Tobias Schmidt Slørdahl

Anti-c-MET Nanobody - a new potential drug in multiple myeloma treatment.

c‐MET is the tyrosine kinase receptor of the hepatocyte growth factor (HGF). HGF‐c‐MET signaling is involved in many human malignancies, including multiple myeloma (MM). Recently, multiple agents have been developed directed to interfere at different levels in HGF‐c‐MET signaling pathway. Nanobodies® are therapeutic proteins based on the smallest functional fragments of heavy‐chain‐only antibodies. In this study, we wanted to determine the anticancer effect of a novel anti‐c‐MET Nanobody in MM.

The phosphatase of regenerating liver-3 (PRL-3) is important for IL-6-mediated survival of myeloma cells

Multiple myeloma (MM) is a neoplastic proliferation of bone marrow plasma cells. PRL-3 is a phosphatase induced by interleukin (IL)-6 and other growth factors in MM cells and promotes MM-cell migration. PRL-3 has also been identified as a marker gene for a subgroup of patients with MM. In this study we found that forced expression of PRL-3 in the MM cell line INA-6 led to increased survival of cells that were depleted of IL-6. It also caused redistribution of cells in cell cycle, with an increased number of cells in G2M-phase. Furthermore, forced PRL-3 expression significantly increased phosphorylation of Signal transducer and activator of transcription (STAT) 3 both in the presence and the absence of IL-6. Knockdown of PRL-3 with shRNA reduced survival in MM cell line INA-6. A pharmacological inhibitor of PRL-3 reduced survival in the MM cell lines INA-6, ANBL-6, IH-1, OH-2 and RPMI8226. The inhibitor also reduced survival in 9 of 9 consecutive samples of purified primary myeloma cells. Treatment with the inhibitor down-regulated the anti-apoptotic protein Mcl-1 and led to activation of the intrinsic apoptotic pathway. Inhibition of PRL-3 also reduced IL-6-induced phosphorylation of STAT3. In conclusion, our study shows that PRL-3 is an important mediator of growth factor signaling in MM cells and hence possibly a good target for treatment of MM.

Src Family Kinases Are Regulated in Multiple Myeloma Cells by Phosphatase of Regenerating Liver-3

Phosphatase of regenerating liver-3 (PTP4A3/PRL-3) is a dual-specificity phosphatase that is upregulated in various types of cancers and is related to poor prognosis and aggressive tumor behavior. The expression level of PRL-3 is elevated in response to several antiapoptotic cytokines, including IL6, in cancer cells from patients with multiple myeloma (MM) and can promote survival and migration. Here, it is demonstrated that PRL-3 activates Src kinase in the IL6-dependent MM cell line INA-6. Inhibition of PRL-3 by a small-molecule inhibitor of PRL-3 or by shRNA resulted in inactivation of Src. In addition to activation of Src, PRL-3 also activated the Src family kinase (SFK) members LYN and HCK in INA-6 cells. Forced expression of catalytically inactive mutant PRL-3 decreased the activation of these three SFK members while the total level of HCK and FYN remained elevated. Inhibitors of Src increased sensitivity of cells overexpressing PRL-3 to the PRL-3 inhibitor through joint downregulation of both PRL-3 and Mcl-1. In conclusion, PRL-3 protected MM cells against apoptosis by dysregulating both the total levels and the activation levels of specific SFK members that are important for IL6 signal transduction in MM cells. Eventually, this led to increased levels of Mcl-1. Implications: This study suggests PRL-3 and SFKs are key mediators of the IL6-driven signaling events and points to both PRL-3 and SFK members as potential targets for treatment of MM. Mol Cancer Res; 15(1); 69–77. ©2016 AACR.

Identification of the source of elevated hepatocyte growth factor levels in multiple myeloma patients

BackgroundHepatocyte growth factor (HGF) is a pleiotropic cytokine which can lead to cancer cell proliferation, migration and metastasis. In multiple myeloma (MM) patients it is an abundant component of the bone marrow. HGF levels are elevated in 50% of patients and associated with poor prognosis. Here we aim to investigate its source in myeloma.MethodsHGF mRNA levels in bone marrow core biopsies from healthy individuals and myeloma patients were quantified by real-time PCR. HGF gene expression profiling in CD138+ cells isolated from bone marrow aspirates of healthy individuals and MM patients was performed by microarray analysis. HGF protein concentrations present in peripheral blood of MM patients were measured by enzyme-linked immunosorbent assay (ELISA). Cytogenetic status of CD138+ cells was determined by fluorescence in situ hybridization (FISH) and DNA sequencing of the HGF gene promoter. HGF secretion in co-cultures of human myeloma cell lines and bone marrow stromal cells was measured by ELISA.ResultsHGF gene expression profiling in both bone marrow core biopsies and CD138+ cells showed elevated HGF mRNA levels in myeloma patients. HGF mRNA levels in biopsies and in myeloma cells correlated. Quantification of HGF protein levels in serum also correlated with HGF mRNA levels in CD138+ cells from corresponding patients. Cytogenetic analysis showed myeloma cell clones with HGF copy numbers between 1 and 3 copies. There was no correlation between HGF copy number and HGF mRNA levels. Co-cultivation of the human myeloma cell lines ANBL-6 and JJN3 with bone marrow stromal cells or the HS-5 cell line resulted in a significant increase in secreted HGF.ConclusionsWe here show that in myeloma patients HGF is primarily produced by malignant plasma cells, and that HGF production by these cells might be supported by the bone marrow microenvironment. Considering the fact that elevated HGF serum and plasma levels predict poor prognosis, these findings are of particular importance for patients harbouring a myeloma clone which produces large amounts of HGF.

