Forms Of Chronic Lymphocytic Leukemia Research Articles

Targeting the NTSR2/TrkB oncogenic pathway in chronic lymphocytic leukemia

Current therapies that target the B-cell receptor pathway or the inhibition of anti-apoptotic proteins do not prevent the progressive forms of chronic lymphocytic leukemia (CLL), have low long-term efficacy and are subject to therapeutic resistance. Deciphering the mechanisms of leukemic cell survival and searching for new specific targets therefore remain major challenges to improve the management of this disease. It was evidenced that NTSR2 (neurotensin receptor 2), through the recruitment of TRKB (tropomyosin related kinase B), induces survival pathways in leukemic B cells. We have investigated the therapeutic potential of this protein complex as a new target. The binding domain of NTSR2 and TRKB was identified and a peptide targeting the latter was designed. The peptide binds TRKB and efficiently decreases the interaction of the two proteins. It is also effectively internalized by CLL-B cells in which it notably affects Src family kinase signaling and anti-apoptotic proteins levels. It demonstrated a cytotoxic effect both in vitro on the MEC-1 cell line and ex vivo on a cohort of 30 CLL patients. Altogether, these results underline the therapeutic potential of the NTSR2/TRKB protein complex as a target in CLL and open new perspectives for the development of targeted therapies.

Abstract 3814: In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways

Abstract 5-10% of patients with the IGHV wild type form of chronic lymphocytic leukemia (CLL) carry a g.3A>C driver mutation in the U1 small nuclear RNA (snRNA). We investigatedthe patterns of mis-splicing and their pathway consequences in three U1-mutant CLL cellline models using long read sequencing in order to better understand the mechanisms ofoncogenicity in tumors carrying this mutation. CLL cell lines (HG3, JVM3 and MEC1) expressing the U1 mutation and their wild-type counterparts were submitted to Oxford Nanopore sequencing to generate full-length RNA transcripts. This transcriptomic datawas evaluated, together with previously obtained short-read data. The g.3A>C mutation occurs at a specific location of the U1 snRNA, a core component of the eukaryotic spliceosome, and acts by altering the 5' splice site (5'SS) recognition sequence to cause consistent patterns of mis-splicing. Long read analysis identified multiple instances of intron retention and suppression of exon skipping, and in silico translation of these mis-splicing events predicted stop-gain and other loss of function mutations in several expressed genes, as well as widespread changes in gene expression levels. We performed a pathway overrepresentation analysis of the altered gene expression patterns in the mutant cells using the Reactome knowledgebase and identified an enrichment in the processes of translation, non-sense mediated decay (NMD) and immune signaling, including interferon signaling. In particular, there was a marked down-regulation of genes related to ribosomal assembly and the translational machinery. A more comprehensiveanalysis of the U1 mutation phenotype in CLL may accelerate the development ofbetter therapeutic and diagnostic approaches in patients. Citation Format: Andrea Senff-Ribeiro, Fatemeh Almodaresi, Quang Trinh, Shimin Shuai, David Spaner, Xose S. Puente, Elias Campo, Lincoln D. Stein. In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3814.

COMPARISON OF INSULIN LIKE GROWTH FACTOR-1 LEVELS IN CHRONIC MYELOCYTIC LEUKEMIA PATIENTS RECEIVING IMATINIB AND NILOTINIB THERAPY WITH HEALTHY POPULATIONS

