Resistance to Bruton's tyrosine kinase (BTK) and BCL2 inhibitors, such as ibrutinib (Ibru) and venetoclax (Ven), remains a major therapeutic challenge in Mantle Cell Lymphoma (MCL), which limits treatment options. The receptor tyrosine kinase ROR1, aberrantly expressed in MCL and other malignancies but largely absent in normal adult tissues, has been implicated in tumour cell proliferation, survival, and migration. This study aimed to elucidate mechanisms underlying Ibru and Ven resistance in MCL and to investigate the therapeutic potential of targeting ROR1 using the small molecule inhibitor KAN571C in Ibru and/or Ven resistant MCL cell lines. Ibru-, Ven-, and Double (Ibru+Ven)-resistant models were generated from the JEKO-1, Granta-519, and Z138 MCL cell lines. The cytotoxic effects of Ibru, Ven, and KAN571C were assessed in both naïve and drug-resistant models using MTT assays. Western blotting was employed to analyse downstream signalling alterations following exposure to KAN571C. KAN571C induced potent cytotoxicity in both naïve and drug-resistant models, preferentially in venetoclax- and ibrutinib/venetoclax-resistant cells. Mechanistically, KAN571C dephosphorylated ROR1 at tyrosine kinase (TK) and proline-rich domains (PRD), suppressed downstream PI3K/AKT and β-catenin signalling, and activated caspase-dependent apoptosis. Notably, long-term exposure (2 weeks) failed to establish KAN571C-resistant cell lines. Our findings identify ROR1 as a convergent mediator of resistance in MCL and highlight KAN571C as a potential therapeutic strategy to overcome treatment refractoriness.

Background/Objectives: Nurse-like cells (NLCs) derived from monocytes in the tumor microenvironment support the growth of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the effects of a CX3CR1 (fractalkine receptor) antagonist (KAND567) on autologous monocytes and their pro-survival effects on CLL cells in vitro. Methods: Plasma concentration of CX3CL1 was determined by ELISA and CX3CR1 expression by flow cytometry. CD19+ cells and autologous monocytes from patients with CLL and healthy donors were treated with KAND567 either in co-culture or alone. The apoptosis of CD19+ cells and monocytes was determined by Annexin V/PI staining and live-cell imaging. Results: Plasma concentration of CX3CL1 (fractalkine) was significantly higher in patients with CLL (n = 88) than in healthy donors (n = 32) (p < 0.0001), with higher levels in patients with active compared to non-active disease (p < 0.01). CX3CR1 was found on monocytes but not B cells in patients and controls. Levels of intermediate and non-classical CX3CR1+ monocytes were higher in patients with CLL than in controls (p < 0.001), particularly in those with active disease (p < 0.0001). Co-culture experiments revealed that autologous monocytes promoted the survival of both malignant and normal B cells and that KAND567 selectively inhibited the growth of CLL cells in a dose-dependent manner but only in the presence of autologous monocytes (p < 0.05). Additionally, KAND567 inhibited the transition of monocytes to NLCs in CLL (p < 0.05). Conclusions: Our data suggest that the CX3CR1/CX3CL1 axis is activated in CLL and may contribute to the NLC-driven growth-promoting effects of CLL cells. KAND567, which is in clinical trials in other disorders, should also be explored in CLL.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1(+) group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1(+) NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue-and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Receptor tyrosine kinases (RTKs) are frequently dysregulated in malignancies and important for the malignant characteristics of tumor cells. RTKs are attractive structures for drug targeting of cancer. The RTK ROR1 is of significance during embryogenesis but downregulated in post-partum tissues. However, ROR1 is overexpressed in several hematological and solid tumors and important for tumor cell proliferation, survival, migration, and metastasis. WNT5a is a main ligand for ROR1. Several clinical trials are ongoing using anti-ROR1 antibody based drugs directed against the external domain (monoclonal antibodies, BiTE, CAR-T). We have produced small molecules (KAN834/1571c) fitting to the ATP pocket of the intracellular tyrosine kinase (TK) domain of ROR1 (TK inhibitor, TKI). These inhibitors of ROR1 prevented ROR1 phosphorylation and inactivated the WNT/β-catenin independent as well as WNT/β-catenin dependent pathways. ROR1-TKI induced apoptosis of ROR1 positive fresh patient derived tumor cells and appropriate cell lines and a dose and time dependent tumor reduction in animal models. In combination with other clinically relevant targeting drugs as venetoclax a synergistic apoptotic effect was seen. Two other small molecules (ARI-1 and strictinin) bound also to ROR1 and inhibited tumor growth. Development of small molecule ROR1 inhibitors is warranted to include this novel therapeutic approach for cancer therapy.

