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.

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia worldwide. Novel treatment strategies are needed to improve the prognosis. Non-malignant cells of the tumor microenvironment (TME) are of importance for the growth of tumor cells. Monocytes expressing various chemokine receptors as CX3CR1 (the fractalkine receptor (FKNR)) have been shown to promote the growth of tumor cells in vitro and in vivo. In CLL, monocyte derived nurse-like cells (NLC) support the growth of CLL cells, promoting leukemic cell proliferation and survival by secretion of chemokines/cytokines. In this study, we studied the role of autologous monocytes on the survival of CLL cells and the effects of a small molecule antagonist of CX3CR1 (KAND567/FKNRa). KAND567 is in clinical trials for diseases with an activated fractalkine system as part of the pathobiology including covid-19 infected patients as well as patients with acute myocardial infarction and ovarian carcinoma. Methods: Primary CLL cells and B cells (CD19 +) of healthy donors as well as autologous monocytes (CD14 +) from CLL patients (n=22) and healthy control donors (n=11) were cultured alone or in co-cultures (isolated B cells together with autologous monocytes) with the FKNRa (250 and 1000 nM) for up to 120 h. Apoptosis of CD19 + cells and monocytes was analysed by Annexin V/PI staining, Western blotting and the IncuCyte® Live Cell Imaging System. The NBT assay was also applied to check morphological changes of monocytes. Plasma concentration of fractalkine (CX3CL1) was determined by ELISA. Results and discussion: CLL patients had a significant (p=0.0001) increase in the plasma concentration of CX3CL1 compared to healthy controls. CLL cells as well as normal B cells did not express surface CX3CR1. However, monocytes from both CLL patients and healthy donors expressed high levels of surface CX3CR1. A significantly higher proportion of pro-inflammatory intermediate (CD14 +/CD16 +) and non-classical (CD14 -/CD16 +) monocytes (p<0.0001 and <0.01 resp.) as well as intermediate and non-classical CX3CR1 + monocytes were observed in patients with active CLL compared to healthy donors (p<0.0001 and <0.01 resp.). Moreover, CX3CR1 + intermediate and non-classical monocytes were significantly higher in CLL patients with active disease compared to those with early-stage disease (p<0.0001 and <0.001 resp.). A growth supportive effect of autologous monocytes was observed on survival of both CLL cells and healthy donor B cells in 120 h cell co-culture experiments (p<0.001 and <0.01 resp.). FKNRa reduced the number of alive CLL cells in a dose-dependent manner in co-cultures with autologous monocytes (p<0.05). No effect was seen on CD19 + B cells of healthy donors co-cultured with autologous monocytes. FKNRa did not induce apoptosis of CLL cells alone. Furthermore, FKNRa also inhibited transformation of CLL monocytes to NLC (p<0.05) which was not the case for normal monocytes. Conclusion: The CX3CR1/CX3CL1 (fractalkine receptor/ligand)) axis appears to be activated in CLLand might be of importance for the NLC-driven growth of CLL cells. This is the first report of a small molecule antagonist of CX3CR1 reducing the growth supportive effect of autologous CLL monocytes on leukemic CLL cells. Disruption of growth promotion induced by monocytes may prevent leukemic cell proliferation and survival. Inhibition of autologous monocytes in the TME might represent a new therapeutic strategy in CLL, complementary to treatment with drugs that directly target the tumor cells.