Introduction. CML is the most common form of chronic leukemia in Indonesia, whereas in Western countries it is more commonly found in the form of chronic lymphocytic leukemia (CLL). Chronic myelocytic leukemia (CML) is a chronic myeloproliferative disease with clonal abnormalities due to genetic changes in stem cell pluripotence. This disease is characterized by proliferation of granulocyte series without differentiation disorders. On the CML, the Philadelphia chromosome (Ph1 chr) is found in a reciprocal translocation 9.22 (t9; 22). Insulin-like Growth Factor-1 is a natural polypeptide in the human body that has similarities with insulin. IGF-1 plays an important role in the growth and development of tissues. As such, several studies have shown an association between IGF-1 and -2 circulation rates. IGF-1 plays an important role in terms of stimulating cell proliferation and inhibition of apoptosis. This affects the regulation of the body's physiological growth as well as pathological growth such as cancer. Until now there has been no research on IGF-1 levels in CML patients with imatinib and nilotinib treatment. Methods. This research is a cross-sectional study, by observing the status of exposure and simultaneous disease in individuals from a single population, in one period. The study was conducted in December 2019 at the Outpatient Clinic of H Adam Malik General Hospital Medan with the approval of the North Sumatera University Research Ethics Commission. Data were analyzed using SPSS where p <0.05 was considered significant. Results. This study showed that there were no differences in IGF-1 levels between CML patients who received imatinib and nilotinib therapy. The mean IGF-1 results were obtained in the imatinib group 107.43±19.70 and nilotinib 107.43±18.09. While the healthy population is 138.60±52.85. Conclusion. No significant differences were found in IGF-1 levels between CML patients receiving imatinib and nilotinib therapy with healthy populations.

Ibrutinib in the Treatment of Refractory Chronic Lymphocytic Leukemia

Background & Aims. This paper presents the results of the observational study of ibrutinib in patients with chronic lymphocytic leukemia (CLL), conducted in SP Botkin Municipal Clinical Hospital. The main objective was the analysis of complications of ibrutinib and identification of factors, influencing the dosage regimen; the secondary objective was the estimation of the total response to treatment, event-free and overall survival. Materials & Methods. The study included 96 patients with CLL with indications for ibrutinib therapy. The median age was 64,9 years (range 32-91 years), the study population consisted of 69 (72 %) men and 27 (28 %) women. The condition of 25 (26 %) patients according to the ECOG scale was of > 3 points. The disease of stage C were diagnosed in 36 (37 %) patients. Deletion of 17p/TP53 mutations were detected in 29 (33 %) of 87 patients. Seventy patients had refractory CLL. The median of the number of the lines of the previous therapy was 3 (range 1-9). Adverse events were assessed in accordance with the CTCAE criteria, version 4.0; the bleeding severity was evaluated using ITP-specific bleeding score; hematological complications were classified according to the recommendations of IWCLL-2008. Results. Ibrutinib was administered at a dosage of 420 mg per day daily until progression or intolerable toxicity. The median duration of ibrutinib therapy was 10.3 months. brutinib was shown to have moderate toxicity, mostly of grade I or II. The bleeding was the most frequent complication. Of the hematological complications, thrombocytopenia was the most common (35 %); neutropenia < 1 <sup>x</sup> 10<sup>9</sup>/L was observed in 4 patients. GIT complications were identified in 51 (53 %) patients. Atrial fibrillation was registered in 5 patients, who initially had sinus rhythm. The total of 144 infections were diagnosed in 64 (66 %) patients. Severe infections (> grade III) developed in 26 % of patients. The treatment response was assessed in 92 patients. The overall response to treatment was 89 %. Complete remission, partial remission and partial remission with lymphocytosis were achieved in 4 (4 %), 57 (62 %), and 21 (23 %) patients, respectively. The event-free survival and overall survival by the month 10 was 90 % and 91 %, respectively. For this observation period, ECOG status and the number of the lines of therapy prior to ibrutinib had the prognostic value. Conclusion. Ibrutinib was shown to have high efficiency in relapsed/refractory forms of CLL. The nature of the ibrutinib toxicity is fundamentally different from that of the conventional chemotherapy. The frequency of ibrutinib therapy complications and patients' non-compliance depends on the intensity of the previous treatment of CLL. Despite a short observation period, it can be concluded that ibrutinib had the greatest impact on the patient's quality of life when administered for the first relapse. The low toxicity of ibrutinib is likely to allow the combination with other antitumor agents.

Achieving Minimal Residual Disease Negative Status in CLL - a Good Point to Start Thinking about Life without Treatment: How to Check MRD Status?

