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Background: ROR1 - a receptor tyrosine kinase (RTK) - is essential for normal embryonic development, but is absent on most normal adult tissues. ROR1 is of importance of cell proliferation, differentiation, survival and metabolism. However, ROR1 is overexpressed in several types of cancer (onco-fetal RTK). MCL is an aggressive and incurable non-Hodgkin lymphoma characterized by translocation (11;14) (q13;q32) and cyclin D overexpression. ROR1 has been described to be highly expressed in MCL cells. We have previously presented results on a small molecule ROR1 inhibitor in CLL (KAN0439834) (Leukemia 32(10):2291, 2018). A second generation ROR1 inhibitor, KAN0441571C, has been developed with improved killing of tumor cells and with longer half-life time (>10h) (PK studies in mice/rats/dogs) compared to KAN0439834. Aim: In this study we examined effects of the ROR1 small molecule inhibitor KAN0441571C in human MCL cells (Granta-519, Jeko-1, JVM-2, Z138, Mino) as part of a pre-clinical evaluation. Methods: ROR1 expression was evaluated by flow cytometry and WB. Cytotoxicity was analysed by MTT and apoptosis by Annexin V/P staining and Western Blot for apoptotic proteins. Cytotoxicity (MTT) was also analysed by combining the ROR1 inhibitor with ibrutinib, acalabrutinib, venetoclax and bendamustine. Effects of KAN0441571C on ROR1 inactivation (dephosphorylation) and signaling pathways were evaluated by WB. Results: All five cell-lines expressed phosphorylated ROR1 (130 kDa) with a varying intensity. Surface expression (flow-cytometry) varied from 0% (JVM-2) to 100% (Mino and JeKo-1). The data indicate expression of also splice variants lacking the extracellular domain. KAN0441571C induced time and dose dependent apoptosis of the five MCL cell-lines which was p53 independent. EC50 varied between 100-250 nM (24h). Apoptosis was confirmed by cleavage of caspase 3 and PARP as well as down-regulation of the MCL-1 and BCL-2 proteins. Moreover, ROR1 was dephosphorylated by KAN0441571C. Downstream of ROR1 both the Wnt canonical and non-canonical pathways were inactivated depending on the cell line. KAN0441571C had in most cell lines a similar cytotoxic effect as ibrutinib, acalabrutinib and venetoclax while bendamustine was inferior. KAN0441571C had an additive effect to ibrutinib, acalabrutinib and venetoclax respectively and KAN0441571C in combination with either of these three agents induced a complete killing of the cell lines. Conclusions: KAN0441571C is a second generation of a novel class of ROR1-tyrosine kinase inhibitor. This small molecule was effective in inducing apoptosis of MCL cells with other mechanisms of action than for drugs in clinical use for MCL. Combination of KAN0441571C with other MCL targeting drugs induced a complete killing of the tumor cell population in a preclinical in vitro model. Our results support the further development of ROR1 small molecule inhibitors as a new therapeutic principle in MCL as well as in other B-cell malignancies with an additive effect to existing targeted therapeutics. Disclosures Schultz: Kancera AB: Employment, Equity Ownership. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment, Equity Ownership. Österborg:Janssen: Research Funding; Kancera AB: Research Funding; BeiGene: Research Funding; Abbvie: Research Funding; Gilead: Research Funding.

Background: Receptor tyrosine kinase (RTK) (ROR1) is normally expressed during embryogenesis but absent in most normal tissues. However, ROR1 is overexpressed in several cancers (onco-fetal RTK) and of importance for various tumor cell functions such as proliferation and survival. In patients with diffuse large B-cell lymphomas (DLBCL) there is a great medical need to develop new treatment alternatives for those not responding to primary treatment as well as for patients with relapse as effective treatments are warranted. Inhibition of ROR1 by a small molecule ROR1 inhibitor (KAN0439834) abrogated downstream kinase activities as well as induced apoptosis of various tumor cells as CLL and pancreatic carcinoma (Leukemia, Oct;32(10):2291-2295, 2018) (PLoS One. 13(6): e0198038, 2018). A 2nd generation of ROR1 inhibitor (KAN0441571C) has been synthesized with the aim to bind to the