Dissecting the Prognostic Significance and Functional Role of Progranulin in Chronic Lymphocytic Leukemia.

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.

Tumor necrosis factor receptor signaling is a driver of chronic lymphocytic leukemia that can be therapeutically targeted by the flavonoid wogonin.

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.

Isolation, structural analyses and biological activity assays against chronic lymphocytic leukemia of two novel cytochalasins - sclerotionigrin A and B.

Two new cytochalasins, sclerotionigrin A (1) and B (2) were isolated together with the known proxiphomin (3) from the filamentous fungus Aspergillus sclerotioniger. The structures and relative stereochemistry of 1 and 2 were determined based on comparison with 3, and from extensive 1D and 2D NMR spectroscopic analysis, supported by high resolution mass spectrometry (HRMS). Compounds 2 and 3 displayed cytotoxic activity towards chronic lymphocytic leukemia cells in vitro, with 3 being the most active.

Bio-activity and dereplication-based discovery of ophiobolins and other fungal secondary metabolites targeting leukemia cells.

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 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.

Synthesis, enzymatic stability and in vitro cytostatic effect of Daunorubicin-GnRH-III derivative dimers.

Bioconjugates containing chemotherapeutic agents attached to peptide hormones, such as gonadotropin-releasing hormone (GnRH), are developed as drug delivery systems for targeted cancer chemotherapy. We report here the synthesis and biochemical characterization of disulfide bond-linked dimeric bioconjugates in which daunorubicin was coupled via an oxime linkage to aminooxyacetylated GnRH-III ([Glp-His-Trp-Ser-His-Asp-Trp-Lys(DauAoa-Cys)-Pro-Gly-NH2]2; where Glp is pyroglutamic acid and Aoa is aminooxyacetyl) and its derivatives modified in position four by N-Me-Ser and Lys(Ac). The in vitro stability/degradation of the bioconjugates was determined in human serum, as well as in the presence of rat liver lysosomal homogenate and digestive enzymes. All compounds were stable at least for 24h in human serum and in the presence of pepsin and trypsin, while they were degraded by lysosomal enzymes. The daunorubicin-GnRH-III derivative dimers were partly digested by α-chymotrypsin; however, they had increased stability compared to the corresponding monomers, making them potential candidates for oral administration. The in vitro cytostatic effect of the compounds was determined on MCF-7 human breast cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All daunorubicin-GnRH-III derivative dimers exerted slightly increased in vitro cytostatic effect (IC50 values in low µM range) than the corresponding monomeric bioconjugates.

Lenalidomide reduces survival of chronic lymphocytic leukemia cells in primary cocultures by altering the myeloid microenvironment.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental stimuli for their survival, provided for example by monocyte-derived nurse-like cells (NLCs). The immunomodulatory drug lenalidomide shows therapeutic effects in subgroups of CLL patients, and is believed to act via the microenvironment. To investigate the effects of lenalidomide on the survival support of NLCs, cocultures of monocytes and CLL cells were treated for 14 days with lenalidomide, which resulted in significantly decreased viability of CLL cells. Among the changes induced by this drug, we observed reduced expression of HLA-DR in NLCs as well as increased secretion of interleukin-10 (IL-10), indicating an altered inflammatory milieu in the cocultures. The increase in IL-10 levels lead to an induction of STAT1 phosphorylation in CLL cells and to enhanced cell-surface expression of intercellular adhesion molecule 1 and altered expression of cytoskeletal and migration-related genes. Chemotaxis assays with lenalidomide-treated CLL cells revealed an impaired migration capability. Our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug affects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide acts on the migratory potential of CLL cells, which may affect circulation and homing of CLL cells in vivo.