Chromatin activation profiling of stereotyped chronic lymphocytic leukemias reveals a subset 8-specific signature.

The chromatin activation landscape of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor immunoglobulin is currently unknown. In this study, we report the results of a whole-genome chromatin profiling of histone 3 lysine 27 acetylation of 22 CLLs from major subsets, which were compared against nonstereotyped CLLs and normal B-cell subpopulations. Although subsets 1, 2, and 4 did not differ much from their nonstereotyped CLL counterparts, subset 8 displayed a remarkably distinct chromatin activation profile. In particular, we identified 209 de novo active regulatory elements in this subset, which showed similar patterns with U-CLLs undergoing Richter transformation. These regions were enriched for binding sites of 9 overexpressed transcription factors. In 78 of 209 regions, we identified 113 candidate overexpressed target genes, 11 regions being associated with more than 2 adjacent genes. These included blocks of up to 7 genes, suggesting local coupregulation within the same genome compartment. Our findings further underscore the uniqueness of subset 8 CLL, notable for the highest risk of Richter's transformation among all CLLs and provide additional clues to decipher the molecular basis of its clinical behavior.

The TΑp63/BCL2 axis represents a novel mechanism of clinical aggressiveness in chronic lymphocytic leukemia.

The TA-isoform of the p63 transcription factor (TAp63) has been reported to contribute to clinical aggressiveness in chronic lymphocytic leukemia (CLL) in a hitherto elusive way. Here, we sought to further understand and define the role of TAp63 in the pathophysiology of CLL. First, we found that elevated TAp63 expression levels are linked with adverse clinical outcomes, including disease relapse and shorter time-to-first treatment and overall survival. Next, prompted by the fact that TAp63 participates in an NF-κB/TAp63/BCL2 antiapoptotic axis in activated mature, normal B cells, we explored molecular links between TAp63 and BCL2 also in CLL. We documented a strong correlation at both the protein and the messenger RNA (mRNA) levels, alluding to the potential prosurvival role of TAp63. This claim was supported by inducible downregulation of TAp63 expression in the MEC1 CLL cell line using clustered regularly interspaced short palindromic repeats (CRISPR) system, which resulted in downregulation of BCL2 expression. Next, using chromatin immunoprecipitation (ChIP) sequencing, we examined whether BCL2 might constitute a transcriptional target of TAp63 and identified a significant binding profile of TAp63 in the BCL2 gene locus, across a genomic region previously characterized as a super enhancer in CLL. Moreover, we identified high-confidence TAp63 binding regions in genes mainly implicated in immune response and DNA-damage procedures. Finally, we found that upregulated TAp63 expression levels render CLL cells less responsive to apoptosis induction with the BCL2 inhibitor venetoclax. On these grounds, TAp63 appears to act as a positive modulator of BCL2, hence contributing to the antiapoptotic phenotype that underlies clinical aggressiveness and treatment resistance in CLL.

RPS15 mutations rewire RNA translation in chronic lymphocytic leukemia.

Recent studies of chronic lymphocytic leukemia (CLL) have reported recurrent mutations in the RPS15 gene, which encodes the ribosomal protein S15 (RPS15), a component of the 40S ribosomal subunit. Despite some evidence about the role of mutant RPS15 (mostly obtained from the analysis of cell lines), the precise impact of RPS15 mutations on the translational program in primary CLL cells remains largely unexplored. Here, using RNA sequencing and ribosome profiling, a technique that involves measuring translational efficiency, we sought to obtain global insight into changes in translation induced by RPS15 mutations in CLL cells. To this end, we evaluated primary CLL cells from patients with wild-type or mutant RPS15 as well as MEC1 CLL cells transfected with mutant or wild-type RPS15. Our data indicate that RPS15 mutations rewire the translation program of primary CLL cells by reducing their translational efficiency, an effect not seen in MEC1 cells. In detail, RPS15 mutant primary CLL cells displayed altered translation efficiency of other ribosomal proteins and regulatory elements that affect key cell processes, such as the translational machinery and immune signaling, as well as genes known to be implicated in CLL, hence highlighting a relevant role for RPS15 in the natural history of CLL.

