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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 also be an interesting oncofetal antigen for active immunotherapy. In this 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.

We have previously demonstrated that ROR1 and FMOD (fibromodulin) are two genes upregulated in chronic lymphocytic leukaemia (CLL) cells compared to normal blood B cells. In this study, siRNAs were used to specifically silence ROR1 and FMOD expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75-95%) in FMOD and ROR1 mRNA. Western blot analysis with specific antibodies for FMOD and ROR1 demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in statistically significant (P ≤ 0·05-0·001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with FMOD and ROR1 siRNA did not undergo apoptosis. This is the first report demonstrating that ROR1 and FMOD may be involved in the survival of CLL cells. ROR1 in particular is further explored as potential target for therapy in CLL.

Gene profiling studies of patients with chronic lymphocytic leukemia (CLL) has revealed increased expression of Ror1, a cell surface receptor tyrosine kinase. The aim of present study was to analyze gene and protein expression of Ror1 in CLL cells and normal blood leukocytes. Gene expression analysis reverse transcriptionpolymerase chain reaction of ROR1 revealed that all patients with CLL (n 5 100) spontaneously expressed ROR1 mRNA whereas enriched blood B and T cells as well as granulocytes from healthy donors (n 5 10) were negative. A strong nonphysiological activation signal (PMA/ionomycin) was required to induce expression in vitro in normal lymphocytes. Major genomic aberrations (mutations or truncation) of ROR1 were not observed. Protein expression was analyzed by Western blot using a panel of polyclonal anti-Ror antibodies as well as flow cytometry. Blood lymphocytes from 18/18 CLL patients, but none of the 10 healthy donors, expressed surface Ror1. The majority of CLL cells exhibited Ror1 surface expression (71% mean; range 36–92%) with a mean fluorescence intensity (MFI) of 20 (range 10–45). The corresponding MFI of CD19 on CLL cells was 26 (range 9–48). There was no difference in the Ror1 protein expression comparing IgVH mutated and unmutated cases as well as progressive and nonprogressive CLL patients. Two different variants of the Ror1 protein, 105 and 130 kDa, were identified. The Ror1 protein expression in patients with CLL but not in normal leukocytes merits further studies of its role in the pathobiology of CLL, which may provide a basis for development of Ror1 directed targeted therapy. ' 2008 Wiley-Liss, Inc.

Purpose: Erythropoietin (EPO) and EPO receptor (EPO-R) expression have been reported in solid tumors and are claimed to regulate tumor growth; however, no data have been published on this issue in B-cell malignancies or normal lymphoid cells. This report describes genomic/protein EPO-R expression and in vitro effects of recombinant human EPO (epoetin) in B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), and multiple myeloma (MM).Experimental Design: Blood samples were obtained from patients with B-CLL, MCL, and healthy volunteers, and bone marrow was obtained from MM patients. EPO-R mRNA was detected by reverse transcription-PCR. EPO-R surface expression was investigated by flow cytometry using digoxigenin-labeled epoetin and polyclonal rabbit anti–EPO-R antibody for intracellular receptor. Tumor cell stimulation was determined in vitro using [3H]thymidine incorporation and CD69 expression after exposure to epoetin α or β or darbepoetin α.Results: EPO-R mRNA was detected in mononuclear cells from 32 of 41 (78%) B-CLL and 5 of 7 (71%) MCL patients, and 21 of 21 (100%) MM samples. Expression was also detected in highly purified T cells from six of eight B-CLL patients, four of four MM patients, and normal donor B and T cells. Surface EPO-R protein was not detected. Intracellular EPO-R staining with anti–EPO-R antibodies was unspecific. No tumor-stimulatory effect was observed with high epoetin concentrations.Conclusions: EPO-R gene is frequently expressed in lymphoid malignancies and normal B and T cells. However, there was no surface protein expression and no epoetin-induced in vitro stimulation of tumor B cells, indicating that epoetin therapy in vivo is likely to be safe in patients with lymphoid malignancies.