The receptor tyrosine kinase ROR1 is absent in most normal adult tissues, but overexpressed in several malignancies. In this study, we explored clinical and functional inhibitory aspects of ROR1 in diffuse large B-cell lymphoma (DLBCL). ROR1 expression in tumor cells was more often observed in primary refractory DLBCL, Richter's syndrome and transformed follicular lymphoma than in relapsed and non-relapsed DLBCL patients (p < 0.001). A survival effect of ROR1 expression was preliminarily observed in relapsed/refractory patients independent of gender and stage but not of age, cell of origin and international prognostic index. A second generation small molecule ROR1 inhibitor (KAN0441571C) induced apoptosis of ROR1+ DLBCL cell lines, similar to venetoclax (BCL-2 inhibitor) but superior to ibrutinib (BTK inhibitor). The combination of KAN0441571C and venetoclax at EC50 concentrations induced almost complete killing of DLBCL cell lines. Apoptosis was accompanied by the downregulation of BCL-2 and MCL-1 and confirmed by the cleavage of PARP and caspases 3, 8, 9. PI3Kδ/AKT/mTOR (non-canonical Wnt pathway) as well as β-catenin and CK1δ (canonical pathway) were inactivated. In zebra fishes transplanted with a ROR1+ DLBCL cell line, KAN0441571C induced a significant tumor reduction. New drugs with mechanisms of action other than those available for DLBCL are warranted. ROR1 inhibitors might represent a novel promising approach.

Abstract Background The chemokine CX3CL1 is associated with many autoimmune diseases. Type 1 diabetes (T1D) is an autoimmune disease characterized by an insufficient insulin production due to T-cells mediated destruction of pancreatic beta cells. CX3CL1/CX3CR1 plays an important role in recruitment and extravasation of T-cells and monocytes to the inflammatory site. Aim To investigate the role of CX3CL1 and its receptor CX3CR1 in children with T1D in acute phase (day 0), 10 days and 3 months after treatment compared to healthy subjects (HS). Methods Blood samples from children (range 2–18 years) with new onset type 1 diabetes (day 0 (n=14), day 10 (n=9) and 3 month (n=14)) and 24 age matched HS was collected. The concentrations of CX3CL1 in plasma (T1D day 0, day 10 and HS) was measured by ELISA. Receptor expression of CX3CR1 on monocytes and T-cells was analyzed by flow cytometry. Results The study showed significant higher concentrations of CX3CL1 in serum from T1D day 0 compared to HS (p<0.05) and T1D day 10 (p<0.01). No significant difference was observed between T1D at day 10 and HS. Furthermore, the level of CX3CR1 positive T-cells was significantly lower in T-cells from T1D day 0 compared to HS (p<0.003) and T1D 3 months (p<0.007). In addition, the amount CX3CR1 positive monocytes were significant lower in T1D day 0 compared to HS (p<0.003). Conclusion The low amount of CX3CR1 positive T-cells and monocytes in peripheral circulation in acute phase T1D, despite the high level of s-CX3CL1, indicate that the T-cells and monocytes might have transmigrated to the inflammatory site. This study indicates that CX3CL1/CX3CR1 axis could play a role in the pathogenesis of T1D.

