The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells.

BACKGROUND: Multiple myeloma (MM) is an incurable malignancy of plasma cells. The serine protease matriptase is frequently dysregulated in human carcinomas, which facilitates tumor progression and metastatic dissemination. The importance of matriptase in hematological malignancies is yet to be clarified. In this study, we aimed to characterize the role of matriptase in MM. MATERIALS AND METHODS: mRNA expression of matriptase and its inhibitors hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 was studied in primary MM cells from patient samples and human myeloma cell lines (HMCLs). We further investigated the effect of matriptase on migration and proliferation of myeloma cells in vitro. By use of the CoMMpass database, we assessed the clinical relevance of matriptase in MM patients. RESULTS: Matriptase was expressed in 96% of patient samples and all HMCLs tested. Overexpression of matriptase in vitro reduced proliferation, and significantly decreased cytokine-induced migration. Conversely, matriptase knockdown significantly enhanced migration. Mechanistically, overexpression of matriptase inhibited activation of Src kinase. CONCLUSIONS: Our findings may suggest a novel role of matriptase as a tumor suppressor in MM pathogenesis.

Paired miRNA- and messenger RNA-sequencing identifies novel miRNA-mRNA interactions in multiple myeloma.

Multiple myeloma (MM) is an incurable cancer of terminally differentiated plasma cells that proliferate in the bone marrow. miRNAs are promising biomarkers for risk stratification in MM and several miRNAs are shown to have a function in disease pathogenesis. However, to date, surprisingly few miRNA-mRNA interactions have been described for and functionally validated in MM. In this study, we performed miRNA-seq and mRNA-seq on CD138 + cells isolated from bone marrow aspirates of 86 MM patients to identify novel interactions between sRNAs and mRNAs. We detected 9.8% significantly correlated miRNA-mRNA pairs of which 5.17% were positively correlated and 4.65% were negatively correlated. We found that miRNA-mRNA pairs that were predicted by in silico target-prediction algorithms were more negatively correlated than non-target pairs, indicating functional miRNA targeting and that correlation between miRNAs and mRNAs from patients can be used to identify miRNA-targets. mRNAs for negatively correlated miRNA-mRNA target pairs were associated with gene ontology terms such as autophagy, protein degradation and endoplasmic stress response, reflecting important processes in MM. Targets for two specific miRNAs, miR-125b-5p and miR-365b-3p, were functionally validated in MM cell line transfection experiments followed by RNA-sequencing and qPCR. In summary, we identified functional miRNA-mRNA target pairs by correlating miRNA and mRNA data from primary MM cells. We identified several target pairs that are of potential interest for further studies. The data presented here may serve as a hypothesis-generating knowledge base for other researchers in the miRNA/MM field. We also provide an interactive web application that can be used to exploit the miRNA-target interactions as well as clinical parameters associated to these target-pairs.

Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells.

Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo. High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism.

TAK1-inhibitors are cytotoxic for multiple myeloma cells alone and in combination with melphalan.

Multiple myeloma (MM) is an incurable cancer caused by malignant transformation of plasma cells. Transforming growth factor-ß activated kinase 1 (MAP3K7, TAK1) is a major regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. Both NF-κB and MAPK control expression of genes with vital roles for drug resistance in MM. TAK1 is an attractive drug target as it switches these survival pathways to cell death. Our analysis showed that patients with high MAP3K7 expression in the tumor had shorter overall and progression free survival. The TAK1-inhibitors NG25 and 5Z-7-oxozeaenol (5Z-7) were cytotoxic to MM cell lines and patient cells. NG25 reduced expression of MYC and E2F controlled genes, involved in tumor cell growth, cell cycle progression and drug resilience. TAK1 can be activated by genotoxic stress. NG25 and 5Z-7 induced both synergistic and additive cytotoxicity in combination with the alkylating agent melphalan. Melphalan activated TAK1, NF-κB, and the MAPKs p38 and c-Jun N-terminal kinase (JNK), as well as a transcriptional UV-response. This was blocked by NG25, and instead apoptosis was activated. MM induce elevated bone-degradation resulting in myeloma bone disease (MBD), which is the main cause of disability and morbidity in MM patients. NG25 and 5Z-7 reduced differentiation and viability of human bone degrading osteoclasts, suggesting that TAK1-inhibition can have a double beneficial effect for patients. In sum, TAK1 is a promising drug target for MM treatment.