The receptor tyrosine kinase orphan receptor 1 (ROR1) is absent in most normal adult tissues but overexpressed in various malignancies and is of importance for tumor cell survival, proliferation, and metastasis. In this study, we evaluated the apoptotic effects of a novel small molecule inhibitor of ROR1 (KAN0441571C) as well as venetoclax (BCL-2 inhibitor), bendamustine, idelalisib (PI3Kδ inhibitor), everolimus (mTOR inhibitor), and ibrutinib (BTK inhibitor) alone or in combination in human MCL primary cells and cell lines. ROR1 expression was evaluated by flow cytometry and Western blot (WB). Cytotoxicity was analyzed by MTT and apoptosis by Annexin V/PI staining as well as signaling and apoptotic proteins (WB). ROR1 was expressed both in patient-derived MCL cells and human MCL cell lines. KAN0441571C alone induced significant time- and dose-dependent apoptosis of MCL cells. Apoptosis was accompanied by decreased expression of MCL-1 and BCL-2 and cleavage of PARP and caspase 3. ROR1 was dephosphorylated as well as ROR1-associated signaling pathway molecules, including the non-canonical WNT signaling pathway (PI3Kδ/AKT/mTOR). The combination of KAN0441571C and ibrutinib, venetoclax, idelalisib, everolimus, or bendamustine had a synergistic apoptotic effect and significantly prevented phosphorylation of ROR1-associated signaling molecules as compared to KAN0441571C alone. Our results suggest that targeting ROR1 by a small molecule inhibitor, KAN0441571C, should be further evaluated particularly in combination with other targeting drugs as a new therapeutic approach for MCL.

ROR1 - a receptor tyrosine kinase - is overexpressed in CLL. Ibrutinib, a Bruton's tyrosine kinase inhibitor, is clinically effective in CLL but patients may develop resistance. We evaluated the effect of an ROR1 inhibitor, KAN0441571C, in CLL cells from six patients obtained before and after developing resistance to ibrutinib. The ROR1 inhibitor induced apoptosis in ibrutinib-resistant CLL cells to the same degree as in ibrutinib-sensitive cells and dephosphorylated ROR1. This was also noted in one patient who became resistant to both ibrutinib and the Bcl-2 inhibitor venetoclax. The combination of ROR1 inhibitor and venetoclax had a synergistic apoptotic effect on ibrutinib-resistant cells.

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.

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent his tone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment.

Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition. Tissue microarrays (TMAs) and formalin-fixed paraffin-embedded (FFPE) SCLC patient samples were utilized to determine the prevalence of ROR1 and BCL2 expression in SCLC. Eight SCLC-derived cell lines were used to determine the antitumor activity of a small molecule ROR1 inhibitor (KAN0441571C) alone and in combination with the BCL2 inhibitor venetoclax. The Chou-Talalay method was utilized to determine synergy with the drug combination. ROR1 and BCL2 protein expression was identified in 93% (52/56) and 86% (48/56) of SCLC patient samples, respectively. Similarly, ROR1 and BCL2 were shown by qRT-PCR to have elevated expression in 79% (22/28) and 100% (28/28) of SCLC patient samples, respectively. KAN0441571C displayed efficacy in 8 SCLC cell lines, with an IC50 of 500 nM or less. Synergy as defined by a combination index of <1 via the Chou-Talalay method between KAN0441571C and venetoclax was demonstrated in 8 SCLC cell lines. We have shown that ROR1 inhibition is synergistic with BCL2 inhibition in SCLC models and shows promise as a novel therapeutic target in SCLC.

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.

In breast cancer, Lysine-specific demethylase-1 (LSD1) and other lysine demethylases (KDMs), such as Lysine-specific demethylase 6A also known as Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), are co-expressed and co-localize with estrogen receptors (ERs), suggesting the potential use of hybrid (epi)molecules to target histone methylation and therefore regulate/redirect hormone receptor signaling. Here, we report on the biological activity of a dual-KDM inhibitor (MC3324), obtained by coupling the chemical properties of tranylcypromine, a known LSD1 inhibitor, with the 2OG competitive moiety developed for JmjC inhibition. MC3324 displays unique features not exhibited by the single moieties and well-characterized mono-pharmacological inhibitors. Inhibiting LSD1 and UTX, MC3324 induces significant growth arrest and apoptosis in hormone-responsive breast cancer model accompanied by a robust increase in H3K4me2 and H3K27me3. MC3324 down-regulates ERα in breast cancer at both transcriptional and non-transcriptional levels, mimicking the action of a selective endocrine receptor disruptor. MC3324 alters the histone methylation of ERα-regulated promoters, thereby affecting the transcription of genes involved in cell surveillance, hormone response, and death. MC3324 reduces cell proliferation in ex vivo breast cancers, as well as in breast models with acquired resistance to endocrine therapies. Similarly, MC3324 displays tumor-selective potential in vivo, in both xenograft mice and chicken embryo models, with no toxicity and good oral efficacy. This epigenetic multi-target approach is effective and may overcome potential mechanism(s) of resistance in breast cancer.