Crosstalk between leukemic cells and the tumor microenvironment is of importance in chronic lymphocytic leukemia (CLL). T cells seem to sustain the survival of CLL cells by various mechanisms. The Krüppel-like family of transcription factors (KLFs) are identified as regulators of proliferation and cell death. In the present study, we analyzed the expression of the wild type (WT) gene KLF6 and the oncogenic splice variant 1 (KLF6-SV1) at the mRNA level in subsets of T cells from CLL patients (n = 29), multiple myeloma patients (n = 6) and normal donors (n = 10). RNA Silencing was used for wtKLF6 and KLF6-SV1. Tumor cell apoptosis was measured. A significant overexpression of wtKLF6 and KLF6-SV1 in T cells of CLL patients compared to normal donors and myeloma patients was noted (p<0.002). Western blot showed that both wtKLF6 and KLF6-SV1 were expressed in purified T cells from CLL patients. KLF6-SV1 siRNA transfection induced a significant down-regulation of KLF6-SV1 in CLL T cells, which lost the capability to sustain the growth of leukemic cells. However, no such a significant effect was seen after wtKLF6 transfection of the autologous T cells. The results suggest that KLF6-SV1 may play a role in the regulation of survival CLL cells.

There is a great unmet medical need in pancreatic carcinoma (PC) for novel drugs with other mechanisms of action than existing. PC cells express the onco-fetal RTK ROR1, absent on most normal post-partem cells. ROR1 is involved in proliferation, survival, EMT and metastasis of tumor cells in various malignancies. A small molecule inhibitor (KAN0439834) (530 Da) targeting the TK domain of ROR1 was developed and the activity in ROR1 expressing human PC cell lines (n = 8) evaluated. The effects were compared to a murine mAb against the external part of ROR1, gemcitabine, erlotinib and ibrutinib. KAN0439834 induced significant apoptosis of the tumor cells. EC50 values for KAN0439834 varied between 250-650 nM depending on the cell line. The corresponding values for erlotinib and ibrutinib were 10-40 folds higher. KAN0439834 was much more effective in inducing tumor cell death than the ROR1 mAb although both inhibited ROR1 phosphorylation and downstream non-canonical Wnt pathway molecules. Combination of KAN0439834 with erlotinib or ibrutinib had significant additive effects on tumor cell death. A first-in-class small molecule ROR1 inhibitor (KAN0439834) showed promising in vitro activity against a number of human PC cell lines. Interesting is the additive effects of erlotinib and ibrutinib which warrants further studies as both these agents are in clinical trials for pancreatic carcinoma.

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200μmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT.

Chronic kidney disease (CKD) is a major risk factor for cardiovascular disease (CVD), partly due to endothelial dysfunction and chronic inflammation. Vitamin D treatment in end stage renal disease is suggested to modulate the immune system and lead to improved outcomes. We and others have demonstrated that treatment with vitamin D or activated vitamin D analogues protects the endothelial function in less severe renal disease as well. Since the endothelial protection might be mediated by vitamin D effects on inflammation, we assessed levels of pro-inflammatory cytokines and micro RNAs (miRs) in patients with moderate CKD, treated with an active vitamin D analogue (paricalcitol).

