Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eμ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6C(low) patrolling or nonclassical phenotype. In addition, the percentage of MHC-II(hi) dendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-α and CXCL9. In vivo myeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

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Abstract:The purpose of this study was to identify and characterize fungal natural products (NPs) with in vitro bioactivity towards leukemia cells. We based our screening on a combined analytical and bio-guided approach of LC-DAD-HRMS dereplication, explorative solid-phase extraction (E-SPE), and a co-culture platform of CLL and stromal cells. A total of 289 fungal extracts were screened and we tracked the activity to single compounds in seven of the most active extracts. The novel ophiobolin U was isolated together with the known ophiobolins C, H, K as well as 6-epiophiobolins G, K and N from three fungal strains in the Aspergillus section Usti. Ophiobolins A, B, C and K displayed bioactivity towards leukemia cells with induction of apoptosis at nanomolar concentrations. The remaining ophiobolins were mainly inactive or only slightly active at micromolar OPEN ACCESSMolecules 2013, 18 14630 concentrations. Dereplication of those ophiobolin derivatives possessing different activity in combination with structural analysis allowed a correlation of the chemical structure and conformation with the extent of bioactivity, identifying the hydroxy group at C3 and an aldehyde at C21, as well as the A/B-cis ring structure, as indispensible for the strong activity of the ophiobolins. The known compounds penicillic acid, viridicatumtoxin, calbistrin A, brefeldin A, emestrin A, and neosolaniol monoacetate were identified from the extracts and also found generally cytotoxic.

The metabolism of CIostridium butyricum DSM 5431 was studied in chemostat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explained the constant proportion of glycerol conversion into 1,3-PD. The production of acetate or butyrate constitutes another important branch point and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, suggesting this enzyme may be the bottleneck for carbon flux to butyrate biosynthesis providing an explanation for the accumulation of large amounts of intracellular acetyl-CoA, and (iv) high levels of NADH were found in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked to the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (9gluc,,se = 11.1 mmol h-' g-') was around four times lower than the specific rate of glycerol catabolism (qglycero, = 57-4 mmol h-' g-'). On glucose-grown cells, reducing equivalents which are released in the glycolytic pathway were reoxidized by the butyric pathway and the low specific formation rate of butyric acid led to an increase in the intracellular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways. INTRODUCTIONOver recent years there has been an increase in research and development efforts involving clostridial fermentation, namely the fermentation of glucose by butyric acid bacteria (Woods, 1993). Clostridia are able to ferment the carbon source to a variety of end-products by means of branched fermentation pathways. Generally, the different branches of fermentation are not equivalent with respect to the generation of energy or reducing equivalents, andAbbreviations: DHA, dihydroxyacetone; fd, ferredoxin; 3-HPA, 3-hydroxypropionaldehyde; PD, propanediol.the amount of reduced versus neutral and oxidized products has the potential for a great deal of natural variation. It was shown (Jungermann et al., 1973; Petitdemange e t d. , 1976) that the reduced electron carrier ferredoxin (fd) plays a pivotal role in electron distribution in the cell in that it can either transfer electrons via hydrogenase to generate hydrogen or transfer electrons to the pyridine nucleotides via the appropriate fd oxidoreductase. The activities of three enzymes, NADH-fd oxidoreductase, NADPH-fd oxidoreductase and hydrogen...

Key Points Lenalidomide treatment of primary CLL/nurse-like cell cocultures resulted in significantly decreased viability of CLL cells. Lenalidomide increased IL-10 levels, activation of STAT1, expression of ICAM-1, and migration-related genes, and reduced CLL cell motility.

Chronic lymphocytic leukemia is a malignancy of mature B cells that strongly depend on microenvironmental factors, and their deprivation has been identified as a promising treatment approach for this incurable disease. Cytokine array screening of 247 chronic lymphocytic leukemia serum samples revealed elevated levels of tumor necrosis factor (TNF) receptor-1 which were associated with poor clinical outcome. We detected a microenvironment-induced expression of TNF receptor-1 in chronic lymphocytic leukemia cells in vitro, and an aberrantly high expression of this receptor in the proliferation centers of patients’ lymph nodes. Stimulation of TNF receptor-1 with TNF-α enhanced nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activity and viability of chronic lymphocytic leukemia cells, which was inhibited by wogonin. The therapeutic effects of wogonin were analyzed in mice after adoptive transfer of Eμ-T-cell leukemia 1 (TCL1) leukemic cells. Wogonin treatment prevented leukemia development when given early after transplantation. The treatment of full-blown leukemia resulted in the loss of the TNF receptor-1 on chronic lymphocytic leukemia cells and their mobilization to blood. Targeting TNF receptor-1 signaling is therefore proposed for the treatment of chronic lymphocytic leukemia.

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Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn−/− mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn−/− chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.