ROR1-TK domain and inhibit ROR1 signaling. Aim: To examine the expression of ROR1 in DLBCL cell lines (RC-KB, SUDHL4, MS, OCL-LY3, U2932) and in patients´ samples at different stages of DLBCL as well as effects of KAN0441571C on survival of DLBCL cells and ROR1 signaling. Methods: Flow cytometry, tissue microarray and immunohistochemistry assays were used to check ROR1 expression. MTT and Annexin V/PI assays were applied to analyse cytotoxicity and apoptosis of KAN0441571C alone or in combination with ibrutinib (BTK inhibitor) and venetoclax (BCL-2 inhibitor) on DLBCL cell lines. Western blot was performed to evaluate ROR1 phosphorylation and associated signaling pathways. DLBCL cells were also cultured with HS-5 stromal cells (ROR1 neg.) to evaluate the apoptosis inhibitory effects of stromal cells. Results: ROR1 expression was significantly more frequently noted in patients with advanced disease (Richter´s, transformation, transformed follicular lymphoma and refractory DLBCL) compared to less advanced disease (recurrent or de novo DLBCL) (p=0.0001). In primary refractory and relapsing DLBCL 5-years survival was 45% in ROR1- patients (n=17) while in ROR1+ patients (n=16) the corresponding figure was KAN0441571C induced a dose-dependent cytotoxic effect in all ROR1+ DLBCL cell lines (EC50=50-100 nM) while no effect could be noted in the ROR1- U2932 cell line (EC50>10000 nM). EC50 for venetoclax in the ROR1+ DLBCL cell lines varied between 100 and 500 and 5000 - 10000 nM for ibrutinib. In comparison to venetoclax, KAN0441571C induced a similar or significantly higher cytotoxic effect. KAN0441571C and venetoclax seemed to be the most promising drug combination approaching 100% killing at the EC50 dose for each drug. Apoptosis was confirmed by Annexin V/PI staining as well as by downregulation of BCL-2 and MCL-1 as well as cleavage of PARP and caspase 3. KAN0441571C dephosphorylated ROR1 as well as the co-receptor LRP6 and the SRC protein which binds to phosphorylated ROR1. The downstream molecules PI3Kδ/AKT/mTOR was also dephosphorylated and the transcription factor CREB. CK1δ and GSK3B were also dephosphorylated and β-catenin downregulated indicating involvement of both the non-canonical and canonical Wnt pathways. When DLBCL and HS-5 cells (ROR1 neg.) were co-cultured, HS-5 cells could partially prevent induction of apoptosis of DLBCL cells at low concentrations of KAN0441571C, while at higher concentrations the presence of stromal cells was less effective. Zebrafish embryos transplanted with the OCI-Ly3 cell line were treated for 3 days with KAN0441571C (25-1000 nM). No toxic effects of the drug could be noted. A significant dose and time-dependent decrease in the tumor area were noted. Conclusion: KAN0441571C is the 2nd generation of a novel class of ROR1-inhibiting small molecule drugs. The molecule was more effective in inducing apoptosis of DCBCL cells than venetoclax or ibrutinib. New anti-cancer drugs with other mechanisms of action than those clinically available for DLBCL are warranted to improve the prognosis. ROR1 inhibitors in combination with other targeted drugs as venetoclax and ibrutinib might improve the therapeutic effects. KAN0441571C may be a novel drug candidate which needs further exploration in DLBCL. Disclosures Lehto: Kancera AB: Employment. Vågberg:Kancera AB: Employment. Olsson:Kancera AB: Employment. Löfberg:Kancera AB: Employment. Norström:Kancera AB: Employment. Schultz:Kancera AB: Employment, Equity Ownership. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment, Equity Ownership. Österborg:Kancera AB: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Gilead: Research Funding; BeiGene: Research Funding. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: The receptor tyrosine kinase (RTK) ROR1 is detected during embryogenesis but downregulated in adult normal tissues. However, it is expressed in several solid tumors and hematological malignancies. Targeting ROR1 with specific siRNAs in chronic lymphocytic leukemia (CLL) induced apoptosis of the leukemic cells. Moreover, ROR1 specific monoclonal antibodies (mAbs) dephosphorylated ROR1 followed by apoptosis of the CLL cells. Furthermore, ROR1 tyrosine kinase inhibitors (ROR1-TKI) (small molecule inhibitors) have been shown to dephosphorylate ROR1, downregulate the activated PI3K/AKT/mTOR signaling pathway and induce specific apoptosis of CLL cells. Aim: In the present report we analysed the effects of a ROR1-TKI drug candidate (KAN0439834) on other intracellular signaling pathways involved in cell survival, differentiation and migration in addition to the PI3K/AKT/mTOR pathway in CLL cells. Methods: KAN0439834 ROR1-TKI was derived from a high-throughput screening (HTS) and a chemical synthesis program, including cellular assays for CLL specific cytotoxicity. The compound was also tested for ADME and in vivo pharmacokinetics characteristics. Peripheral blood mononuclear cells (PBMC) were derived from patients with CLL and normal healthy donors. Intracellular signaling molecules were analysed by Western blot (WB) after 30 min incubation of the cells with the ROR1-TKI (50-1000 nM). Apoptosis/necrosis was determined by the MTT cytotoxicity assay and Annexin V/PI staining in flowcytometry after 24 h of incubation. Results: CLL cells expressed ROR1 as determined by WB and flowcytometry. ROR1 was shown to be phosphorylated using a polyclonal anti-phospho-ROR1 (pROR1) antibody (WB). After 30 min of incubation with 50-1000 nM of KAN0439834, ROR1 was dephosphorylated in a dose-dependent manner. KAN0439834 also dephosphorylated LRP6, GSK3β, JNK, MAPK/ERK/p42,44, PKC, Src, and c-Jun and decreased the β-catenin concentration as well as deactivated BCL-2 and Bax proteins. KAN0439834 had no effect on Bruton tyrosine kinase (Btk) phosphorylation involved in B-cell receptor (BCR) signaling. Incubation of CLL cells with KAN0439834 (50-1000 nM) showed a dose dependent induction of apoptosis/necrosis of leukemic cells with more than 80% specific killing of CLL cells after 24 h and an IC50 value of 250 nM. Conclusions: Our data show that KAN0439834 downregulated the activity of various signaling pathways in CLL cells suggested to be connected with ROR1 signaling, including the Wnt-canonical associated molecule as LRP6, GSK3β and β-catenin as well as several Wnt non-canonical associated proteins as Src, MAPK/ERK p42,44, JNK, and PKC and inactivation of c-Jun that was followed by apoptosis of the CLL cells in a dose dependent manner. Further studies are ongoing to study the effects of the ROR1 specific TKIs on ROR1 downstream signaling as well as in preclinical in vivo animal models using human fresh tumors and cell lines to evaluate the anti-tumor effects. Available data suggest a specific ROR1-mediated cytotoxic effect of KAN0439834 on CLL cells, which represents a first-in-class of a novel CLL drug candidate targeting ROR1. Disclosures Moshfegh: Kancera AB: Employment. Vågberg:Kancera AB: Employment. Styrbjörn:Kancera AB: Employment. Schultz:Kancera AB: Employment. Olsson:Kancera AB: Employment. Löfberg:Kancera AB: Employment. Norström:Kancera AB: Employment. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment, Equity Ownership. Österborg:Janssen Cilag: Research Funding.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for several malignant and non-malignant diseases. Busulphan (Bu) is an alkylating agent that is used in high doses as a part of the conditioning regime prior to HSCT. Busulphan is metabolized mainly in the liver by conjugation with glutathione (GSH) by glutathione transferases (GST), in particular, GSTA1. Busulphan interactions with drugs metabolized by hepatic enzymes other than GSH/GST have been reported. The aim of the present study was to investigate the role of other enzymes than GSH/GST in the busulphan metabolism including FMO3 and cytochrome P450 enzymes (CYPs). In our in vitro studies, human liver microsomes were incubated with busulphan first core metabolite, tetrahydrothiophene (THT). The incubation results showed a rapid disappearance of THT and gradual formation of the other metabolites. THT incubation with recombinant enzymes showed FMO3 to give the highest Initial THT disappearance rate (v0, 6.87 µmol/min/mL) and the highest intrinsic clearance(CLint, 0.26 µL/min/mg protein) followed by other CYPs. Comparing CYPs based on the initial THT disappearance rate (v0) per POR/CYP ratio, CYP2C8 had the highest rate for THT metabolism (5.03 nmol/min/(POR/CYP ratio) followed by CYPs 2C9, 2C19, 2E1 and 3A4. These results showed that THT is