Patients diagnosed with chronic lymphocytic leukemia (CLL) do not necessarily have to undergo intensive treatment. The implementation of the therapy depends on patient condition and the form of CLL which is determined using predictive and prognostic factors. Both indolent and aggressive forms of CLL should be monitored. To improve survival and the quality of life it is very important to know how the organism responds to the treatment. For this purpose, the estimation of the minimal residual disease (MRD) - the pool of persistent leukemia cells after or at the time of treatment was introduced. These cells resistant to treatment can lead to recurrence of the disease. There are several methods for MRD assessment, however in every case it is necessary to know the initial status of the tested alterations (chromosome aberrations, surface protein expression, etc.). It allows for assessment whether the treatment eliminated the leukemic cells. The most common method for MRD evaluation in CLL is flow cytometry. Intensive development of molecular biology techniques can contribute to implementation of other extremely sensitive and specific methods. This review presents the most recent state of the art concerning minimal residual disease in CLL, including methods that are used for MRD assessment.

Prognostic value of thymidine kinase activity in patients with chronic lymphocytic leukemia.

Thymidine kinase (TK) activity is a marker of biological activity that allows the indolent and aggressive forms of chronic lymphocytic leukemia (CLL) to be distinguished. The aims of the study were to determine the relationship between TK activity and clinical status and prognosis, as well as to compare its activity with that of other prognostic and predictive factors. TK activity was measured in patient sera at the time of diagnosis using the DiviTum method, with the mean value being 439 Du/L. A correlation was found between TK activity and risk of disease progression (p=0.045). The optimal discriminative value of TK activity in the prediction of CLL progression was found to be 600 Du/L. TK activity significantly differed between the patients who achieved complete remission and those who only partially responded to therapy. In 93% of patients without any response to treatment and 18 out of 20 patients with progressive disease, TK activity over 600 Du/L was noted. In addition, all of the 10 patients with 17p13 deletion displayed TK activity of over 600 Du/L (p=0.0004). High TK activity also correlated with elevated levels of LDH (p=0.001) and β2‑microglobulin (p=0.03) in the study group. The results of the study indicated the importance of TK activity as a prognostic factor in patients with CLL.

Global and MYC-Specific Translation Is Enhanced in Activated Chronic Lymphocytic Leukemia Cells Carrying NOTCH1 C.7541_7542delct Mutations

Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D’Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay (’Click-iT’ assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLLΔCT cohort" of U-CLL with NOTCH1ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19+CD5+ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLLWT cohort" of U-CLL with no NOTCH1ΔCT (VAF<1% in all cases) or additional genetic lesion other than 13q deletion. CLL cells were incubated with 7.5 μg/ml CpG-ODN 2006 for 24 hours and assays were performed at baseline, 3 and 24 hours. NOTCH1 pathway γ-secretase inhibition was performed with DAPT GSi. Results: The CLLΔCT cohort had higher sIgM levels (range 31-372 MFI, median 81 MFI) and signaling capacity (Fab’2 anti-IgM induced intracellular calcium mobilization sIgM [iCa2+] range 47-54%, median 51) than the CLLWT cohort (sIgM levels range 19-288 MFI, median 47 MFI; IgM iCa2+ range 2-78%, median 25%). Following TLR9-mediated cell activation, the CLLΔCT cohort had sustained NICD (NOTCH1-intracellular cleaved domain) protein accumulation for up to 24 hours and expressed higher NOTCH1 target gene HES1 (hairy enhancer of split) transcript levels than in the CLLWT cohort. These data indicated NOTCH1 canonical pathway sustainment in the CLLΔCT upon activation. Global mRNA translation after 24 hours in the presence of CpG was 11.5 fold higher than that without CpG in the CLLΔCT cohort and only 4 fold higher in the CLLWT cohort, revealing significantly higher levels of translation in CLLΔCT than in CLLWT (p=0.03). CpG-induced global mRNA translation in the CLLWT cohort was similar to that in the CLLΔCT cohort treated with CHX. By using CpG-induced global mRNA translation in the presence of CHX inhibitor as background levels for each group, DAPT GSiat 2.5 to 10 μM showed from 47% to 63% inhibition of the residual CpG-induced global translation in CLLΔCT (p<0.05), but no effect in CLLWT. Remarkably, c-MYC mRNA translation after 3 hour culture with CpG was higher in CLLΔCT than in CLLWT (p= 0.02), and a similar trend was maintained in the cases investigated at 24 hour. Treatment of CLLΔCT cells with DAPT GSi decreased expression of c-MYC in a dose-dependent manner. Conclusion: NOTCH1ΔCT mutations associate with a very aggressive clinical behavior in CLL. These results now indicate that pro-activating mutations of NOTCH1 pathway associate with increased global mRNA translation and c-MYC expression. They highlight a mechanism by which NOTCH1 pathway may induce c-MYC overexpression in CLL, likely leading to increased proliferation and survival. The association of increased NOTCH1 variant allele frequency with sIgM levels and signaling capacity indicate that these mechanisms are predominant in the less anergic subgroup of U-CLL and make NOTCH1 mediated c-MYC translation an attractive target for therapeutic inhibition. DisclosuresSteele:Portola Pharmaceuticals: Honoraria. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding.