Mn2+ regulates myeloma cell adhesion differently than the proadhesive cytokines HGF, IGF‐1, and SDF‐1α

Adhesion of multiple myeloma (MM) cells in the bone marrow (BM) is important for the growth and survival of the myeloma cells. Very late antigen‐4 (VLA‐4) is one of the main adhesion receptors that mediate MM cell binding to fibronectin (FN). In this study we have examined the effect of divalent cations on adhesion of MM cells to FN, and compared this type of adhesion with the adhesion induced by the cytokines HGF, IGF‐1 and SDF‐1α. Mn2+ induced adhesion in all cell lines tested. Cytokine‐ and Mn2+‐induced VLA‐4‐mediated adhesion were different in many respects, including binding specificity, adhesion kinetics and the activation state of VLA‐4. To study a potential role of divalent cations in vivo, we measured the concentrations of divalent cations in BM plasma from 14 MM patients. We also found that Mn2+‐mediated adhesion to FN activated the MAPK pathway, indicating that the interaction of MM‐cells with FN mediated by Mn2+ could play a critical role for growth and proliferation. In conclusion, this study shows a potential important role of divalent cations in MM cell biology and supports earlier studies pointing to activated VLA‐4 as a key for homing of MM cells to the BM.

Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance

Phosphatase of regenerating liver-3 (PRL-3/PTP4A3) is upregulated in multiple cancers, including BCR-ABL1- and ETV6-RUNX-positive acute lymphoblastic leukemia (ALL). With this study, we aim to characterize the biological role of PRL-3 in B cell ALL (B-ALL). Here, we demonstrate that PRL-3 expression at mRNA and protein level was higher in B-ALL cells than in normal cells, as measured by qRT-PCR or flow cytometry. Further, we demonstrate that inhibition of PRL-3 using shRNA or a small molecular inhibitor reduced cell migration towards an SDF-1α gradient in the preB-ALL cell lines Reh and MHH-CALL-4. Knockdown of PRL-3 also reduced cell adhesion towards fibronectin in Reh cells. Mechanistically, PRL-3 mediated SDF-1α stimulated calcium release, and activated focal adhesion kinase (FAK) and Src, important effectors of migration and adhesion. Finally, PRL-3 expression made Reh cells more resistance to cytarabine treatment. In conclusion, the expression level of PRL-3 was higher in B-ALL cells than in normal cells. PRL-3 promoted adhesion, migration and resistance to cytarabine. PRL-3 may represent a novel target in the treatment of B-ALL.

Expression of phosphatase of regenerating liver (PRL)-3, is independently associated with biochemical failure, clinical failure and death in prostate cancer

Background Prostate cancer (PC) stratification needs new prognostic tools to reduce overtreatment. Phosphatase of regenerating liver (PRL-3) is a phosphatase found at high levels in several cancer types, where its expression is associated with survival. A recent PC cell line study has shown it to be involved in PC growth and migration. Methods We used a monoclonal antibody to evaluate the expression of PRL-3 in PC tissue of patients in an unselected cohort of 535 prostatectomy patients. We analyzed associations between PRL-3 expression and biochemical failure-free survival (BFFS), clinical failure-free survival (CFFS) and PC death-free survival (PCDFS). Results Cytoplasmic PRL-3 staining in tumor cells was significantly correlated to expression of molecules in the VEGFR-axis, but not to the clinicopathological variables. High PRL-3 was not significantly associated with survival in the univariate analysis for BFFS (p = 0.131), but significantly associated with CFFS (p = 0.044) and PCDFS (p = 0.041). In multivariate analysis for the various end points, PRL-3 came out as an independent and significant indicator of poor survival for BFFS (HR = 1.53, CI95% 1.10–2.13, p = 0.012), CFFS (HR = 2.41, CI95% 1.17–4.98, p = 0.017) and PCDFS (HR = 3.99, CI95% 1.21–13.1, p = 0.023). Conclusions PRL-3 is independently associated with all PC endpoints in this study. Since high PRL-3 expression also correlates with poor prognosis in other cancers and functional studies in PC support these findings, PRL-3 emerges as a potential treatment target in PC.