metabolized by microsomal enzymes and FMO3 is the main enzyme. For in vivo studies, we determined the kinetics of Bu in mice in the presence or absence of phenylthiourea (PTU), an inhibitor of FMO3. Moreover, the effect of THT accumulation on Bu metabolism was also determined in the same species. Treatment with PTU resulted in increase of Bu plasma concentrations as expressed as AUC. Furthermore, synchronized treatment of mice with THT and Bu also enhanced the plasma levels of Bu as compared to animals receiving Bu alone. After THT dosing, THT concentrations and AUC significantly (P < 0.05) increased after concurrent PTU injection. THT concentrations and AUC for mice treated with Bu and PTU also significantly (P < 0.05) increased compared to mice injected with Bu alone. To evaluate the role of FMO3 in the kinetics of Bu in the clinical settings, we investigated twelve patients undergoing HSCT and conditioned with busulphan / cyclophosphamide. A significant up-regulation (P < 0.05) of mRNA was found for FMO3 after Bu conditioning as confirmed using qRT-PCR. The up-regulation observed for FMO3 was similar to what was observed for GSTA1 (P < 0.05). To further confirm the involvement of FMO3 on in vivo Bu kinetics in humans, blood samples were drawn routinely from a patient treated with high dose busulphan and concomitant administration of voriconazole. Voriconazole is an antimycotic drug is wieldy used in hematology and HSCT and is known to be metabolized via FMO3. High busulphan levels that were not relevant to the administered dose were detected in this patient. Measuring THT in the samples showed that THT levels detectable with high accumulation and slow oxidation rate. In conclusion, our results showed that FMO3 and, to a lesser extent, CYPs are involved in busulphan metabolism through metabolism of its first core metabolite, THT. FMO3 is up regulated during Bu conditioning. FMO3 inhibition affects Bu kinetics. Our findings may offer valuable explanations into several drug interactions involving busulphan during HSCT conditioning therapy and thus, lower treatment related toxicity reported in HSCT. Disclosures No relevant conflicts of interest to declare.

In this study we aimed to identify biomarkers predictive of clinical response in acute myeloid leukemia (AML). For this purpose mRNA was isolated from diagnostic samples from 42 AML patients (“training cohort”) and subjected to Affymetrix® gene expression analysis. All patients entered complete remission (CR) after high-dose induction chemotherapy, reaching a median CR duration of 161 (range 12-3701) days. Samples from patients with “short CR duration” (6 months n=18), respectively, were pooled and compared. Gene expression analyses revealed 383 genes to be up-regulated and 610 genes down-regulated more than two fold in samples from patients with short, as compared to those with long CR duration. Ten genes were found to be up-regulated >30 times, with the runt-related transcription factor 1; translocated to 1 (cyclin D-related) (RUNX1T1) gene showing the highest differential expression (116-fold), while annexin 1 (ANXA1) was the most down-regulated gene (58-fold). Significantly higher transcript levels of RUNX1T1 were confirmed in the poor outcome group when performing quantitative real time polymerase chain reaction (qRT-PCR) on individual samples (n=20, p Our training cohort data were validated in silico extracting information from an independent study by Metzeler et al, publicly available from Oncomine® (www.oncomine.org) and encompassing diagnostic samples from 162 AML patients. Among genes differentially and similarly regulated in poor responders in both the training and validation cohorts we observed TCF3, chemokine (C-X-C motif) ligand 3 (CXCL3), Zinc finger, MIZ-type containing 1 (ZMIZ1) (up-regulated) and Peroxiredoxin 2 (PRDX2) (down-regulated). Analyses of clinical outcome revealed that AML patients with a high ZMIZ1 expression had a significantly decreased overall survival (OS) as compared to that of patients with a low ZMIZ1 expression (p In conclusion, we report clear differences in gene expression in diagnostic samples from AML patients with subsequent poor vs. better long-term clinical outcome to therapy, thus to constitute possible novel predictive