Fractalkine (CX3CL1) is a chemokine constitutively expressed in many hematopoietic and non-hematopoietic tissues (Bazan J.F. et al., Nature, 1997). It mediates different biological activities by binding to its receptor CX3CR1, expressed on different cell types such as natural killer (NK) cells, monocytes, cytotoxic effector T cells and B cells. The CX3CL1/CX3CR1 axis is involved in the pathogenesis of several cancers including various B-cell malignancies (Ferretti E. et al.,Mediators Inflamm, 2014). CX3CR1 was found both at the mRNA and protein levels in several subtypes of lymphoma (Andreasson, U. et al.,Cancer Lett, 2008). In this study, we analysed the expression of the fractalkine receptor (CX3CR1) in peripheral blood mononuclear cells (PBMC) of chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) patients (pts) with active disease (n=22), pts with newly diagnosed diffuse large B-cell lymphoma (DLBCL) (n=21) as well as classical Hodgkin lymphoma (cHL) (n=19) and compared with healthy donors (n=14). PBMC populations were assessed by flow-cytometry including T cells, NK cells and monocytes. Statistical comparisons between groups were done using the non-parametric Mann-Whitney U test. CLL/SLL pts had a median age of 75 years with equal sex distribution. Thirteen pts had high-risk and 9 intermediate-risk disease according to the modified Rai staging system; 1/21 had 17p deletion. Median lymphocyte count was 103 x 109/L (range 1.5-312). Of the 19 cHL pts, 12 had limited (stage 1-2A) and 7 had advanced (stage 2B-4) disease. Of the 21 DLBCL pts, 10 had stage 1, 4 had stage 2, 2 had stage 3 and 5 had stage 4 disease. CLL/SLL pts had significantly higher absolute number of CD4+and CD8+ T cells compared to controls, DLBCL and cHL patients (p=0.02, p=0.0001 and p<0.0001 for CD4+ T cells and p=0.0007, p<0.0001 and p=0.0002 for CD8+ T cells, respectively). DLBCL and cHL pts had lower CD4+ numbers compared to controls (p<0.005), while no difference was observed for CD8+ cells (Fig. 1A). The absolute numbers of CD4+ and CD8+ cells expressing the CX3CR1 were also significantly higher in CLL pts compared to controls and the other patient groups (p≤0.0001 for all comparisons). No significant difference was observed between either DLBCL or cHL pts and controls (Fig. 1B). Both the CD56bright and the CD56dim NK cell populations were also studied. CLL pts were found to have a significantly higher numbers of NK cells (p=0.001) and CD56dim NK cells (p=0.0027) compared to controls, while no difference was found for the other patient groups. No difference was observed between CLL pts and controls with regard to the CD56bright NK cells, which, however, were lower in both DLBCL and cHL pts than in controls (p<0.05) (Fig. 2A). The absolute numbers of NK cells expressing CX3CR1 was also significantly higher in CLL compared to controls (p=0.002). This was observed in both the CD56dim (p=0.004) and the CD56bright (p=0.04) subpopulations. In the other patient subgroups, no significant difference was observed with the exception of CX3CR1+CD56bright NK cells, which were lower in both DLBCL and cHL pts compared to controls (Fig. 2B). The median monocyte count was not significantly different among the patient groups or controls. Monocyte sub-populations were then defined by flow-cytometry: classical (CD14highCD16low), intermediate (CD14highCD16high) and non-classical (CD14lowCD16high). The median percentage of monocytes expressing CX3CR1 was >95% in all the patient groups and did not significantly differ from controls. No significant difference was observed for the monocyte subpopulations either. In conclusion, CLL/SLL pts with active disease have significantly higher numbers of T and NK cells expressing the fractalkine receptor (CX3CR1) compared to both controls and pts with DLBCL and cHL. No difference was observed for the monocytes. CX3CR1 might be a therapeutic target in CLL/SLL. Small molecule inhibitors of this axis are in clinical development. Disclosures Palma: Takeda: Research Funding. Olin:Kancera AB: Employment, Equity Ownership. Schultz:Kancera AB: Employment, Equity Ownership. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Österborg:Kancera AB: Research Funding; Abbvie: Research Funding; Janssen: Research Funding; BeiGene: Research Funding; Gilead: Research Funding.