Diabetes mellitus is characterized by hyperglycemia and capillary hypoxia that causes excessive production of free radicals and impaired antioxidant defense, resulting in oxidative stress and diabetes complications such as impaired wound healing. We have previously shown that modified forms of tocotrienols possess beneficial effects on the biosynthesis of the mevalonate pathway lipids including increase in mitochondrial CoQ. The aim of this study is to investigate the effects of mono-epoxy-tocotrienol-α on in vitro and in vivo wound healing models as well as its effects on mitochondrial function. Gene profiling analysis and gene expression studies on HepG2 cells and human dermal fibroblasts were performed by microarray and qPCR, respectively. In vitro wound healing using human fibroblasts was studied by scratch assay and in vitro angiogenesis using human dermal microvascular endothelial cells was studied by the tube formation assay. In vivo wound healing was performed in the diabetic db/db mouse model. For the study of mitochondrial functions and oxygen consumption rate Seahorse XF-24 was employed. In vitro, significant increase in wound closure and cell migration (p<0.05) both in normal and high glucose and in endothelial tube formation (angiogenesis) (p<0.005) were observed. Microarray profiling analysis showed a 20-fold increase of KIF26A gene expression and 11-fold decrease of lanosterol synthase expression. Expression analysis by qPCR showed significant increase of the growth factors VEGFA and PDGFB. The epoxidated compound induced a significantly higher basal and reserve mitochondrial capacity in both HDF and HepG2 cells. Additionally, in vivo wound healing in db/db mice, demonstrated a small but significant enhancement on wound healing upon local application of the compound compared to treatment with vehicle alone. Mono-epoxy-tocotrienol-α seems to possess beneficial effects on wound healing by increasing the expression of genes involved in cell growth, motility and angiogenes as well as on mitochondrial function.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL) in adults, accounts for approximately 30-40% of newly diagnosed lymphomas worldwide. Environmental factors, such as viruses and bacteria, may contribute to cancer development through chronic inflammation and the integration of oncogenes, and have previously been indicated in cervical cancer, hepatocellular carcinoma, gastric cancer and lymphoproliferative disorders. In the present study, the presence of microbial agents was analyzed in the lymphoma tissue of patients with activated B-cell like (ABC) DLBCL. The present study compared two groups of patients from geographically varied regions that possess a difference in the prevalence of viral and other microbial agents. The patient populations were from Sweden (a low endemic infectious disease region) and Egypt (a high endemic infectious disease region). A differential expression of several viruses in lymphoma tissues was noted when comparing Swedish and Egyptian patients. JC polyomavirus (JCV) was detected in Swedish and Egyptian patients and, uniquely, the complete hepatitis B virus (HBV) genome was detected only in Egyptian lymphoma patients. None of these viruses were detected in control lymph tissues from Sweden or Egypt. In total, 38% of the Egyptian patients were found to have HBV surface antigens (HBsAgs) in their serum; however, HBsAgs were not found in any of the Swedish patients. The percentage of serum HBsAgs in Egyptian patients with ABC DLBCL was significantly increased compared with the general Egyptian population (P<0.05). The present study may support a notion that viral agents, including JCV and HBV, may be involved in the tumorigenesis of DLBCL in regions of high infectious disease.

Dishevelled (DVL) proteins are components of the Wnt signalling pathways, and increased expression is associated with various malignancies. Information on DVLs in chronic lymphatic leukaemia (CLL) is limited. The aim of the present study was to investigate the role of DVLs in CLL cells and association with Wnt pathways downstream of ROR1. DVL1, 2 and 3 were exclusively expressed in CLL cells as compared to normal peripheral blood mononuclear cells (PBMCs). The expression of DVL1 and DVL3 proteins was significantly more pronounced in progressive than in non-progressive disease (p < 0.01), whereas the level of DVL2 was significantly higher in non-progressive as compared to progressive disease (p < 0.001). Treatment of CLL cells with anti-ROR1 specific monoclonal antibodies induced dephosphorylation of ROR1 as well as of tyrosine and serine residues of both DVL2 and DVL3. However, gene silencing of DVLs in the CLL cell line (EHEB) did not induce detectable apoptosis. Non-progressive CLL patients had a different protein activity pattern with regard to Wnt signalling pathway proteins as GSK-3β, β-catenin and AKT as compared to progressive disease. The DVL2 protein may play a role in the activation of signalling pathways in CLL during early stages of the disease, while DVL1 and 3 may have a role in later phases of the leukaemia.