A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.

Total Proteome Analysis Identifies Migration Defects as a Major Pathogenetic Factor in Immunoglobulin Heavy Chain Variable Region (IGHV)-unmutated Chronic Lymphocytic Leukemia

The mutational status of the immunoglobulin heavy chain variable region defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and unmutated (UM-CLL). To elucidate the molecular mechanisms underlying the adverse clinical outcome associated with UM-CLL, total proteomes from nine UM-CLL and nine M-CLL samples were analyzed by isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry. Based on the expression of 3521 identified proteins, principal component analysis separated CLL samples into two groups corresponding to immunoglobulin heavy chain variable region mutational status. Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells underexpressed proteins associated with cytoskeletal remodeling and overexpressed proteins associated with transcriptional and translational activity. Taken together, our findings indicate that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes, where they are exposed to proliferative stimuli. In keeping with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed a strong and specific association between UM-CLL and lymphadenopathy. Our study illustrates the potential of total proteome analysis to elucidate pathogenetic mechanisms in cancer.

Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development.

We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.

Total Proteome Analysis Identifies Migration Defects As a Major Pathogenetic Factor in IGHV-Unmutated Chronic Lymphocytic Leukemia

The mutational status of the immunoglobulin heavy chain variable region (IGHV) defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and un-mutated (UM-CLL). Patients with M-CLL usually have a favourable outcome whereas those with UM-CLL develop progressive disease and have shorter survival. However, the molecular mechanisms responsible for the more aggressive clinical behaviour associated with UM-CLL are not well understood. Here we describe the application of isobaric tags for relative and absolute quantification (iTRAQ) based mass spectrometry (MS) to analyse the total proteome of M-CLL and UM-CLL samples. This has enabled us to generate the largest quantity of proteomic information for CLL to date and, in particular, to directly compare the functions of differentially expressed proteins between UM-CLL and M-CLL cells through a systems biology approach.We isolated CLL cells from the peripheral blood from 18 CLL patients (9 UM-CLL, 9 M-CLL) and prepared cellular protein extracts which were digested and subjected to labelling with iTRAQ reagents, as previously described (Kitteringham et al, J Proteomics, 2010;73(8):1612-1631). Principal component analysis was used to assess variance across the data set generated by iTRAQ-MS. Statistical significance of the difference in the levels of expression of proteins between UM-CLL and M-CLL samples was determined using student T-test (2-tailed). Several differentially expressed proteins identified by iTRAQ-MS were also validated by immunoblotting. Computational analysis was performed to examine the functions of the differentially expressed proteins and their associated signalling pathways using the GeneGo pathway maps in the Metacore database (Thomson Reuters, NY, USA).Unsupervised clustering, based on the expression of 3521 identified proteins, separated CLL samples into two groups corresponding to IGHV mutational status. We identified 274 proteins that were differentially expressed between UM-CLL and M-CLL subgroups (p<0.05, Figure 1A). Hierarchical clustering based on the relative expression of differentially expressed proteins also separated individual CLL cases into two distinct clusters according to their IGHV status (Figure 1B). Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched (p<0.05) by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells under-expressed proteins associated with cytoskeletal remodelling and over-expressed proteins associated with transcriptional and translational activity. Taken together, these findings indicated that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes where they are exposed to proliferative stimuli. In agreement with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed that twice as many patients with UM-CLL than M-CLL had documented lymphadenopathy (50% v 24%; P<0.01). The association between UM-CLL and lymphadenopathy was not simply a reflection of increased tumour burden as there was no significant difference in the leukocyte count between the two groups (medians of 37 x 109/L and 28 x 109/L, respectively; P>0.05).In addition, other pathways that promote cell survival and proliferation in UM-CLL cells were also enriched by the differentially expressed proteins. These include the immune response pathway involving B-cell receptor (BCR) signalling (P=0.006), the endoplasmic reticulum (ER) stress response pathway (P=0.035) and the Wnt signalling pathway (P=0.006).Our study has shown that quantitative analysis of the total proteome by iTRAQ-MS was able to separate individual CLL cases according to IGHV status and explained the more aggressive clinical behaviour of UM-CLL and its particular sensitivity to novel therapeutic agents that induce anatomical displacement from the lymph node microenvironment, such as ibrutinib and idelalisib. Moreover, in keeping with the ability of proteomics to detect alterations in gene expression resulting from both transcriptional and post-transcriptional mechanisms, the study illustrates the considerable potential of iTRAQ-MS coupled with computational analysis to elucidate pathogenetic mechanisms and indicate therapeutic strategies in cancer. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-CD44 mAb for the treatment of B-cell chronic lymphocytic leukemia and other hematological malignancies: evaluation of WO2013063498