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

Despite the recent reductions in the global burden of malaria, this disease remains a devastating cause of death in tropical and subtropical regions. As there is no broadly effective vaccine for malaria, prevention and treatment still rely on chemotherapy. Unfortunately, emerging resistance to the gold standard artemisinin combination therapies means that new drugs with novel modes of action are urgently needed. In this context, Plasmodium histone modifying enzymes have emerged as potential drug targets, prompting us to develop and optimize compounds directed against such epigenetic targets. A panel of 51 compounds designed to target different epigenetic enzymes were screened for activity against Plasmodium falciparum parasites. Based on in vitro activity against drug susceptible and drug-resistant P. falciparum lines, selectivity index criterion and favorable pharmacokinetic properties, four compounds, one HDAC inhibitor (1) and three DNMT inhibitors (37, 43 and 45), were selected for preclinical studies in a mouse model of malaria. In vivo data showed that 37, 43 and 45 exhibited oral efficacy in the mouse model of Plasmodium berghei infection. These compounds represent promising starting points for the development of novel antimalarial drugs.

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.

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.

The sirtuins form a superfamily of evolutionarily conserved NAD+-dependent protein N-ϵ-acyl-lysine (AcK) deacylases with roles in a variety of key cellular processes. Sirtuins have a broadly conserved overall structure with a catalytic site formed by a hydrophobic channel between the NAD+-binding Rossmann fold domain and a smaller Zn2+-binding domain. Schistosomes express five members of the sirtuin family and generic sirtuin inhibitors induce apoptosis and death in schistosome larvae, the disruption of adult worm pairs, inhibition of egg laying and damage to the male and female worm reproductive systems. Sirtuins in schistosomes and other parasitic flatworms present structural differences from their human orthologues that should allow the development of selective inhibitors that can be developed as drug leads.

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.

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 is ROR receptors. ROR1 is of importance during embryogenesis but down-regulated in most adult human tissues. ROR1 has been shown to be overexpressed in hematological malignancies and solid tumors as pancreatic carcinoma. ROR1 siRNA transfection of CLL cells and pancreatic carcinoma cells induced apoptosis. Antibodies against the CRD region of ROR1 evoked specific apoptosis of CLL cells and dephosphorylation of ROR1 (PLOS One 8(10) e78339, 2013). In the present report we have explored the activity of a new class of compound, ROR1 tyrosine kinase inhibitors (ROR1-TKI), inhibiting phosphorylation of the TK domain of ROR1. Methods: ROR1 kinase inhibitorswere derived from a high-through-put screening (HTS) using HTRF assay based on recombinant intracellular kinase domain of ROR1. One chemical lead series was optimized for activity on target, physico-chemical properties and in vitro ADME properties as hERG and CYP inhibition, bioavailability and metabolic stability in plasma and hepatocytes. In the present study IN0439365 was used. Results: PaCa2 and PANC1 pancreatic carcinoma cells expressed ROR1 as determined by flow-cytometry and Western blot (WB). In both cell lines ROR1 was constitutively phosphorylated (pROR1 antibody) (WB). After 72 h of incubation with 2-3 µM IN0439365, 70% of PaCa2 cells were dead and 40% of PANC1. ROR1 TKI induced a strong dephosphorylation of ROR1 (<2h) as well as of src, PI3K, AKT, mTOR and of CREB. (As a control gemcitabine induced cell death but not dephosphorylation of ROR1.) Conclusions: The TKR ROR1 is overexpressed in pancreatic carcinoma and of importance for survival. Targeting ROR1 by a TKI induced a strong killing of the tumor cells. The drug dephosphorylated ROR1 as well as PI3K/AKT/mTOR. This pathway might be a survival signal in pancreatic cancer cells associated with an aberrant expression of ROR1. This is a first report of a novel class of drugs targeting ROR1 (ROR1-TKI). The drug candidates will be further evaluated in preclinical in vivo models before proceeding to clinical evaluation.

Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ϵ-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.