biomarkers for response. In our training set RUNX1T1 was the most differentially expressed gene, while ZMIZ1 was upregulated in both the training and validation sets and significantly associated with survival. Further, functional studies of differentially expressed genes in clinical subsets of AML patients appear warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract 1769 Background: Phosphoinositide 3-kinase (PI3K)/AKT cascade regulates cell survival, proliferation and differentiation in a variety of cells. In CLL cells PI3K pathway is constitutively activated leading to AKT activation and phosphorylation of cAMP response element-binding protein (CREB). CREB is a transcription factor overexpressed and constitutively phosphorylated in a variety of cancers and seems to have a role in tumor pathobiology. There is a great need to develop novel strategies for targeted therapy in CLL. Monoclonal antibodies (mAbs) specifically targeting leukemic cells might be a rewarding approach. ROR1 is a type I transmembrane receptor tyrosine kinase belonging to one of the twenty families of receptor tyrosine kinases (RTKs). ROR1 is overexpressed on CLL cells but not in white blood cells of healthy donors. ROR1 is constitutively phosphorylated in CLL and siRNA transfection induced apoptosis. We have developed a unique anti-ROR1 mAb directed against CRD (cysteine-rich domain) of the extracellular region of ROR1 capable of inducing direct apoptosis of primary CLL cells. Our anti-CRD mAb induced dephosphorylation of the ROR1 molecule. Aims: To study the apoptotic effect of an anti-ROR1 CRD mAb and effects on downstream signaling pathways involved in CLL, specially the PI3-kinase/AKT/CREB pathway using primary CLL cells. Methods: Using a peptide-based mouse mAb generation method we produced several mAbs against the three extracellular domains of ROR1. In the current study we used one of the best anti-ROR1 antibodies, an anti-CRD mAb raised against the CRD region of ROR1 (Daneshmanesh et al., Leukemia. 2012 Jun;26(6):1348-55). Flow cytometry was used for surface staining of ROR1. Primary CLL cells were incubated with the anti-ROR1 CRD mAb and apoptosis was detected by the MTT assay and Annexin V/propidium iodide (flow cytometry) methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, CREB, p-CREB, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-2 h. Results: ROR1 surface expression was detected on 80–85% of the CLL cells. The frequency of apoptotic cells induced by the anti-CRD mAb was in the range of 45–50% which is in accordance with our previous reports (see above). Time kinetics experiments using anti-ROR1 CRD mAb incubated with primary CLL cells revealed dephosphorylation of ROR1 downstream signaling molecules. We analysed the following molecules known to be involved in CLL: PKC, PI3-kinase and ERK1/2. After co-culturing CLL cells with the anti-ROR1 CRD mAb, Western blot analysis showed decreased level of phosphorylated AKT in treated compared to untreated samples. No changes in the phosphorylation levels of ERK1/2 and PKC proteins were seen. Furthermore, we analysed the PI3-kinase protein which is upstream of AKT, and noticed that in CLL cells treated with the anti-ROR1 CRD mAb, the phosphorylation intensity of PI3-kinase p85 isoform has decreased but not p55 isoforrn. Moreover, we also studied the CREB phosphorylation in treated and untreated CLL samples and detected dephosphorylation of CREB in treated as compared to untreated samples. Conclusion: Incubation of CLL cells with an anti-ROR1 CRD mAb induced apoptosis of primary CLL cells. Apoptosis was preceded by dephosphorylation within 2 h of PI3-kinase, AKT and CREB proteins indicating deactivation of these signaling proteins by the anti-ROR1 mab. In untreated CLL cells no effect on phosphorylation of these proteins was noted. Furthermore our ROR1 mAb did not dephosphorylate PKC or ERK. Our data may suggest that activation of CREB molecule might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. The constitutive phosphorylation of PKC and ERK1/2 seen in CLL might not be related to the overexpression of ROR1. Further studies are warranted for a better understanding of signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4583 Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world