Examining the educational impact of the mini-CEX: a randomised controlled study.

BACKGROUND: The purpose of this study is to evaluate the mini-Clinical Evaluation Exercise (mini-CEX) as a formative assessment tool among undergraduate medical students, in terms of student perceptions, effects on direct observation and feedback, and educational impact. METHODS: Cluster randomised study of 38 fifth-year medical students during a 16-week clinical placement. Hospitals were randomised to provide a minimum of 8 mini-CEXs per student (intervention arm) or continue with ad-hoc feedback (control arm). After finishing their clinical placement, students completed an Objective Structured Clinical Examination (OSCE), a written test and a survey. RESULTS: All participants in the intervention group completed the pre-planned number of assessments, and 60% found them to be useful during their clinical placement. Overall, there were no statistically significant differences between groups in reported quantity or quality of direct observation and feedback. Observed mean scores were marginally higher on the OSCE and written test in the intervention group, but not statistically significant. CONCLUSIONS: There is considerable potential in assessing medical students during clinical placements and routine practice, but the educational impact of formative assessments remains mostly unknown. This study contributes with a robust study design, and may serve as a basis for future research.

Phosphatase of regenerating liver-3 regulates cancer cell metabolism in multiple myeloma.

Cancer cells often depend on microenvironment signals from molecules such as cytokines for proliferation and metabolic adaptations. PRL-3, a cytokine-induced oncogenic phosphatase, is highly expressed in multiple myeloma cells and associated with poor outcome in this cancer. We studied whether PRL-3 influences metabolism. Cells transduced to express PRL-3 had higher aerobic glycolytic rate, oxidative phosphorylation, and ATP production than the control cells. PRL-3 promoted glucose uptake and lactate excretion, enhanced the levels of proteins regulating glycolysis and enzymes in the serine/glycine synthesis pathway, a side branch of glycolysis. Moreover, mRNAs for these proteins correlated with PRL-3 expression in primary patient myeloma cells. Glycine decarboxylase (GLDC) was the most significantly induced metabolism gene. Forced GLDC downregulation partly counteracted PRL-3-induced aerobic glycolysis, indicating GLDC involvement in a PRL-3-driven Warburg effect. AMPK, HIF-1α, and c-Myc, important metabolic regulators in cancer cells, were not mediators of PRL-3's metabolic effects. A phosphatase-dead PRL-3 mutant, C104S, promoted many of the metabolic changes induced by wild-type PRL-3, arguing that important metabolic effects of PRL-3 are independent of its phosphatase activity. Through this study, PRL-3 emerges as one of the key mediators of metabolic adaptations in multiple myeloma.

Targeting phosphoglycerate dehydrogenase in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a hematological malignancy characterized by the clonal expansion of plasma cells in the bone marrow. To date, this disease is still incurable and novel therapeutic approaches are required. Phosphoglycerate dehydrogenase (PHGDH) is the first and rate-limiting enzyme in the de novo serine synthesis pathway, and it has been attributed to bortezomib-resistance in MM. METHODS: Two different PHGDH inhibitors, CBR5884 and NCT-503, were tested against human myeloma cell lines, primary MM cells from patients, and peripheral blood mononuclear cells isolated from healthy donors. The PHGDH inhibitors were then tested in combination with proteasome inhibitors in different MM cell lines, including proteasome-resistant cell lines. Furthermore, we confirmed the effects of PHGDH inhibition through knocking down PHGDH and the effect of NCT-503 in vivo in the 5T33MM mouse model. RESULTS: All the tested myeloma cell lines expressed PHGDH and were sensitive to doses of NCT-503 that were tolerated by peripheral blood mononuclear cells isolated from healthy donors. Upon testing bortezomib in combination with NCT-503, we noticed a clear synergy in several HMCLs. The sensitivity to bortezomib also increased after PHGDH knockdown, mimicking the effect of NCT-503 treatment. Interestingly, targeting PHGDH reduced the intracellular redox capacity of the cells. Furthermore, combination treatment with NCT-503 and bortezomib exhibited a therapeutic advantage in vivo. CONCLUSIONS: Our study shows the therapeutic potential of targeting PHGDH in MM, and suggest it as a way to overcome the resistance to proteasome inhibitors.