Introduction: The CD44 transmembrane glycoprotein family mediates cellular responses to the microenvironment through binding of hyaluronic acid (HA) and other proteins of the extracellular matrix. These interactions start intracellular signaling cascades that foster tumor growth, survival and spread.Areas covered: The patent concerns the use of a humanized anti-CD44 mAb (RG7356) to treat patients with aggressive forms of chronic lymphocytic leukemia (CLL). The RG7356 humanized antibody was designed to bind the constant region of CD44, preventing binding with HA. The interruption of this circuit in vitro is followed by the induction of caspase-dependent apoptosis in leukemic cells. In agreement with a functional association between CD44 and zeta-associated protein of 70 kDa (ZAP-70) in activating intracellular signaling, the strongest effects were observed in ZAP-70+ CLL cells, generally associated to a poor prognosis. Furthermore, these effects were confirmed using in vivo models, where CLL cells were xenografted in immunocompromised mice.Expert opinion: In summary, these studies suggest that this new humanized mAb may provide additional clinical benefit to CLL patients receiving current standard treatments, specifically to those with a more aggressive disease.

PPARα and fatty acid oxidation mediate glucocorticoid resistance in chronic lymphocytic leukemia

AbstractHigh-dose glucocorticoids (GCs) can be a useful treatment for aggressive forms of chronic lymphocytic leukemia (CLL). However, their mechanism of action is not well understood, and resistance to GCs is inevitable. In a minimal, serum-free culture system, the synthetic GC dexamethasone (DEX) was found to decrease the metabolic activity of CLL cells, indicated by down-regulation of pyruvate kinase M2 (PKM2) expression and activity, decreased levels of pyruvate and its metabolites, and loss of mitochondrial membrane potential. This metabolic restriction was associated with decreased size and death of some of the tumor cells in the population. Concomitant plasma membrane damage increased killing of CLL cells by DEX. However, the nuclear receptor peroxisome proliferator activated receptor α (PPARα), which regulates fatty acid oxidation, was also increased by DEX, and adipocyte-derived lipids, lipoproteins, and propionic acid protected CLL cells from DEX. PPARα and fatty acid oxidation enzyme inhibitors increased DEX-mediated killing of CLL cells in vitro and clearance of CLL xenografts in vivo. These findings suggest that GCs prevent tumor cells from generating the energy needed to repair membrane damage, fatty acid oxidation is a mechanism of resistance to GC-mediated cytotoxicity, and PPARα inhibition is a strategy to improve the therapeutic efficacy of GCs.