and is characterized by progressive accumulation of CD5+, CD19+, and CD23+ B cells in the blood, bone marrow, and secondary lymphoid organs. The progressive accumulation of malignant B cells is partly due to defective apoptosis. An important constitutively activated signaling pathway in CLL cells might be the Wnt signaling cascades that include the Wnt/b-catenin pathway and the non-canonical Wnt pathway. These pathways might be activated downstream of the constitutively phosphorylated ROR1. Dishevelled family proteins (Dvl1, Dvl2, and Dvl3) are important cytoplasmic mediators of Wnt signaling and have recently been shown to be expressed in many cancer types. The expression and precise roles of individual Dvl proteins are however not clear. The aim of the study was to characterize the expression of Dvl1, Dvl2 and Dvl3 proteins in progressive and non-progressive CLL and compare to normal white blood cells (PBMC) and to assess the expression of other important proteins of the canonical pathway as b-Catenin and GSK-3β as well as the non-canonical pathways as GSK-3α, PKC, ERK and AKT respectively. Leukemic cells from progressive (n=9) and non-progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. Dvl 1, Dvl 2, Dvl 3 proteins were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3α, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Immunoprecipitation was performed to check the phosphorylation status of Dvl proteins. Overexpression of Dvl1, Dvl2, and Dvl3 was detected in all progressive and non-progressive CLL patients and the six cell lines. There was a significantly higher expression of Dvl1 and Dvl3 in progressive compared to non-progressive CLL patients (p In summary Dvl1, Dvl2 and Dvl3 are highly upregulated in CLL patients and CLL cell lines but the expression in normal PBMC is almost negligible. Dvl1, Dvl2 and Dvl3 may have a key role in the transduction of Wnt mediated signals in CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3885 Background: Receptor tyrosine kinases (RTK) play crucial roles for different normal cellular processes as cell proliferation/differentiation, apoptosis and survival, as well as for the malignant phenotype of many types of cancer. ROR1, as a member of twenty RTKs families, has important functions during normal embryogenesis. ROR1 has shown to be overexpressed in chronic lymphocytic leukemia (CLL), mantle cell lymphoma and other hematological malignancies, as well as in solid tumors. ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. In this study, we investigated the effects of an anti-ROR1 mAb against the extracellular CRD domain for specific dephosphorylation at the tyrosine kinase domain of ROR1 in CLL cells. The CRD domain contains a frizzle receptor, which is considered to be the ligand-binding region for e.g. Wnt5a. Wnt5a has been suggested to stimulate growth of CLL cells. Aims: To investigate phosphorylation of tyrosine and serine residues, within the tyrosine kinase domain of ROR1, of importance for survival of CLL cells. Methods: Bioinformatic analysis of the ROR1 structure revealed that three amino acid residues in the tyrosine kinase domain might be critically phosphorylated. Based on this prediction, a 19 amino acid long peptide, phosphorylated at two tyrosine (tyrosine 641, 646) and one serine (serine 652) residues was designed and used for immunization of rabbits. An anti-phospho-ROR1 (pROR1) polyclonal antibody (pAb) with high titers of anti-pROR1 pAb was purified, using phospho-peptide affinity chromatography. The specificity of anti-pROR1 pAb was determined by ELISA, immunoprecipitation (IP) and western blot experiments. An anti-ROR1 mAb (IgG) (CRD 1D8 clone) was used to analyse the effects on ROR1 phosphorylation in CLL cells at tyrosine 641, 646 and serine 652 residues preceding apoptosis. ROR1 phosphorylation was investigated by western blot and IP of ROR1 probed with anti-pROR1 pAb, from untreated and CLL cells treated with the anti-CRD 1D8 mAb. Quantitative intracellular staining of ROR1 by flowcytometry in time kinetics experiment after treatment with anti-CRD 1D8 mAb was also used to check phosphorylation of ROR1. Annexin V/PI staining (flowcytometry), MTT assay, PARP and caspase 8 cleavage as well as MCL-1 protein (western