Olga V. Danilova

Chromosomal instability substantiates poor prognosis in patients with Diffuse Large B-Cell Lymphoma

Purpose: The specific role of chromosomal instability (CIN) in tumorigenesis has been a matter of conjecture. In part, this is due to the challenge of directly observing chromosome mis-segregation events as well as the inability to distinguish the role of CIN, which consists of increased rates of chromosome mis-segregation, from that of aneuploidy, which is a state of nondiploid chromosome number. Experimental Design: Here, we examine the contribution of CIN to the prognosis of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) by directly surveying tumor cells, fixed while undergoing anaphase, for evidence of chromosome mis-segregation. Hematoxylin and eosin–stained samples from a cohort of 54 patients were used to examine the relationship between frequencies of chromosome mis-segregation and patient prognosis, overall survival, and response to treatment. Results: We show that a two-fold increase in the frequency of chromosome mis-segregation led to a 24% decrease in overall survival and 48% decrease in relapse-free survival after treatment. The HR of death in patients with increased chromosome mis-segregation was 2.31 and these patients were more likely to present with higher tumor stage, exhibit tumor bone marrow involvement, and receive a higher International Prognostic Index score. Conclusions: Increased rates of chromosome mis-segregation in DLBCL substantiate inferior outcome and poor prognosis. This is likely due to increased heterogeneity of tumor cells leading to a larger predilection for adaptation in response to external pressures such as metastasis and drug treatments. We propose that targeting CIN would yield superior prognosis and improved response to chemotherapeutic drugs. Clin Cancer Res; 17(24); 7704–11. ©2011 AACR.

Molecular Pathogenesis of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is unique among malignancies since it represents an accumulation of B-lymphocytes resistant to apoptosis. Several factors are thought to confer this unusual feature to a CLL B-cell. Misbalance between cytoplasmic pro-survival and pro-death molecules, such as Bcl-2, Mcl-1 and alike, appears to be one of the key factors defining B-cell longevity. Autocrine pathways, such as vascular endothelial growth factor-receptor pathway, also contribute to survival. The role of B-cell receptor (BCR) is less straightforward. In the last decade it became clear that CLL does not constitute a uniform disease, but, based on the prevalence of mutations in the BCR heavy chain (IgVH), can be classified into two distinct subgroups. Several molecular markers correlate with IgVH mutations. Some of them, like zeta-chain associated protein kinase, are also involved in BCR signaling and influence cell cycle. Yet the primary pathogenic event leading to increased proliferation and survival in CLL is difficult to ascertain. Molecules involved in BCR signaling pathways and cytoplasmic pro-survival players probably act in concert to confer resistance to apoptosis. In this respect, the role of the B-CLL environment, which includes nurse-like cells and T-cells, cannot be underestimated. Nurse-like cells provide stimuli necessary for perpetuation of life in CLL. On the other hand, abnormal T-cell function, whether it is excessive immunosuppression delivered by regulatory T-cells or insufficient anti-tumor immunity rendered by T-helpers, allows malignant CLL cells to go unnoticed by the cellular immune system.

Pro-apoptotic TP53 homolog TAp63 is repressed via epigenetic silencing and B-cell receptor signalling in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is an accumulative disorder marked by deficient apoptosis. The TP53 homolog TAp63 promotes apoptosis and chemosensitivity in solid tumours and its deregulation may contribute to CLL cell survival. We found that TAp63α was the most prevalent TP63 isoform in CLL. Compared to healthy B cells, TAp63 mRNA was repressed in 55·7% of CLL samples. TP63 promoter methylation was high in CLL and inversely correlated with TP63 protein expression in B‐cell lymphoma cell lines. siRNA‐mediated knockdown of TP63 resulted in partial protection from spontaneous apoptosis accompanied by reductions in PMAIP1 (NOXA), BBC3 (PUMA), and BAX mRNA in CLL cells and increased proliferation of Raji lymphoma cells. TAp63 mRNA levels were higher in CLL with unmutated IGHV. B‐cell receptor (BCR) engagement led to repression of TP63 mRNA expression in malignant B cells, while pharmacological inhibition of BCR signalling prevented TP63 downregulation. MIR21, known to target TAp63, correlated inversely with TAp63 expression in CLL, and BCR‐mediated downregulation of TP63 was accompanied by MIR21 upregulation in most CLL samples. Our data illustrate the pro‐apoptotic function of TP63, provide insights into the mechanisms of BCR‐targeting agents, and establish a rationale for designing novel approaches to induce TP63 in CLL and B‐cell lymphoma.

