as a transcription factor [74].

Theassociation of differentROR1 isoformsto themalignant phe-notype of tumor cells needs further evaluation. However, the fully

mature and glycosylated ROR1 isoform (130 kDa) was reported to

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Fig.3. Strategiesto target 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 aswell as inductionof cell death by apoptosisor necrosis.Dual blockage by mAbs andTKI may further augment the anti-tumor effects as has been shown for anti-EGFRmAb/gefitinib and anti-HER2 mAb/lapatinib combinations [8890].

be morefrequently seen in CLL patientswithprogressive comparedto non-progressive disease [28]. It should be noted that also HER2isoforms were related to clinical characteristics in breast cancer

[82].

3.6. RTKs and targeted cancer therapies

Oncogenic RTKs are multi-target proteins. Strategies to inhibitRTK signaling include mAbs and TKI (Fig. 3). MAbs targeting theextracellularpartoftheRTKmay disruptTKsignalingbyneutraliza-tionoftheligand,hinderingligandbinding,receptorinternalizationand degradation but may also activate the immune system to killthe tumor cells. MAbs have been developed against different RTKsand their ligands fora variety of cancers as HER-2,EGFR,VEGFR andVEGF. Trastuzumab was the first anti-HER2 mAb approved for thetreatment of HER2positivebreast cancer patients[83]. Pertuzumabis another approved anti-HER2 mAb preventing dimerization of

HER2 with other members of the EGFR family.TKIs targetingthe intracellular TK domainof the RTKs have been

developed against EGFR/HER2, VEGFR and PDGFR family membersand approved for clinical use. Gefitinib [84] and erlotinib [85] arepotent TKIs of EGFR and lapatinib of HER2 [86].

Dual blockade of extra- and intracellular parts of RTKs by mAbsand TKIs is likely to represent an advancement in the treatment.Combination of trastuzumab and lapatinib in xenografted micewith HER2 overexpressing cell lines showed significant inhibitionof tumor growth [87]. Dual targeting of EGFR using cetuximab andgefitinib showed synergistic effects on prevention of proliferationand induction of apoptosis in colon cancer cell lines [88]. Combi-nation of trastuzumab and lapatinib has been examined in severalclinical 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 andKNGdomainsof theextracellularpartof ROR1 have been produced.The mAbs could induce direct apoptosis or kill tumor cells throughactivation of complement or immune effector cells in vitro [28,30].

Most effective anti-ROR1 mAbs were those against the CRD andKNG domains [30]. Treatment of pancreatic cancer cell lines with

anti-ROR1 CRD mAb induced programmed cell death. Apoptosiswas preceded by dephosphorylation of ROR1, the PI3K isoform(p110), AKT and mTOR proteins indicating inactivation of thesesignaling proteins by the anti-ROR1 mAb. However, no change inthe phosphorylation levels of ERK and PKC proteins was observed.The findings might suggest that ERK and PKC signaling pathwaysare not involved in ROR1 signaling in pancreatic carcinoma celllines, but the PI3K/AKT/mTOR signaling axis through ROR1 acti-vation [79].

Anti-ROR1 mAbs could also kill melanoma cell lines [63]. Threeof four anti-ROR1 mAbsinduceda significant direct apoptosisof theESTDAB049, ESTDAB112, DFW and A375 melanoma cells as wellas by activating CDC and ADCC. Two cell lines (ESTDAB081 and094) were resistant to direct apoptosis by the mAb but not to mAbmediated CDC or ADCC. Transfection of ESTDAB081 cellswithROR1siRNA downregulated ROR1 at the mRNA and protein levels andinduced apoptosis.

Anti-ROR1 antibodies might also prevent metastasis by down-regulation of proteins involved in cell motility. ROR1 expressionwas associated with epithelialmesenchymal transition of tumorcells [73]. Treatment of breast cancer cells with anti-ROR1 mAbsdown-regulated proteins involved in metastasis. These cells losttheir ability to migrate and invade in vitro and to form metastasisin vivo [73].

Antibodies labeled with cytotoxic agents including radioiso-topes or toxins [antibody drug conjugates (ADC)] have shownclinical benefits in cancer treatment [91]. Anti-ROR1 antibodiesmay be used as a carrier of toxic compounds. A ROR1-immunotoxinconjugate (BT-1) consisting of a toxin from Pseudomonas exotoxin

(PE38) andthe variable fragments of ananti-ROR1 mAb(clone 2A2)showed a dose-dependent and selective binding to leukemic cellsfromCLL and MCLpatients. Theimmunotoxin was internalized andinduced a strong apoptosis in vitro [92]. The apoptotic effects weredue to exposure of inner-membrane phosphatidylserine to the cellsurface, changes in mitochondrial membranes and activation ofcaspase 3.

Invivoeffects of an unconjugatedanti-ROR1 mAbwere analyzedin a ROR1xTCL1 transgenic mice model crossing ROR1+ and TCL1transgenic mice [93]. These mice developed ROR1+/CD5+/B220low

leukemic B-cells with high levels of phosphorylated AKT and seemto be a relevant model for in vivo studies. Anti-ROR1 mAbs againstdistinct epitopes of ROR1 had different effects in vivo. The activityof two anti-ROR1 mAbs, D10 and 4A5 was evaluated. Treatment of

ROR1xTCL1 leukemia cells with the D10 anti-ROR1 mAb reducedthe expression of phosphorylated AKT, but not by the 4A5 mAb.Intravenous injections of D10 or 4A5 to ROR1 transgenic miceengrafted with CD5+/B220low/ROR1+ leukemia cells showed thatonly the D10 mAb was able to clear leukemic cells from the bloodand spleen [93].

A humanized anti-ROR1 mAb, cirmtuzumab (UC-961) devel-oped from the D10 anti-ROR1 mAb had a high specificity andaffinity (Kd=4 nM) for ROR1. Intravenous injection of cirm-tuzumab followed by infusion of human ROR1+CD5+B220low

murine leukemia cells from ROR1xTCL1 transgenic mice, as wellas of human ROR1+ CLL cells into the peritoneal cavity of Rag-2//c/ immunedeficientmice, induced clearanceof leukemiccells in the spleen and peritoneal cavity. Cirmtuzumab had not

only a direct killing effect of tumor cells, but was also internalized

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by malignant B cells. An ADC using this mAb showed promisingresults with enhanced cytotoxic activity against ROR1 expressingcells. Cirmtuzumab-ADC cleared ROR1 expressing CLL cells in vivoin xenografted mice and in vitrousing adenocarcinoma cell lines ofthe 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 efficiently kill CLL cells with a high speci-ficity. The best compounds killed 50 times more CLL cells thannormal blood lymphocytes. ROR1 was specifically dephosphory-lated as well as inactivated PI3K/AKT/mTOR proteins [95].

Interestingly, ROR1-TKIs killed not only treatment naive CLLcells (EC50

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