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Composite lymphoma (CL) is defined as the simultaneous presentation of two histologically distinct types of malignant lymphoma in the same patient, frequently in the same organ or tissue. With a fr...

Imbalances in the composition of BCL2 family proteins contribute to tumourigenesis and therapy resistance of mantle cell lymphoma (MCL), making these proteins attractive therapy targets. We studied the efficiency of dual targeting the NOXA/MCL1 axis by combining fatty acid synthase inhibitors (NOXA stabilization) with the CDK inhibitor Dinaciclib (MCL1 reduction). This combination synergistically induced apoptosis in cell lines and primary MCL cells and led to almost complete inhibition of tumour progression in a mouse model. Apoptosis was NOXA-dependent and correlated with the NOXA/MCL1 ratio, highlighting the importance of the NOXA/MCL1 balance for effective cell death induction in MCL.

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of B-cell receptor (BCR) signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 complex that links BCR signaling to the NF-κB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls an MYC-driven gene expression network predominantly through increasing MYC protein stability. Thus, our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1-induced MYC regulation is not restricted to MCL, but represents a common mechanism. MYC itself is pivotal for MCL survival because its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy of targeting the MALT1-MYC axis in MCL patients.

Glioblastoma (GBM), the most common malignant brain tumor, is among the most lethal neoplasms, with a median survival of approximately 1 year. Prognosis is poor since GBMs possess a strong migratory and highly invasive potential, making complete surgical resection impossible. Reduced expression of carboxypeptidase E (CPE), a neuropeptide-processing enzyme, in a cell death-resistant glioma cell line and lower CPE expression levels in the cohort of GBM samples of The Cancer Genome Atlas compared to normal brain control specimens prompted us to analyze the function of CPE as a putative tumor suppressor gene. In our samples, CPE was also reduced in GBM compared to normal brain with the strongest loss in cells surrounding hypoxic tumor areas as well as in most glioma cell lines and primary glioma cells. In our cohort of glioma patients, loss of CPE predominantly occurred in glioblastomas and was associated with worse prognosis. In glioma cells, CPE overexpression was significantly reduced, whereas knockdown or inhibition enhanced glioma cell migration and invasion. The decreased migratory potential following CPE overexpression was paralleled by altered cellular morphology, promoting a transition to focal adhesions and associated stress fibers. In contrast to the decreased migration, high CPE levels were associated with higher proliferative rates. As microenvironmental regulation cues, we identified CPE as being downregulated upon hypoxia or glucose deprivation. Our findings indicate an oxygen- and nutrition-dependent anti-migratory, but pro-proliferative role of CPE in gliomas with prognostic impact for patient survival, thereby contributing to the understanding of the "go or grow" hypothesis in gliomas.