Breast cancer is globally the most common malignancy in women. Her2-targeted monoclonal antibodies are established treatment modalities, and vaccines are in late-stage clinical testing in patients with breast cancer and known to promote tumour-killing through mechanisms like antibody-dependent cellular cytotoxicity. It is therefore increasingly important to study immunological consequences of conventional treatment strategies. In this study, functional tests and four-colour flow cytometry were used to detect natural killer (NK)-cell functions and receptors as well as T-cell signal transduction molecules and intracellular cytokines in preoperative breast cancer patients, and patients who had received adjuvant radiotherapy or adjuvant combined chemo-radiotherapy as well as in age-matched healthy controls. The absolute number of NK cells, the density of NK receptors as well as in vitro quantitation of functional NK cytotoxicity were significantly higher in preoperative patients than the post-treatments group and controls. A similar pattern was seen with regard to T-cell signalling molecules, and preoperative patients produced significantly higher amounts of cytokines in NK and T cells compared to other groups. The results indicate that functions of NK and T cells are well preserved before surgery but decrease following adjuvant therapy, which may speak in favour of early rather than late use of immunotherapeutic agents such as trastuzumab that may depend on intact immune effector functions.

We have previously demonstrated that ROR1 and FMOD (fibromodulin) are two genes upregulated in chronic lymphocytic leukaemia (CLL) cells compared to normal blood B cells. In this study, siRNAs were used to specifically silence ROR1 and FMOD expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75-95%) in FMOD and ROR1 mRNA. Western blot analysis with specific antibodies for FMOD and ROR1 demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in statistically significant (P ≤ 0·05-0·001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with FMOD and ROR1 siRNA did not undergo apoptosis. This is the first report demonstrating that ROR1 and FMOD may be involved in the survival of CLL cells. ROR1 in particular is further explored as potential target for therapy in CLL.

Fibromodulin is an extracellular matrix protein normally produced by collagen-rich tissues; the fibromodulin gene has been found to be the most overexpressed gene in B-cell chronic lymphocytic leukemia. In this study, fibromodulin was expressed at the gene level (reverse transcription-polymerase chain reaction [RT-PCR]) in all patients with B-CLL (n = 75) and in most (5 of 7) patients with mantle cell lymphoma (MCL). No mutations in the fibromodulin gene were detected. Fibromodulin was also detected at the protein level in the cytoplasm of the B-CLL cells and in the supernatant after in vitro cultivation, but not at the cell surface. Fibromodulin was not found in patients with T-cell chronic lymphocytic leukemia (T-CLL), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL), hairy cell leukemia, follicular lymphoma, lymphoplasmacytic lymphoma, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), or chronic myelogenous leukemia (CML) or in 36 hematologic cell lines. Normal blood mononuclear cells (T and B lymphocytes, monocytes), tonsil B cells, and granulocytes did not express fibromodulin. Activation (phorbol 12-myristate 13-acetate [PMA]/ionomycin) of normal T and B lymphocytes induced weak fibromodulin gene expression, but not to the extent seen in freshly isolated B-CLL cells. The reason for the exclusive ectopic expression of fibromodulin in B-CLL and MCL is unknown. However, its unique protein expression makes it likely that fibromodulin is involved in the pathobiology of B-CLL and MCL. (Blood. 2005;105:4828-4835)