blot) were used for detection of apoptosis. To investigate phosphorylation and localization of 64–130 kDa ROR1 isoforms in various compartments of CLL cells, lysates were prepared from the nucleus and cytoplasmic proteins of CLL cells. Results: Two tyrosine (641, 646) residues and one serine (652) residue of the tyrosine kinase domain were phosphorylated in CLL cells. As previously described (Mellstedt et al, Abstract No: 1771, 53th ASH annals meeting, 2011), the 64, 105 and 130 kDa ROR1 isoforms were shown to be constitutively phosphorylated at tyrosine and serine residues in CLL leukemic cells. Treatment of CLL cells with an anti-ROR1 mAb against the CRD domain induced rapid dephosphorylation of ROR1 at tyrosine 641, 646 and serine 652 residues within 20 min and gradually increased up to 4 hours. The phosphorylated 64 kDa ROR1 isoform was localized to the nucleus of CLL cells and probably represents an intracellular part of ROR1, while the ROR1 130 kDa isoform was presented both in cytoplasm and nucleus of CLL cells. Conclusion: Our data show that the ROR1 molecule is phosphorylated at tyrosine 641, 646 and serine 652 residues. The presence of 64 and 130 kDa ROR1 isoforms in the nucleus of CLL cells may suggest a role of these isoforms as transcription factors. Collectively, the data might suggest that phosphorylated ROR1 may be an important protein for the growth of CLL cells as well as an interesting structure to target in a therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Abstract 667 Background: Targeted tyrosine kinase inhibitor (TKI) therapy efficiently induces rapid hematologic and cytogenetic responses in most CML patients. In vitro studies have suggested that CML stem cells are resistant to TKIs and therefore the treatment may need to be life-long. However, the in vivo effects of TKIs on the leukemic stem cell pool in a patient population have not been prospectively assessed. In addition, the biological impact and prognostic value of leukemic stem cell burden at diagnosis is unknown. Aim: To analyze the proportions of Ph+ cells in the stem cell compartment in newly diagnosed CP CML patients at diagnosis, and correlate the initial leukemic stem cell burden to biological variables and hematological toxicity during first 3 months of TKI therapy. Patients and Methods: 42 newly diagnosed CP CML patients within the Nordic countries were randomized to receive either dasatinib 100 mg (n=21) or imatinib 400 mg (n=21) once daily. Stem cell assays were performed at diagnosis and at 1, 3, and 6 months from start of TKI therapy. After pre-selection of CD34+ cells from large volume bone marrow (BM) aspirates with paramagnetic beads, the CD34+ cells were fractionated into CD38 positive and negative pools using a sorting flow cytometer. The proportion of Ph+ cells in the stem cell fractions was assayed by counting 1000 cells with interphase FISH for BCR-ABL1. Results: Measurement of Ph+ stem cells was feasible in most patients at diagnosis and results from 36 evaluable patients will be presented. The median volume of BM aspirate was 28, 37, 40 and 36 ml at diagnosis, 1, 3, and 6 months after therapy start, respectively. The median yield of BM mononuclear cells was 1000, 110, 93 and 79 ×106, respectively. The median proportion of Ph+ cells was significantly lower in the more primitive CD34+CD38neg fraction when compared to the CD34+CD38+ fraction or to unfractionated BM (79%, range 0.6–100%; 96%, 50–100%; and 96%, 57–100%, respectively, p=0.0001). The proportion of Ph+ cells in the CD34+CD38neg fraction at diagnosis correlated with high leukocyte count (r=0.59, p Conclusions: The proportion of Ph+ stem cells at the time of diagnosis varied from 1 to 100% between individual CML patients. It was correlated with hemoglobin concentration, leukocyte count, blast percentage and spleen size at diagnosis and with hematological toxicity during early course of treatment, mirroring paucity of healthy hematopoietic stem cell reservoir. The size of the leukemic stem cell pool at diagnosis may be a powerful prognostic marker and a major biological determinant for the high Sokal risk group. The effect of TKI therapy on the malignant stem cell pool size and correlation to therapy responses will be evaluated when all patients have reached the primary study endpoint of 6 months. Disclosures: Mustjoki: BMS, Novartis: Honoraria. Richter:BMS, Novartis: Consultancy, Honoraria. Simonsson:BMS, Novartis: Consultancy, Honoraria, Research Funding. Porkka:BMS, Novartis: Consultancy, Honoraria, Research Funding. Hjorth-Hansen:BMS, Novartis: Consultancy, Honoraria, Research Funding.