Neurogenin 3-Specific Dipeptidyl Peptidase-2 Deficiency Causes Impaired Glucose Tolerance, Insulin Resistance, and Visceral Obesity

The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. Here we introduce the N-terminal dipeptidase, DPP2, as a novel regulator of the glucose metabolism. We generated mice with a neurogenin 3 (NGN3)-specific DPP2 knockdown (kd) to explore a possible role of DPP2 in maintaining metabolic homeostasis. These mice spontaneously developed hyperinsulinemia, glucose intolerance, and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in DPP2 kd mice, which was associated with a significant increase in adipose tissue mass and enhanced liver steatosis but no difference in body weight. In accordance with these findings, the mutant mice had a higher rate of respiratory exchange than the control littermates. This phenotype was exacerbated with age and when challenged with a high-fat diet. We report, for the first time, that DPP2 enzyme activity is essential for preventing hyperinsulinemia and maintaining glucose homeostasis. Interestingly, the phenotype of NGN3-DPP2 kd mice is opposite that of DPP4 knockout mice with regard to glucose metabolism, namely the former have normal glucagon-like peptide 1 levels but present with glucose intolerance, whereas the latter have increased glucagon-like peptide 1, which is accompanied by augmented glucose tolerance.

Cardiac non‐Hodgkin's lymphoma: clinical characteristics and trends in survival

The purpose of this study was to describe the clinical characteristics and outcomes in cardiac non‐Hodgkins lymphoma (NHL).

Dipeptidyl Peptidase 2 apoptosis assay determines the B-cell activation stage and predicts prognosis in chronic lymphocytic leukemia

OBJECTIVE Dipeptidyl peptidase 2 (DPP2/DPP7) is a regulator of quiescence as inhibition of DPP2 results in apoptosis of resting, but not activated lymphocytes. The purpose of the present study was to investigate the prognostic value of DPP2 inhibition and the role of DPP2 in cell cycle in chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS We screened 152 peripheral blood samples from patients with CLL in an apoptosis assay with AX8819, a DPP2-specific inhibitor. The apoptotic response was correlated with B-cell receptor signaling and cell cycle and molecular prognostic factors. RESULTS We categorized CLL into two prognostic subgroups. Inhibition of DPP2 induced apoptosis in 60% of CLL, while 40% were resistant to apoptosis. Resistance to apoptosis correlated with unmutated IgV(H) and increased ZAP-70 expression and was associated with unfavorable clinical outcomes. Sensitive CLL B cells expressed high p27, low c-Myc protein levels and decreased Syk phosphorylation, indicative of a resting phenotype. DPP2 inhibition in those cells resulted in apoptosis accompanied by enhanced phosphorylation of Syk, degradation of p27 and p130, and upregulation of c-Myc, indicative of activation and inappropriate cell cycle entry. Resistant CLL demonstrated baseline low p27 and high c-Myc protein levels and increased pSyk, indicative of an activated phenotype. Inhibition of heat shock protein 90 in this subset of CLL partially reversed apoptosis resistance. CONCLUSIONS The DPP2 apoptosis assay provides a reliable prognostic factor in CLL. CLL B cells sensitive to DPP2 inhibition are in true G(0), while resistant CLL B-cells are partially activated. DPP2 inhibition alone or with concomitant inhibition of heat shock protein 90 warrants investigation as a therapeutic modality in CLL.

Cell cycle control and adhesion signaling pathways in the development of metastatic melanoma

Metastatic melanoma is a fatal malignancy which is remarkably resistant to treatment. It is not entirely clear what determines transition from primary local to metastatic melanoma. Recent gene profiling studies shed light onto the complexity of pathogenesis of melanoma progression. An interaction between cell cycle signaling, adhesion pathways and epithelial–mesenchimal transition program appears to be critical in the development of metastatic disease. An isolated deregulation of either of those pathways may not be sufficient to initiate tumor evolution towards an aggressive phenotype. Here we review how they act in concert to make such a transition possible.