PD1 is expressed on exhausted T cells as well as virus specific memory CD8+ T cells in the bone marrow of myeloma patients

Characterization of CD8+ T cells in the tumor microenvironment (TME) is important to predict responses to checkpoint therapy. The TME in multiple myeloma is the bone marrow, which also is an immune organ where immune responses are generated and memory cells stored. The presence of T cells with other specificities than the tumor in the bone marrow may affect the search for biomarkers to predict responses to immunotherapy in myeloma. Here, we found similar proportions of PD1+ CD8+ T cells and similar levels of PD1 expression on CD8+ T cells in the bone marrow of myeloma patients and healthy controls. PD1 expression on CD8+ T cells did not correlate with tumor load suggesting that at least some of the PD1+ CD8+ T cells were specific for non-myeloma antigens. Indeed, PD1+ EBV-specific CD8+ T cells were detected it the bone marrow of patients. Terminal effectors (Teff), effector memory (Tem) and central memory (Tcm) cells as well as exhausted T cells were all found in the myeloma bone marrow. However, myeloma patients had more terminal effectors and fewer memory cells than healthy controls suggesting that the tumor generate an immune response against myeloma cells in the bone marrow. The presence of CD8 EOMEShigh Tbetlow T cells with intermediate levels of PD1 in myeloma patients suggests that T cell types, that are known to be responsive to checkpoint therapy, are found at the tumor site.

Phosphatase of regenerating liver-3 (PRL-3) is overexpressed in classical Hodgkin lymphoma and promotes survival and migration

BackgroundPhosphatase of regenerating liver-3 (PRL-3) is implicated in oncogenesis of hematological and solid cancers. PRL-3 expression increases metastatic potential, invasiveness and is associated with poor prognosis. With this study, we aimed to show a possible oncogenic role of PRL-3 in classical Hodgkin lymphoma (cHL).MethodsPRL-3 expression was measured in 25 cHL patients by immunohistochemistry and gene expression was analyzed from microdissected malignant cells. We knocked down PRL-3 in the cHL cell lines L1236 and HDLM2 and used small molecular inhibitors against PRL-3 to investigate proliferation, migration and cytokine production.ResultsPRL-3 protein was expressed in 16% of patient samples. In three different gene expression datasets, PRL-3 was significantly overexpressed compared to normal controls. PRL-3 knockdown reduced proliferation, viability and Mcl-1 expression in L1236, but not in HDLM2 cells. Thienopyridone, a small molecule inhibitor of PRL-3, reduced proliferation of both L1236 and HDLM2. PRL-3 affected IL-13 secretion and enhanced STAT6 signaling. IL-13 stimulation partially rescued proliferation in L1236 cells after knockdown of PRL-3. PRL-3 knockdown reduced migration in both L1236 and HDLM2 cells.ConclusionPRL-3 was overexpressed in a subset of cHL patients. Inhibition of PRL-3 increased IL-13 cytokine production and reduced migration, proliferation and viability. The effects could be mediated through regulation of the anti-apoptotic molecule Mcl-1 and a feedback loop of IL-13 mediated activation of STAT6. This point to a role for PRL-3 in the pathogenesis of Hodgkin lymphoma, and PRL-3 could be a possible new drug target.

Hvordan kan strykprosenten ved eksamen stabiliseres

BACKGROUND The study programme in medicine at the Norwegian University of Science and Technology (NTNU) holds written examinations once annually. The limit to achieving a pass grade is at least 65 % correct answers. The failure rate varies from one year to the next. Our hypothesis was that the variations in the failure rate were caused by a varying degree of difficulty in the examination questions. We investigated whether relative standard-setting methods would reduce the variation in the failure rate without lowering the average limit for a pass grade. MATERIAL AND METHOD Cohen’s relative standard-setting methods correct for the degree of difficulty in the examination questions. They are easy to apply and provide an alternative to setting an absolute limit of 65 % for a pass grade. We used data from 34 examinations for medical studies at the Norwegian University of Science and Technology (NTNU) from the period 2010–2015 and compared the failure rates estimated using the existing assessment method with those produced by Cohen’s methods. RESULTS Using the existing 65 % limit for a pass grade, the failure rate varied from 0 % to 13.7 %, with a falling rate at later stages of the studies. With the exception of the examination held in the first year of study, the failure rate was lower and there was less variation in the failure rate with the original as well as the modified Cohen method when compared to the existing method. One of the Cohen methods resulted in a failure rate of 0 % to 10.4 % INTERPRETATION In our data material, an absolute limit of 65 % for a pass grade can be defended because the failure rate was generally low. Cohen’s methods could be an alternative in medical schools that have a high failure rate or where there are major variations in the failure rate from one year to the next in the same examination in the course of study.