Background: The PI3-kinase/AKT/mTOR pathway is a frequently dysregulated pathway in cancer and of importance for tumor cell growth, differentiation and survival. This pathway may be activated in various cancers due to aberrant activation of receptor tyrosine kinases (RTKs). The PI3-kinase/AKTmediated activation of mTOR, stimulates cell proliferation and regulation of protein translation. In pancreatic cancer, PI3-kinase and AKT are constitutively activated. ROR1 is a type I transmembrane RTK belonging to one of the twenty families of RTKs. ROR1 is overexpressed and constitutively phosphorylated in pancreatic carcinoma. A unique anti-ROR1 mAb directed against the CRD (cysteine-rich domain) of the extracellular region of ROR1 was capable of inducing apoptosis as well as dephosphorylating the ROR1 molecule in CLL cells. Aim: To investigate the apoptotic effect of the anti-ROR1 CRD mAb and effects on the downstream signaling pathways PI3-kinase/AKT/mTOR using the ROR1+ pancreatic cancer cell line PaCa-2. Methods: The MTT assay and Annexin V/PI methods were used to detect apoptosis in a 24-96 hr assay. Cell lysates were prepared from antibody treated and untreated samples and subjected to Western blot analysis for identification of the signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. The total and phosphorylated levels of the following signaling proteins were analysed: ROR1, p-ROR1, PI3-kinase, p-PI3-kinase, AKT, p-AKT, mTOR, p-mTOR, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 30 min-2 h. Results: ROR1 was expressed on the surface of 40-45% of the pancreatic carcinoma cell line PaCa-2. ROR1 was constitutively phosphorylated. The frequency of apoptotic cells after incubation with the anti-ROR1 mAb ranged from 10% at 24 hr to 40% at 96 hr. Western blot analysis showed decreased levels of phosphorylated ROR1 and the PI3-kinaseδ isoform (p110δ) in treated compared to untreated samples. Phosphorylation intensity of AKT and mTOR also decreased in treated compared to untreated cells. There was no change in the phosphorylation levels of ERK and PKC proteins. Conclusion: Incubation of pancreatic cancer cells with an anti-ROR1 CRD mAb induced apoptosis. Apoptosis was preceded by dephosphorylation of ROR1, PI3-kinaseδ isoform (p110δ), AKT and mTOR proteins indicating deactivation of these signaling proteins by the anti-ROR1 mAb. Activation of mTOR molecule was related to activation of the PI3-kinase/AKT pathway. This pathway might be a survival signal in pancreatic cancer cells associated with an aberrant expression of ROR1. Further studies are warranted to better understand the signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs, which might be a potential therapeutic option. Citation Format: Amir Hossein Daneshmanesh, Mohammad Hojjat-Farsangi, Ali Moshfegh, Eva Mikaelsson, Abdul Salam Khan, Anders Österborg, Håkan Mellstedt. Anti-ROR1 monoclonal antibodies induce apoptosis in pancreatic cancer cells via the PI3-kinase/AKT/mTOR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4770. doi:10.1158/1538-7445.AM2014-4770

e13561 Background: There is a medical need to develop novel compounds for treatment of pancreatic carcinoma. Members of the TKR families are attractive therapeutic candidates. One of the families i...

Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.