Multiple myeloma is a disease requiring multidisciplinary approach–mostly oncologists and nephrologists are involved. Here we describe a 62-year-old male patient with multiple myeloma (IgG Kappa) (diagnosis 2006). Treatment with Vincristin, Doxorubicin, and Dexamethason was started. Restaging revealed evidence of progression of myeloma. Therefore, autologous hematopoietic stem cell transplantation was performed, leading to complete remission. Two years later, there was progression of myeloma and multiple chemotherapy regimens were administered up to now (cyclophosphamide, thalidomide, lenalidomide, bendamustine). At time of presentation in the outpatient clinic, the patient had no complaints except diarrhea for a few days. Abnormal laboratory findings at presentation were strongly increased phosphorus level, elevated serum creatinine of 2.3 mg/dL (estimated glomerular filtration rate (GFR), calculated using MDRD (Modification of Diet in Renal Disease) Study equation was 31 mL/min per 1.73 m2), and pancytopenia. Chronic kidney disease due to myeloma was diagnosed several months ago. Serum immunofixation electrophoresis revealed elevated IgG and Κ:λ ratio was 185.2 (normal range adapted to patients with impaired kidney function 0.37–3.1). In addition, total serum protein concentration was strongly increased. Urinary studies showed spot urine protein to creatinine ratio of 0.13 g/g (normal range #x003C;0.25 g/g). Significant laboratory findings are shown in Table​Table1.1. The patient did not report symptoms or signs of hyperphosphatemia and he denied neither using any dietary protein supplements or phosphate soda preparation nor having excessive dairy product intake in the recent past. At this stage, a nephrologist was contacted with the question whether renal replacement therapy due to severe acute hyperphosphatemia should be started. In steady state, the kidney primarily determines serum phosphate concentration by excretion of dietary phosphate. There are three general circumstances in which phosphate entry into the extracellular fluid exceeds the degree to which it can be excreted resulting in hyperphosphatemia: acute or chronic kidney disease, massive acute phosphate load, and primary increase in tubular phosphate reabsorption. Reduction in GFR results in diminished phosphate filtration and excretion. Initially, phosphate balance could be maintained by decreased proximal phosphate reabsorption. The GFR of our patient was 31 mL/min and kidney function had remained stable over the last few months. Therefore, severe hyperphosphatemia could not exclusively be explained by impaired kidney function. Either exogenous or endogenous sources might cause massive acute phosphate load. Phosphate is an intracellular ion and therefore it could be released into extracellular fluid due to marked tissue breakdown, causing symptomatic hypocalcemia. The phosphorus concentration in malignant cells is higher than in normal cells and rapid tissue breakdown often leads to acute hyperphosphatemia including an increased risk of calcium phosphate precipitation in the renal tubules leading to acute kidney failure 1. It is noteworthy, that tumor lysis syndrome (TLS) is less common in multiple myeloma 2 and in our patient, uric acid was only slightly elevated, lactate dehydrogenase and calcium levels were within the normal range, arguing against TLS as reasons for hyperphosphatemia (Table​(Table22 shows differential diagnosis of hyperphosphatemia). Due to these findings, additional investigations were performed. The patient's initial blood sample was tested as previously described 3 using a clinical analyzer in which inorganic phosphorus reacts with ammonium molybdate in the presence of sulfuric acid to form an unreduced phosphomolybdate complex. This complex absorbs light and is quantified photometrically in the ultraviolet range (wavelength 340 nm). Light absorbance is directly proportional to the inorganic phosphorus concentration 3. Due to concern about pseudohyperphosphatemia the samples were analyzed in a second step using simple dilution of the samples to eliminate turbidity and interference (1:5 and 1:10) as previously described 4–6. Afterward, phosphate levels decreased to still slightly elevated levels of 1.82 mmol/L (normal range 0.68–1.68 mmol/L), which is explained by the chronic kidney disease. During follow-up, the phosphate levels remained stable. Table 1 Laboratory findings at outpatient clinic. Table 2 Differential diagnosis of hyperphosphatemia and pseudohyperphosphatemia. Two explanations exist regarding pseudohyperphosphatemia. The first explanation comprised the hypothesis that pseudohyperphosphatemia is caused by direct binding of paraproteins to phosphorus, whereas the other hypothesis postulated that paraproteins bind to molybdate resulting in turbidity. During analysis, the turbid reaction mixture scatters and adsorbs light while reducing its transmittence. This might be interpreted as falsely high phosphorus level 3,7,8. Most reported cases of patients with myeloma and pseudohyperphosphatemia had normal kidney function, which leads to the diagnosis of pseudohyperphosphatemia much more easily 3,9, whereas our patient had impaired kidney function. In general, treatment is not necessary if the true phosphate level is within the reference range and the patient is asymptomatic 3. This case illustrates the course of pseudohyperphosphatemia in a patient with myeloma and chronic kidney disease. Especially oncologists and nephrologists should be aware of pseudohyperphosphatemia leading to prompt identification of these patients and mitigate unnecessary treatment.

Mantle cell lymphoma (MCL) is a rare B-cell-neoplasm with an aggressive clinical course characterized by the hallmark translocation t(11;14)(q13;q32) resulting in overexpression of cyclin D1. Furthermore, genomic profiling revealed numerous secondary genetic alterations and recurrent mutations contributing to MCL pathogenesis. Among them, mutations in the NOTCH1 gene have been described with a frequency of ~10% and are associated with significantly shorter survival rates. Therefore, further investigation of the biological impact of this mutation in MCL and its potential as a target for a specific inhibitory antibody therapy is of great interest. Here we investigated the role of the Notch receptor ligands Jagged1 (JAG1), Jagged2 (JAG2), Delta-like canonical Notch ligand 1 (DLL1) and Delta-like canonical Notch ligand 4 (DLL4) in activating the Notch1-signaling pathway in NOTCH1-mutated Mino and NOTCH1-wt Jeko1 MCL cell lines and evaluated the effects of the novel and specific monoclonal Notch1-antibody OMP-52M51. Mino cells are characterized by a single nucleotide substitution in exon 34 of the NOTCH1 gene, leading to truncation of the C-terminal PEST-domain. These mutations are known to remove the recognition site from the ubiquitin ligase degradation complex, resulting in increased stability and transcriptional activity of Notch1 upon ligand-stimulation. We confirmed the predicted truncation of Notch1 in Mino cell lysates and demonstrated that the expression of cleaved Notch1 was potently stimulated by recombinant DLL4 protein. In contrast, in the NOTCH1-wt cell line Jeko1, no stable overexpression of cleaved Notch1 could be observed upon any ligand-stimulation. Treatment of the NOTCH1-mutated cells with OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed transcriptional induction of well-known Notch1-target genes HES1 (hairy and enhancer of-split 1), DTX1 (deltex E3 ubiquitin ligase 1) and c-MYC as observed by real-time quantitative PCR. Gene expression profiling furthermore revealed that OMP-52M51 significantly impeded DLL4-induced upregulation of numerous genes involved in lymphoid biology and lymphomagenesis, as well as genes related to the cell cycle and the MAP-Kinase signaling pathway. Western blot analysis confirmed Notch1-antibody-mediated abrogation of enhanced expression of p-ERK and p-MEK upon DLL4-ligand-stimulation. Further investigations of functional effects revealed inhibitory effects of OMP-52M51 on enhanced tumor cell migration of DLL4-stimulated cells. As the DLL4-Notch-axis is known to be involved in regulating angiogenesis, we investigated the impact of activating NOTCH1-mutations in MCL on HUVEC tube formation and observed increased vessel sprouting upon DLL4-stimulation that could be abolished by treatment with OMP-52M51. Importantly, all these effects were specific for NOTCH1-mutated cells and did not occur in the NOTCH1-wt cell line Jeko1. In conclusion, we demonstrate for the first time that DLL4 is the most important ligand to activate Notch1 in MCL harbouring NOTCH1-mutations in the PEST-domain. Our findings indicate that enhanced Notch1 activity promotes a more aggressive behavior of the disease and specific antibody-inhibition of the Notch1-signaling pathway might provide an interesting therapeutic alternative for a subset of MCL patients, warranting further investigation. Disclosures No relevant conflicts of interest to declare.