Abstract 3466 Poster Board III-354 Although high dose chemotherapy induces complete remission (CR) in a majority of treated acute myeloid leukemia (AML) patients, most of these will develop relapse within 1-2 years. Improved predictive biomarkers for response and response duration are clearly needed. We have studied peripheral blood cell samples from 58 AML patients (median age 63 yrs, range 18-86) with the intention to relate RNA and protein expression patterns to clinical outcome. The samples were obtained at diagnosis prior to therapy and stored in our biobank. All patients were treated with anthracycline and cytarabine based induction regimens and all entered CR. Median CR duration was 223 days (range 12 - 3701). The patients were stratified into two groups depending on their CR duration: > 6 months (“long”, n=33) and < 6 months (“short”, n=25). In an initial analysis of protein extracts derived from the diagnostic peripheral blood cells, using chip-based high throughput proteomics (SELDI-MS), we were able to identify a collection of 21 protein/peptide peaks significantly associated with remission duration (Forshed, J Proteome Res 2008, 7, 2332). We have now proceeded to perform global genomic profiling, using gene array (Affymetrix, Santa Clara, CA, USA), followed by pathway analysis (Ingenuity System Inc, USA) to elucidate differences in signaling networks between the two, clinically different cohorts. In cells from patients with short CR duration, as compared to those with long duration, gene array analysis revealed 10 genes to be up-regulated >30 times, which was confirmed by qPCR. Among the most up-regulated genes were: 1. RUNX1T1 (part of the Runt-related transcription factor family of genes, also called core binding factor-α), up-regulated 116 fold, known to be activated in AML including the M2 subtype, chromosomal translocations involving this gene are well documented in AML. 2. TKTL1 (transketolase 1), up-regulated 53 fold, overexpression reported in various human cancers and claimed to predict a poor patient's survival. 3. U2AF1 (U2 small nuclear RNA auxiliary factor 1), up-regulated 40 fold, reported to play a critical role in both constitutive and enhancer-dependent splicing by mediating protein-protein interactions and protein-RNA interactions. Conversely, among the 10 most down-regulated (by at least 23 fold) genes in cells from patients with short, as compared to long, CR were: 1. ANXA1 (annexin 1), -58 fold, has an inhibitory effect on phospholipase A2 which is required for the biosynthesis of potent mediators of inflammation, such as prostaglandins and leukotrienes. Induction of ANXA1 expression has been proposed as a mechanism of action for the treatment of AML with HDAC inhibitors. 2. FLRT3 (member of the fibronectin leucine rich transmembrane protein family), -48 fold, has a function in cell adhesion and receptor signaling. 3. TLR8 (Toll-like receptor 8), -23 fold, plays a fundamental role in pathogen recognition and activation of innate immunity and promotes NF-kappa-B activation, cytokine secretion and inflammatory response. Synthetic agonists of TLR8 are already in anti-cancer trials as immune response enhancers and TLR8 was recently shown to increase the immunostimulatory capacity also of AML cells. Further data on protein expression of deregulated genes from Western blot and immunophenotypical characterization of cytospin preparations are pending and will be presented. In addition, data on other genes in these signaling networks will be examined and reported. Observed major protein aberrations will be validated in a larger patient cohort available at our center. In summary, comparing AML patient cohorts with short vs. long CR duration our data demonstrate major differences in the RNA-expression of genes known to be involved in important regulatory events in normal and leukemic hematopoiesis. Disclosures No relevant conflicts of interest to declare.