MicroRNA expression in chronic lymphocytic leukemia developing autoimmune hemolytic anemia

Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune hemolytic anemia (AIHA). However, the mechanisms governing the association between CLL and AIHA are poorly understood. MicroRNAs (miRNAs) have been associated with different clinico-biological forms of CLL and are also known to play a substantial role in autoimmunity. However, there are no studies correlating miRNA expression with the likelihood that patients with CLL will develop AIHA. In this study, we found that malignant B-cells from patients with CLL subsequently developing AIHA present nine down-regulated (i.e. miR-19a, miR-20a, miR-29c, miR-146b-5p, miR-186, miR-223, miR-324-3p, miR-484 and miR-660) miRNAs. Interestingly, two of these miRNAs (i.e. miR-20a and miR-146b-5p) are involved in autoimmune phenomena, and one (i.e. miR-146b-5p) in both autoimmunity and CLL. Furthermore, we demonstrated that miR-146b-5p modulates CD80, a molecule associated with the B–T-cell synapse and in restoration of the antigen presenting cell capacity of CLL cells.

Quantitative detection of zeta-chain-associated protein 70 expression in chronic lymphocytic leukemia

Overexpression of zeta-chain-associated protein 70 (ZAP-70) was recently recognized as an independent prognostic marker for the aggressive form of chronic lymphocytic leukemia (CLL). The objective of this study was to demonstrate the feasibility and implementation of quantitative detection of ZAP-70 protein in B cells to clearly distinguish patients with CLL with the aggressive form of the disease. B cells were isolated from patient blood and lysed. Released ZAP-70 protein was detected using an immunomagnetic fluorescence assay. The assay protocol was developed using Jurkat cells and recombinant ZAP-70 (rZAP-70). The limit of detection was determined to be lower than 125 Jurkat cells and 39 pg of rZAP-70 protein. The signal response was linear over a wide dynamic range, from 125 to 40 000 Jurkat cells per test (R2 = 0.9987) and from 0 to 40 000 pg rZAP-70 protein per test (R2 = 0.9928). The results from 20 patients with CLL correlated strongly with flow cytometry analysis. Concordance between the two methods for positive and negative results was 100% (7/7) and 92% (12/13), respectively, while the overall concordance between the two methods was 95%. The assay reported here is a simple, reliable and reproducible method for quantitative detection of ZAP-70 in patient leukemic cells, without the need for cell fixation or permeabilization. The ZAP-70 signal was linear over a wide dynamic range, which we believe enables quantitative assessment of small changes in ZAP-70 expression over the course of the disease and in response to therapeutic intervention.

Novel and Emerging Drugs for Rarer Chronic Lymphoid Leukaemias

Rarer chronic lymphoid leukaemias represent a challenge to the clinicians due to the limited information on their pathogenesis, difficulties on setting up prospective clinical trials and to their refractoriness to drugs used in the most common form of chronic lymphocytic leukaemia (CLL). In this review all these issues are addressed in three B-cell leukaemias: B-cell prolymphocytic leukaemia (B-PLL), hairy cell leukaemia (HCL) and HCL-variant and three T-cell leukaemias: T-cell prolymphocytic leukaemia (T-PLL), T-cell large granular lymphocytic leukaemia (T-cell LGLL) and adult T-cell leukaemia lymphoma (ATLL). Data will be presented on the natural history, current therapies and emerging drugs potentially useful in the treatment of patients with these leukaemias. Emphasis is made on: 1- the novel agents targeting a variety of B and T-cell antigens expressed on the surface of the leukaemic cells; these are either unconjugated monoclonal antibodies (McAb) such as Rituximab (anti-CD20), the second and third generation of anti-CD20 McAbs, Alemtuzumab (anti-CD52), Siplizumab (anti-CD2), Daclizumab (anti-CD25) and KW-0761, an anti-chemokine receptor 4 (CCR4) or McAbs conjugated to toxins such as CD22 linked to the pseudomonas exotoxin or radiolabelled McAb; 2- the use of new purine nucleosides such as nelarabine and 3- agents targeting deregulated genes in the leukaemic cells from these diseases such as the Poly (ADP-ribose) polymerase (PARP) Olarapib in T-PLL with deregulation of the ataxia telangiectasia mutated (ATM) gene. Data of phase I and II clinical studies with these agents as well as the potential and current use of other drugs are outlined.