The receptor tyrosine kinase (RTK) ROR1 is of importance during embryogenesis for the central nervous, musco-skeletal and cardio-pulmonary systems. ROR1 is practically not present in normal adult tissues. ROR1 is however re-expressed in several hematological and non-hematological malignancies and involved in tumor cell proliferation, survival, invasiveness and metastases. The unique characteristics of this onco-fetal RTK should make it a suitable candidate for targeted therapy. A chemical synthesis program has been initiated based on a chemical library and phenotypic screening programme. Small molecules have been produced which specifically target the phosphorylated TK domain of ROR1. Tumor cell death in vitro is measured by Annexin V/PI and MTT. Effects on signaling molecules by WB for phosphorylated and non-phosphorylated proteins. Xenotransplanted non-scid mice and zebra fishes are used for in vivo testing of anti-tumor effects. Phosphorylated ROR1 is expressed in e.g. CLL, triple negative breast cancer, pancreatic and lung cancer cells. A lead compound has been synthesized, KAN 0439834. In biochemical assay IC50 is 5 nM. The compound kills specifically with high efficiency chronic lymphocytic leukemia cells, triple negative breast cancer cells, pancreatic carcinoma cells, lung cancer cells. The ligands for ROR1 is Wnt5a and 3a which both are expressed in tumor cells. The drug specifically dephosphorylated ROR1 as well as downstream Wnt canonical (e.g. GSK3&bgr;) and non-canonical (e.g. PI3K, AKT, mTOR) molecules. In non-scid mice transplanted with human CLL cells a significant reduction of CLL cells was noted as well as inhibition in the zebra fish model of triple negative breast cancer cells of tumor growth and metastases. KAN 0439834 is well tolerated in the mice. ROR1 is an activated onco-fetal RTK expressed in various malignancies. Small chemical molecules specifically targeting ROR1 could be shown in vitro and in vivo preclinical models to specifically and efficiently kill tumor cells at a low concentration. The oral available KAN 0439834 is the first generation of a ROR1 TKI with promising characteristic to be further developed for first-in-man clinical trials.

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.

8556 Background: RTK families are attractive therapeutic candidates. One of the families is the ROR receptors. ROR1 is of importance during embryogenesis but down-regulated in most adult human tissues.ROR1 has been shown to be expressed in CLL as well as in other hematological malignancies and solid tumors and is constitutively phosphorylated. ROR1 siRNA transfection of CLL cells induced apoptosis. We have produced and explored the activity of a new class of compounds, acting as inhibitors of phosphorylation of the tyrosine kinase domain of ROR1 (ROR1-TKI). We describe for the first time the activity of a lead ROR-TKI, KAN0439834. Methods: ROR1 inhibitors were derived from a high-through-put screen (HTS) using HTRF assay based on recombinant intracellular kinase domain of ROR1. A chemical lead series was optimized for activity on target, in vitro ADME and in vivo pharmacokinetic properties. Results: KAN0439834 induced apoptosis in vitro of fresh CLL cells from patients with non-progressive and progressive disease as well as from patients with fludarabine resistant disease with and without 17p abnormalities (EC50 for CLL cells < 200 nM and for normal PBMC > 14 µM). In vitro incubation resulted in dephosphorylated ROR1, Src, PIKδ, AKT, mTOR and CREB. 6 h incubation was sufficient to induce full apoptosis at 24 h. In NOD-SCID mice xenografted with human CLL cells orally bioavailable KAN0439834 induced a significant reduction of ROR1+cells, dephosphorylated pROR1, downregulated Mcl-1 and upregulated cleaved PARP. PK analyses showed a blood concentration of KAN0439834 to be sufficient to induce apoptosis of CLL cells. Minimal animal toxicity could be noted. Conclusions: The RTK ROR1 is expressed in CLL cells and of importance for survival. Targeting ROR1 by a TKI induced a strong killing of leukemic cells in vitro. The drug dephosphorylated ROR1 as well as PI3K/AKT/mTOR. In an animal CLL model the lead ROR1 inhibitor compound significantly reduced xenotransplanted CLL cells with minimal animal toxicity. This is a first report of a novel class of candidate drugs targeting ROR1. The candidate drug will be further evaluated in preclinical in vivo efficacy and safety models before proceeding to clinical evaluation.