Abstract 694 Background: Small leucine rich proteoglycans (SLRPs) are a family of glycosylated proteins normally expressed in the extracelluar matrix (ECM) of collagen rich tissues. The biological role of the SLRPs is multifactoral, but mostly these proteins are secreted and bind to membrane receptors or ECM proteins affecting cell proliferation and cell migration. Some SLRPs (eg. Decorin, Lumican) have been reported to be expressed in cancer, but the expression as well as the glycosylation pattern differ. Microarray studies have revealed that the SLRP family member fibromodulin (FMOD) gene is overexpressed in chronic lymphocytic leukemia (CLL). We later reported the specific expression of FMOD in CLL and mantle cell lymphoma (MCL). FMOD is located on chromosome 1q32 adjacent to two other members of the SLRP family, proline/arginine-rich end leucine-rich repeat protein (PRELP) and opticin (OPTC). Aims: To analyse 1) the expression of PRELP and OPTC in CLL and other hematological malignancies. 2) the glycosylation pattern of PRELP and OPTC, and cellular localization of OPTC in CLL cells. Methods: PRELP: Gene expression was tested by RT-PCR and realtime-PCR. Protein expression was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the PRELP protein. OPTC: Cell fractionation was performed using four different methods. Protein expression (molecular weight and cellular location) was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the OPTC protein. Results: PRELP was expressed at the gene level (RT-PCR) in all CLL patients tested (n=30) and in 3/5 patients with mantle cell lymphoma, but not in normal leukocytes (n=10) or in other hematological malignancies (7 different types, n=35). The PRELP protein was detected in all CLL samples tested but not in leukocytes of healthy donors (western blot). Molecular analysis of the CLL PRELP protein revealed a unique unglycosylated 38 kDa core protein, with an intact signal peptide. This protein was not detected in serum that, in combination with the uncleaved signal peptide, suggests cellular retention. A “normal” OPTC protein (50 kDa) was detected in cell lysates of both CLL tumor cells (n=30) and normal leukocytes (n=10). However, the CLL cells also expressed a 37 kDa OPTC that was not detected in healthy controls. This 37 kDa OPTC was detected in all CLL samples and molecular analysis revealed a unique unglycosylated core protein that was located in the cell nucleus and endoplasmatic reticulum of the CLL cells. Conclusion: The unique expression of the SLRPs PRELP and OPTC in CLL (in addition to the previously reported FMOD) is unexpected and merits further studies since the specific expression of three closely related SLRP genes may indicate a role in the pathobiology of the disease. Disclosures: No relevant conflicts of interest to declare.

Background The biological effects of erythropoietin (EPO), the principal regulator of erythropoiesis, may extend to several non-hematopoietic tissues and cells. Recent studies suggest that EPO and erythropoietin receptors (EPO-R) may be expressed by solid tumors including breast, prostate, ovarian, and renal cancers. Studies also suggest that the EPO/EPO-R pathway might regulate survival and growth of cancer cells. Since recombinant human EPO (Epoetin) is frequently used to treat anemia in hematological malignancies, we studied the EPO-R expression as well as the in vitro effects of epoetin on tumor cells from patients with B-cell chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM). Methods The genomic expression of EPO-R was analyzed in highly purified tumor cells by RT-PCR. Flow cytometry was applied to detect EPO-R protein expression on the cell surface (using EPO-dig + anti-dig FITC, F. Hoffmann-La Roche, Basel, Switzerland) and intracellularly (in permeabilized cells, using Santa Cruz polyclonal anti-EPO-R antibody). The functional capability of epoetin alfa, epoetin beta, and darbepoetin alfa to activate tumor cells in vitro (alone, and in combination with CD40L-transfected fibroblasts as co-stimulators) was assessed by proliferation assay (DNA synthesis) and by flow cytometry (CD69). Results EPO-R mRNA was detected in peripheral blood mononuclear cells in 32/41 (78%) B-CLL and 5/7 (71%) MCL patients. The positive results were confirmed in highly purified (>95%) tumor cells from 7 B-CLL patients. In MM patients, bone marrow mononuclear cells from 13/14 (93%) patients and highly purified (>98%) plasma cells from 3/3 (100%) patients were positive for EPO-R mRNA. However, surface EPO-R protein could not be detected in any of 9 tested B-CLL patients (5 of which were positive and 4 negative for EPO-R mRNA). In contrast, intracellular EPO-R protein was detected in 8/8 (100%) PCR-positive patients with B-CLL including 5 patients who had a negative surface EPO-R staining. Intracellular EPO-R protein was also found in 4/4 (100%) tested patients with MCL. CD69 expression and [3H]-thymidine incorporation were assessed in 8 patients with B-CLL and 4 patients with MCL after stimulation of tumor cells with epoetin. CD69 expression was not induced and no epoetin-induced proliferation was observed following in vitro stimulation with any of the 3 epoetin preparations. Conclusion The results indicate that EPO-R mRNA is expressed by tumor cells in most patients with B-CLL, MCL or MM. However, it is unclear whether the gene expression translates into a fully functional protein. EPO-R protein could be detected intracellularly in most samples but not on the cell surface (as assessed by different flow cytometry techniques). Furthermore, in vitro tumor cell stimulation even with high concentrations of epoetin did not induce proliferation/activation.