Tcl1 interacts with Atm and enhances NF-κB activation in hematologic malignancies

AbstractThe T-cell leukemia/lymphoma 1 (TCL1) oncogene is a target of chromosomal translocations and inversions at 14q31.2, and its rearrangement in T cells causes T-cell prolymphocytic leukemias. TCL1 dysregulation in B cells is responsible for the development of an aggressive form of chronic lymphocytic leukemia (CLL), the most common human leukemia. We have investigated the mechanisms underlying the oncogenic functions of Tcl1 protein using a mass spectrometry approach and have identified Atm (ataxia-telangiectasia mutated) as a candidate Tcl1-interacting protein. The Tcl1-Atm complex formation was validated by coimmunoprecipitation experiments. Importantly, we show that the association of Atm with Tcl1 leads to enhanced IκBα phosphorylation and ubiquitination and subsequent activation of the NF-κB pathway. Our findings reveal functional cross-talk between Atm and Tcl1 and provide evidence for a novel pathway that could be targeted in leukemias and lymphomas.

Expression of myelomonocytic antigens in Egyptian B-cell chronic lymphocytic leukemia ( impact on prognosis)

Background B-cell chronic lymphocytic leukemia (CLL) is a clonal malignancy of mature B cells that displays immense clinical heterogeneity. Therefore, there has been considerable interest in identifying prognostic markers that can be used to distinguish those patients who may have an aggressive form of CLL. Clinical staging systems lack the ability to predict disease progression. Numerous cellular and molecular markers with potential prognostic and therapeutic significance have been identified. Among these markers are the myelomonocyte-associated markers whose expression on neoplastic lymphocytes in CLL has been reported to correlate with an unfavorable prognosis. Aim of the study We aimed to study the expression of the myelomonocytic markers CD13 and CD11b in Egyptian CLL patients in comparison with a healthy control group and assess their relation to other prognostic factors and patient outcomes. This study also aimed to reveal the association of CD38 and FMC7 with prognosis and disease progression. Participants and methods Expressions of CD13 and CD11b were detected by flow cytometry in 30 newly diagnosed CLL cases and 10 healthy individuals. Percentages of CD38-positive and FMC7-positive cells were also measured. Results CD13 was expressed in three cases (10%), whereas CD11b was expressed in four cases (13.3%) of CLL. CD38 was expressed in 13 cases (43.3%) and FMC7 in 11 cases (36.6%). None of the individuals in the healthy control group expressed CD13 or CD11b. The expression of CD13 and CD38 was significantly correlated with adverse results of complete blood count ( P P =0.04). Expression of all studied markers (CD13, CD11b, CD38, and FMC7) was significantly correlated with the presence of 11q deletion (11qdel) ( P =0.004, P P =0.001, and P P =0.005, 0.01, P P P =0.002, 0.01, P P P =0.02 and P P =0.01). Conclusion CD13 and CD11b expressions were found to be correlated with the standard adverse prognostic factors in CLL and with shorter survival, which questions their future possible role as prognostic markers in CLL. FMC7 and CD38 expression was also associated with unfavorable prognosis and poor outcome.

