Oxidative stress as candidate therapeutic target to overcome microenvironmental protection of CLL.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6-8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P = 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.

Targeting inhibitor of apoptosis proteins by Smac mimetic elicits cell death in poor prognostic subgroups of chronic lymphocytic leukemia.

Inhibitor of apoptosis (IAP) proteins are highly expressed in chronic lymphocytic leukemia (CLL) cells and contribute to evasion of cell death and poor therapeutic response. Here, we report that Smac mimetic BV6 dose-dependently induces cell death in 28 of 51 (54%) investigated CLL samples, while B-cells from healthy donors are largely unaffected. Importantly, BV6 is significantly more effective in prognostic unfavorable cases with, e.g., non-mutated VH status and TP53 mutation than samples with unknown or favorable prognosis. The majority of cases with 17p deletion (10/12) and Fludarabine refractory cases respond to BV6, indicating that BV6 acts independently of p53. BV6 also triggers cell death under survival conditions mimicking the microenvironment, e.g., by adding CD40 ligand or conditioned medium. Gene expression profiling identifies cell death, NF-κB and redox signaling among the top pathways regulated by BV6 not only in CLL but also in core-binding factor (CBF) acute myeloid leukemia (AML). Consistently, BV6 stimulates production of reactive oxygen species (ROS), which are contributing to BV6-induced cell death, since antioxidants reduce cell death. While BV6 causes degradation of cellular inhibitor of apoptosis (cIAP)1 and cIAP2 and nuclear factor-kappaB (NF-κB) pathway activation in primary CLL samples, BV6 induces cell death independently of caspase activity, receptor-interacting protein (RIP)1 activity or tumor necrosis factor (TNF)α, as zVAD.fmk, necrostatin-1 or TNFα-blocking antibody Enbrel fail to inhibit cell death. Together, these novel insights into BV6-regulated cell death in CLL have important implications for developing new therapeutic strategies to overcome cell death resistance especially in poor prognostic CLL subgroups.

Epigenetic upregulation of lncRNAs at 13q14.3 in leukemia is linked to the In Cis downregulation of a gene cluster that targets NF-kB.

Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.

TCL1A and ATM are co-expressed in chronic lymphocytic leukemia cells without deletion of 11q.

Chronic lymphocytic leukemia is characterized by the accumulation of B cells that are resistant to apoptosis. This resistance is induced by pro-survival stimuli from the microenvironment. TCL1 and ATM are central to the pathogenesis of the disease and associated with more aggressive disease. Their protein products have recently been shown to physically interact in leukemic cells and to impact on NF-κB signaling, which is a key regulator of apoptosis. In the present study we show that TCL1 and ATM are significantly co-expressed and up-regulated in malignant cells compared to non-malignant B cells, and that expression of TCL1 is partially deregulated by aberrant DNA-methylation. In addition, complex external stimuli induce essentially similar TCL1 and ATM time-course kinetics. In line with a coordinative regulation of NF-κB signaling by TCL1, its knockdown induced apoptosis in primary leukemia cells. These findings suggest that both genes functionally cooperate to modulate similar apoptosis-related cellular pathways.

Immune modulatory agents in hematopoietic malignancies.

Immune modulatory drugs have been successfully used to treat patients with multiple myeloma (MM), myelodysplastic syndromes displaying loss of 5q (del5q MDS) and chronic lymphocytic leukemia (CLL). Immune modulatory drugs are used in first-line therapy in combination with functionally complementary compounds, but have also shown efficacy in refractory disease. However, their exact mode of action remains unclear. Here we describe the clinical impact of these compounds on MM, del5q MDS and CLL, discuss their mode of action with respect to intracellular targets, focus on the phenotypic changes that immune modulatory compounds induce in the tumor microenvironment and how they modulate the immune response.

Nurse-like cells show deregulated expression of genes involved in immunocompetence.

Chronic lymphocytic leukaemia (CLL) cells convert CD14(+) cells from patients into 'nurse-like' cells (NLCs). CLL cells can also convert CD14(+) peripheral blood mononuclear cells (PBMCs) from healthy donors into cells with morphological similarities to NLCs (CD14(CLL) -cells). However it is unclear whether only CLL cells induce this conversion process. This study showed that CD14(+) PBMCs from healthy donors could also be converted into differentiated cells (CD14(B) -cells) by non-malignant B-cells. In order to identify changes specifically induced by CLL cells, we compared gene expression profiles of NLCs, CD14(CLL) -cells and CD14(B) -cells. CD14(+) cells cultured with CLL cells were more similar to NLCs than those cultured with non-malignant B-cells. The most significant changes induced by CLL cells were deregulation of the antigen presentation pathway and of genes related to immunity. NLCs had reduced levels of lysozyme activity, CD74 and HLA-DR in-vitro while expression of inhibitory FCGR2B was increased. These findings suggest an impaired immunocompetence of NLCs which, if found in-vivo, could contribute to the immunodeficiency in CLL patients.

High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay.

Contemporary research on cellular signaling has undergone a shift of focus from qualitative measurements of single signaling pathways to high-throughput quantitation of comprehensive signaling networks. Notably, nuclear factor-kappaB (NFkappaB) is a family of transcription factors involved in immune and inflammatory responses, developmental processes, cellular growth and apoptosis and is deregulated in a number of disease states. We have established a chemiluminescent oligonucleotide-based enzyme-linked immunosorbent assay (co-ELISA) that is simple and quantitative. In contrast to currently used assays, it allows quantitation of all NFkappaB components (i.e., RelA, p50, p52, RelB and c-Rel). In addition, it can make use of whole extract and does not require cumbersome nuclear/cytosolic fractionation, saving time and resources. Co-ELISA has a 3.5- to 43-fold higher signal-over-noise ratio than currently available assays, whereas the percent relative standard deviation is 3- to 6-fold lower. Furthermore, the novel method is faster than electrophoretic mobility shift assay, not restricted to transfectable cells as is the case for luciferase reporter assays and 10 times more cost efficient than commercially available ELISA assays. Co-ELISA is a sensitive, fast and cost-efficient quantitation method for all DNA-binding NFkappaB proteins that can be used in high-throughput experimentation.

Chronic lymphocytic leukemia and 13q14: miRs and more.

Loss of a critical region in 13q14.3 [del(13q)] is the most common genomic aberration in chronic lymphocytic leukemia (CLL), occurring in more than 50% of patients (Stilgenbauer et al., Oncogene 1998;16:1891 - 1897, Dohner et al., N Engl J Med 2000;343:1910 - 1916). Despite extensive investigations, no point mutations have been found in the remaining allele that would inactivate one of the candidate tumor suppressor genes and explain the pathomechanism postulated for this region. However, the genes in the region are significantly down-regulated in CLL cells, more than would be expected by gene dosage, and recently a complex epigenetic regulatory mechanism was identified for 13q14.3 in non-malignant cells that involves asynchronous replication timing and monoallelic expression of candidate tumor suppressor genes. Here, we propose a model of a multigenic pathomechanism in 13q14.3, where several tumor suppressor genes, including the miRNA genes miR-16-1 and miR-15a, are co-regulated by the two long non-coding RNA genes DLEU1 and DLEU2 that span the critical region. Furthermore, we propose these co-regulated genes to be involved in the same molecular pathways, thereby also forming a functional gene cluster. Elucidating the molecular and cellular function of the 13q14.3 candidate genes will shed light on the underlying pathomechanism of CLL.