Histone deacetylase inhibitors (HDACi) have been described as multifunctional anticancer agents. The failure of conventional therapy for glioblastoma (GBM) renders this tumor an attractive target for immunotherapy. Innate immune cells, such as natural killer (NK) cells, play a crucial role in antitumor immune responses. Here, we describe how the HDACi trichostatin A (TSA) promotes apoptosis of tumor cells, as well as augments anti-GBM innate immune responses. In vitro treatment of GBM cells with TSA results in an up-regulation of the natural killer group-2 member-D (NKG2D) ligands major histocompatibility complex class I-related chain (MIC)-A and UL16 binding protein (ULBP)-2 at both mRNA and protein levels, rendering them susceptible to NK cell-mediated lysis. In vivo, TSA delays tumor growth of GBM xenografts. Both the in vitro and in vivo antitumor effect of TSA was significantly reduced by blocking NK cell activity. Our data suggest that HDACi, especially in combination with other clinical immunotherapeutical approaches, may be considered in a combined therapeutic approach for GBM.

Mantle cell lymphoma (MCL) cells are characterized by a discrepancy between high mRNA and low protein levels of the pro-apoptotic BH3-only protein NOXA. Modulation of NOXA protein expression has been described as a major mechanism of cell death induction in MCL cells. However, the efficiency of induction of apoptosis at lower concentrations is limited despite effective stabilization of NOXA. We therefore investigated whether the main binding partner of NOXA, the anti-apoptotic protein MCL-1 is co-regulated by these agents and whether dual targeting of NOXA and MCL-1 could be a promising strategy to enhance effectiveness of cell death induction in MCL cell lines. Screening a panel of compounds supposed to lead to NOXA stabilization in MCL, we identified the proteasome inhibitor Bortezomib, the fatty acid synthase inhibitor Orlistat and the ROS inducing agents Helenalin, a sesquesterpenone lactone from Arnica and the naphtoquinone derivative Menadione to kill MCL cells very effectively in a NOXA -dependent manner. Investigating the NOXA-/MCL-1-protein expression upon treatment with these agents, we could observe that at lower, sublethal concentrations of Bortezomib and Orlistat, NOXA protein was increased to a certain extent but also the anti-apoptotic MCL-1 was highly induced, counteracting induction of apoptosis. From this observation it has to be concluded that MCL-1 limits the apoptotic activity of NOXA stabilizing agents and dual targeting of MCL-1 and NOXA is required for optimal killing of MCL cells. In search for MCL-1 regulating agents we could identify the cdk-inhibitor Dinaciclib to be the most effective one. This compound rapidly downregulates Mcl-1 protein in a dose- and time-dependent manner presumably due to cdk 9-mediated inhibition of phosphorylation of the RNA-Polymerase II-subunit RPB1. To study the efficiency of dual targeting of NOXA/MCL-1 in killing of MCL cells we combined sublethal doses of Dinaciclib with NOXA stabilizing agents and observed a synergistic induction of apoptosis in MCL cells. Western Blot analysis showed that combination treatment decreased Mcl-1 and increased NOXA protein expression compared to single-agents. It could be shown that induction of cell death by treatment with the combined agents depends on NOXA as transfection of cells with NOXA-siRNA rescued cells from induction of apoptosis. Cell death upon treatment with Dinaciclib and ROS-inducing agents Helenalin and Menadione could furthermore be rescued by preincubation with the antioxidant GSH. Interestingly, combined treatment of cells with Orlistat and Dinaciclib killed most effectively when Dinaciclib was added for 8 hours after 16 hours of preincubation with the NOXA stabilizing agent. This observation contributes to the hypothesis that stabilization of NOXA protein leads to priming of MCL cells to induction of apoptosis by pharmacological downregulation of Mcl-1 with Dinaciclib. In summary, the NOXA-MCL-1 balance is critical for survival of MCL cells. Dual targeting of MCL-1 and NOXA efficiently kills MCL cells and appears to be of particular importance especially at lower concentrations of these compounds. These culture conditions most likely resemble the limited exposure after in vivo treatment. Therefore the combination of NOXA stabilizing agents with Dinaciclib appears to be a promising strategy to be tested in clinical trials. Disclosures No relevant conflicts of interest to declare.