Expanded and highly active proliferation centers identify a histological subtype of chronic lymphocytic leukemia ("accelerated" chronic lymphocytic leukemia) with aggressive clinical behavior

The concept of "accelerated" chronic lymphocytic leukemia is frequently used by both pathologists and clinicians. However, neither histological criteria to define this form of chronic lymphocytic leukemia nor its clinical correlates and prognostic impact have been formally defined in large series of patients. Tissue biopsies from 100 patients with chronic lymphocytic leukemia were analyzed for the size of proliferation centers and their proliferation rate as assessed by mitosis count and Ki-67 immunostaining. Histological patterns were correlated with main clinico-biological features and outcome. A suspicion of disease transformation was the main reason for carrying out tissue biopsy, which was performed at a median time of 14 months (range, 0 to 204 months) after the diagnosis of chronic lymphocytic leukemia. The biopsy showed histological transformation to diffuse large B-cell lymphoma in 22 cases. In the remaining 78 patients, the presence of expanded proliferation centers (broader than a 20x field) and high proliferation rate (either >2.4 mitoses/proliferation center or Ki-67 >40%/proliferation center) predicted a poor outcome and were selected to define a highly proliferative group. Thus, 23 patients with either expanded proliferation centers or high proliferation rate were considered as having "accelerated" chronic lymphocytic leukemia. These patients displayed particular features, including higher serum lactate dehydrogenase levels and more frequently elevated ZAP-70 than "non-accelerated" cases. The median survival from biopsy of patients with "non-accelerated" chronic lymphocytic leukemia, "accelerated" chronic lymphocytic leukemia and transformation to diffuse large B-cell leukemia was 76, 34, and 4.3 months, respectively (P<0.001). The presence of expanded and/or highly active proliferation centers identifies a group of patients with "accelerated" chronic lymphocytic leukemia characterized by an aggressive clinical behavior.

Abstract 4091: Epigenetic silencing of microRNA in the pathobiology of CLL

Abstract MicroRNAs (miRs, miRNA) are a class of non-coding RNA that control gene expression at a post-transcriptional level. Specific and aberrant miRNA signatures distinguish indolent and relapsed forms of chronic lymphocytic leukemia (CLL). In this study, we focused on the expression and cellular targets of a set of microRNA (miR15a, miR-16, miR-29b and miR-101) that are differentially expressed in indolent and refractory disease. The tumor suppressor microRNAs, miR-15a and miR-16, are downregulated in 68% of all CLL and associated with an indolent course. These miRNA are located on an intron of the dleu2 gene within a genomic locus (chr13q) that is frequently deleted in leukemia. However, deletions in 13q usually involve only one allele and do not adequately account for the low expression levels of miR-15a and miR-16. We hypothesized that aberrant recruitment of the histone deacetylases could be a novel mechanism that drives the silencing of miR-15a and miR-16 in CLL. Correspondingly, exposure to a variety of small molecule histone deacetylase inhibitors led to the re-expression of miR-15a and miR-16 in 30% of samples that undergo silencing. We then evaluated the cellular targets of miR-15a and miR-16 in CLL. Ectopic expression of miR-15 and miR-16 in primary leukemia cells led to a loss of Bcl-2 and Mcl-1. However, pharmacological re-expression of these miRNA led to decreases in Mcl-1 protein, with no loss in Bcl-2. Losses in the levels of Mcl-1 were associated with induction of cell death in these leukemia cells. In contrast to miR-15a and miR-16, the microRNAs miR-29b and miR-101 are well expressed in indolent disease but become silenced in relapsed/refractory disease by unknown mechanisms. We identified that exposure to a variety of histone deacetylase inhibitors induced the re-expression of miR-29b and miR-101 in 30-50% of the downregulated samples. Chromatin immunoprecipitation experiments demonstrated that deacetylase inhibition led to an increase in permissive chromatin marks at the gene promoters for these miRNA. miR-29b is known to target Mcl-1, Tcl-1 as well as the DNA methyltransferases 3a and 3b. Ectopic expression of miR-29b in CLL cells led to the decrease of Mcl-1 protein. In parallel, expression of miR-101 led to the inhibition of EZH2 expression. Correspondingly, pharmacological re-expression of miR-29b and miR-101 was associated with losses in Mcl-1 and EZH2 in CLL cells and apoptotic death of these leukemia cells. These results identify a role for epigenetic mechanisms in the pathological silencing of miRNA in both indolent and relapsed CLL. They also offer a novel therapeutic strategy by which to reverse epigenetic silencing of miRNA for therapeutic benefit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4091.