Monoclonal immunoglobulins promote bone loss in multiple myeloma.

Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.

Conversion of ATP to adenosine by CD39 and CD73 in multiple myeloma can be successfully targeted together with adenosine receptor A2A blockade.

BACKGROUND: PD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its immunosuppressive receptor should be considered. CD39 and CD73 convert extracellular ATP to adenosine, which inhibits T-cell effector functions via the adenosine receptor A2A (A2AR). We set out to investigate whether blocking the adenosine pathway could be a therapy for MM. METHODS: Expression of CD39 and CD73 on BM cells from patients and T-cell proliferation were determined by flow cytometry and adenosine production by Liquid chromatograpy-mass spectrometry (HPCL/MS). ENTPD1 (CD39) mRNA expression was determined on myeloma cells from patients enrolled in the publicly available CoMMpass study. Transplantable 5T33MM myeloma cells were used to determine the effect of inhibiting CD39, CD73 and A2AR in mice in vivo. RESULTS: Elevated level of adenosine was found in BM plasma of MM patients. Myeloma cells from patients expressed CD39, and high gene expression indicated reduced survival. CD73 was found on leukocytes and stromal cells in the BM. A CD39 inhibitor, POM-1, and an anti-CD73 antibody inhibited adenosine production and reduced T-cell suppression in vitro in coculture of myeloma and stromal cells. Blocking the adenosine pathway in vivo with a combination of Sodium polyoxotungstate (POM-1), anti-CD73, and the A2AR antagonist AZD4635 activated immune cells, increased interferon gamma production, and reduced the tumor load in a murine model of MM. CONCLUSIONS: Our data suggest that the adenosine pathway can be successfully targeted in MM and blocking this pathway could be an alternative to PD1/PDL1 inhibition for MM and other hematological cancers. Inhibitors of the adenosine pathway are available. Some are in clinical trials and they could thus reach MM patients fairly rapidly.

Express Team-Based Learning (eTBL): A Time-Efficient TBL Approach in Neuroradiology.

RATIONALE AND OBJECTIVES: Team-based learning (TBL) is a student-centred, teacher-directed instructional method that promotes active learning. The application phase of TBL stimulates group discussion and critical thinking, which could be useful for learning radiology. We designed and evaluated two modified TBL-sessions on computed tomography and magnetic resonance imaging diagnostics in neuroradiology. Our aim was to examine what effects engaging students in in-class team application tasks had on student learning. MATERIALS AND METHODS: A cross-over study was conducted, including 105 third-year medical students using two modified TBL sessions as the active learning intervention compared with two traditional lectures as a control. Student learning was assessed by results on the neuroradiology part of the end-of-year written examination. Student engagement and perceptions were assessed using the Student Self-Report of Engagement Measure and an additional four Likert-type items. RESULTS: There were no statistically significant differences in student scores on the examination. Students reported high levels of engagement, and reported being more satisfied overall with the TBL sessions than traditional lectures. Students rated the TBL sessions higher than lectures on ability to make difficult material comprehensible, ability to engage students and to give them feedback. CONCLUSION: The modified TBL sessions halved in-class teaching time and by omitting the readiness assurance tests, there was more in-class time to focus on problem-solving of real clinical cases. Moreover, shorter sessions may ease implementation of TBL in the curriculum and allow for more frequent sessions. Students were more satisfied with eTBL than lectures, and reported high levels of engagement.

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 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.

How can the examination failure rate be stabilised? / 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 2010­2015 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.

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.