Development of targeted therapies against B-CLL is dependent on the identification of molecules that are essential for the proliferation and survival of the leukemic cells. One such molecule investigated by us as a putative leukemia-associated target is Fibromodulin (FIM). FIM is an extracellular matrix molecule belonging to the leucin-rich proteoglycan family. Our laboratory studies have verified that expression of FIM is upregulated in B-CLL cells. Moreover, this molecule is specifically overexpressed in B-CLL cells and not on normal peripheral blood mononuclear cells. Analysis of FIM expression on various other hematological tumor cells have revealed that with the exception of mantle cell lymphoma, FIM is not expressed in other hematological malignancies. RNAi technology has recently emerged as a powerful method to specifically silence the expression of a gene. We have generated three siRNA against various segments of FIM and tested the effects of these siRNA on B-CLL cells. Transfection of B-CLL cells with these siRNA significantly diminished, or completely abrogated the expression of FIM mRNA as detected by RT-PCR. The figure below shows silencing of the FIM gene following siRNA transfection, as assayed by RT-PCR. “Untrans” and “ctrl” represents untransfected and control siRNA-transfected B-CLL cells respectively. As noted, transfection with each of the three siRNA against FIM completely abrogated the expression of FIM siRNA. 24–48 hours after siRNA transfection, a substantial fraction (30–70%) of the B-CLL cells, compared to cells transfected with control non-silencing siRNA, went into apoptosis as assayed by Annexin-V-propidium iodide staining. Time kinetic studies revealed that siRNA mediated silencing and apoptosis occurred between 18 and 48 hours. No such effect was noted when peripheral blood mononuclear cells of healthy donors or FIM-positive fibroblast cell lines were transfected with siRNA. In reconstitution experiments, siRNA treated B-CLL cells were co-cultured with various numbers of FIM-positive fibroblasts. The presence of these fibroblasts greatly diminished the apoptosis of B-CLL cells induced after siRNA treatment. Our results indicate that overexpression of FIM in B-CLL cells are critical for their survival and FIM may serve as a leukemia-specific target for B-CLL therapy. Figure Figure

Background Opticin (OPTC) is a member of the small leucine-rich proteoglycan (SLRP) family and is localized particularly in certain extracellular matrices. We have previously reported the unique expression of another SLRP, fibromodulin (FMOD) in the leukemic cells of patients with chronic lymphocytic leukemia (CLL). OPTC is located in the same region as FMOD on chromosome 1 (1q32.1). Cluster up-regulation of genes may be observed in malignancies and the aim of the present study was to analyze the expression of OPTC in CLL cells. Methods The expression of OPTC was tested by RT-PCR and realtime qPCR in PBMC from CLL patients, other hematological malignancies and healthy controls. The presence of OPTC protein, and its subcellular localization, was investigated using fractionation methods where the obtained lysate fractions were analyzed by Western blotting. Deglycosylation experiments were performed to investigate the glycosylation status of the CLL OPTC. Results OPTC was expressed at the gene level in all patients with CLL (n = 90) and in 2/8 patients with mantle cell lymphoma (MCL) but not in blood mononuclear cells of healthy control donors (n = 20) or in tumor samples from nine other types of hematological malignancies. OPTC was detected by Western blot in all CLL samples analyzed (n = 30) but not in normal leukocytes (n = 10). Further analysis revealed a CLL-unique unglycosylated 37 kDa core protein that was found to be located preferentially in the cell nucleus and endoplasmic reticulum (ER) of the CLL cells. Conclusions A 37 kDa unglycosylated OPTC protein was detected in ER and in the nucleus of CLL cells and not in healthy control donors. The function of this OPTC core protein remains unclear but its CLL-specific expression and subcellular localization warrants further investigations in the pathobiology of CLL.