Targeting neddylation effectively antagonizes nuclear factor-κB in chronic lymphocytic leukemia B-cells

Chronic lymphocytic leukemia (CLL) B-cells demonstrate both constitutive and stroma-mediated activation of nuclear factor-κB (NF-κB). NEDD8, a ubiquitin-like protein, regulates activity of Cullin-RING ubiquitin ligases (CRLs) and thus indirectly controls NF-κB activity. Inhibition of CRLs with MLN4924, an investigational agent that targets the NEDD8-activating enzyme, induces accumulation of CRL substrates, including inhibitor of NF-κB (IκB), a negative pathway modulator. We demonstrate that both continuous and pulse treatments with MLN4924 abrogate NF-κB activity in CLL B-cells ex vivo in a CD40L-expressing stromal co-culture system and identify pathways potentially responsible for resistance to MLN4924. To achieve long-lasting therapeutic effects in CLL, combination strategies are likely necessary.

FASN and CD36 predict survival in rituximab-treated diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma is the most common lymphoid malignancy, as it accounts for approximately one third of all patient cases of non-Hodgkin’s lymphoma. Patients with diffuse large B-cell lymphoma have markedly different treatment outcomes, suggesting a need for reliable prognostic factors and novel therapeutic approaches. De novo fatty acid synthesis is an important metabolic driver of tumor in multiple malignancies. In this retrospective study, we analyzed expression of fatty acid synthase (a key enzyme in de novo fatty acid synthesis), Spot 14 (thyroid hormone responsive Spot 14, a nuclear protein that promotes expression of genes involved in fatty acid synthesis), and CD36 (the cell surface channel for exogenous fatty acid uptake) in patients with diffuse large B-cell lymphoma and their clinical significance. We observed that overexpression of fatty acid synthase is negatively associated with overall survival (p = 0.001) and progression-free period (p = 0.004) in patients with diffuse large B-cell lymphoma. Multivariate analysis showed that fatty acid synthase overexpression is an independent prognostic marker of aggressive clinical course. For the first time, we report CD36 as an independent protective factor in patients treated with rituximab. Thus, fatty acid synthase and CD36 expression may serve as prognostic markers to predict response to treatment and survival in diffuse large B-cell lymphoma patients. Fatty acid synthase may also be a potential therapeutic target in lymphoid malignancies.

Abstract 4600: Examining the selective contribution of chromosomal instability to tumor evolution and prognosis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The specific role of Chromosomal Instability (CIN) in tumorigenesis has been a matter of conjecture. In part, this is due to the challenge of directly observing dynamic whole-chromosome mis-segregation events as well as the lack of firm understanding of the mechanisms that lead to CIN in cancer. These shortcomings have long prevented the ability to distinguish the role of CIN, which consists of increased rates of chromosome mis-segregation, from that of aneuploidy, which is a state of non-diploid chromosome number. To overcome these limitations, we use human-derived cancer cell lines to understand the biological basis of chromosome mis-segregation. We then extend these findings to tumor samples from patients diagnosed with Diffuse Large B-Cell Lymphoma to examine the selective contribution of CIN to tumor prognosis and evolution. We show that, in human cancer cell lines, chromosome mis-segregation occurs primarily as a result from defective microtubule dynamics, which prevent the correction of erroneous attachments of microtubules to chromosomes during mitosis. These attachment errors lead to the formation of lagging chromosomes and chromatin bridges during anaphase. Strikingly, restoring normal microtubule dynamics significantly decreases chromosome mis-segregation frequencies and suppresses CIN. We then use lagging chromosomes and chromatin bridges as morphological features to examine the selective contribution of CIN to tumor prognosis. Hematoxylin and Eosin-stained samples from a cohort of 54 patients diagnosed with Diffuse Large B-Cell Lymphoma (DLBCL) are used to examine the relationship between frequencies of chromosome mis-segregation and patient prognosis, overall survival, and response to treatment. We show that a two-fold increase in the frequency of chromosome mis-segregation leads to a 24% decrease in overall survival and 48% decrease in relapse-free survival after treatment. The hazard ratio (HR) of death in patients with increased chromosome mis-segregation is 2.31 and these patients are more likely to present with higher tumor stage, exhibit tumor bone marrow involvement, and receive a higher International Prognostic Index (IPI) score. In summary, this work demonstrates that CIN primarily arises due to defects in the attachments of microtubule to chromosomes. Furthermore, increased rates of chromosome mis-segregation in DLBCL substantiate inferior outcome and poor prognosis. This is likely due to increased heterogeneity of tumor cells leading to a larger predilection for adaptation in response to external pressures such as metastasis and drug treatments. We propose that targeting CIN would yield improved prognosis and enhanced response to chemotherapeutic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4600. doi:1538-7445.AM2012-4600