Control of PD-L1 expression in CLL-cells by stromal triggering of the Notch-c-Myc-EZH2 oncogenic signaling axis.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Emerging data suggest that CLL-cells efficiently evade immunosurveillance. T-cell deficiencies in CLL include immuno(metabolic) exhaustion that is achieved by inhibitory molecules, with programmed cell death 1/programmed cell death ligand 1 (PD-L1) signaling emerging as a major underlying mechanism. Moreover, CLL-cells are characterized by a close and recurrent interaction with their stromal niches in the bone marrow and lymph nodes. Here, they receive nurturing signals within a well-protected environment. We could previously show that the interaction of CLL-cells with stroma leads to c-Myc activation that is followed by metabolic adaptations. Recent data indicate that c-Myc also controls expression of the immune checkpoint molecule PD-L1. Therefore, we sought out to determine the role of stromal contact for the CLL-cells' PD-L1 expression and thus their immuno-evasive phenotype.To do so, we analyzed PD-L1 expression on CLL cell (subsets) in untreated patients and on healthy donor-derived B-cells. Impact of stromal contact on PD-L1 expression on CLL-cells and the underlying signaling pathways were assessed in well-established in vitro niche models. Ex vivo and in vitro findings were validated in the Eµ-TCL1 transgenic CLL mouse model.We found increased PD-L1 expression on CLL-cells as compared with B-cells that was further enhanced in a cell-to-cell contact-dependent manner by stromal cells. In fact, circulating recent stromal-niche emigrants displayed higher PD-L1 levels than long-time circulating CLL-cells. Using our in vitro niche model, we show that a novel Notch-c-Myc-enhancer of zeste homolog 2 (EZH2) signaling axis controls PD-L1 upregulation. Ultimately, elevated PD-L1 levels conferred increased resistance towards activated autologous T-cells.In summary, our findings support the notion that the CLL microenvironment contributes to immune escape variants. In addition, several targetable molecules (eg, Notch or EZH2) could be exploited in view of improving immune responses in patients with CLL, which warrants further in-depth investigation.

Stem cell factor is implicated in microenvironmental interactions and cellular dynamics of chronic lymphocytic leukemia.

The inflammatory cytokine stem cell factor (SCF, ligand of c-kit receptor) has been implicated as a pro-oncogenic driver and an adverse prognosticator in several human cancers. Increased SCF levels have recently been reported in a small series of patients with chronic lymphocytic leukemia (CLL), however its precise role in CLL pathophysiology remains elusive. In this study, CLL cells were found to express predominantly the membrane isoform of SCF, which is known to elicit a more robust activation of the c-kit receptor. SCF was significantly overexpressed in CLL cells compared to healthy tonsillar B cells and it correlated with adverse prognostic biomarkers, shorter time-to-first treatment and shorter overall survival. Activation of immune receptors and long-term cell-cell interactions with the mesenchymal stroma led to an elevation of SCF primarily in CLL cases with an adverse prognosis. Contrariwise, suppression of oxidative stress and the BTK inhibitor ibrutinib lowered SCF levels. Interestingly, SCF significantly correlated with mitochondrial dynamics and hypoxia-inducible factor-1a which have previously been linked with clinical aggressiveness in CLL. SCF was able to elicit direct biological effects in CLL cells, affecting redox homeostasis and cell proliferation. Overall, the aberrantly expressed SCF in CLL cells emerges as a key response regulator to microenvironmental stimuli while correlating with poor prognosis. On these grounds, specific targeting of this inflammatory molecule could serve as a novel therapeutic approach in CLL.

Increased proportion and altered properties of intermediate monocytes in the peripheral blood of patients with lower risk Myelodysplastic Syndrome.

Immune deregulation has a critical role in the pathogenesis of lower risk myelodysplastic syndromes (MDS). The cells of the macrophage/monocyte lineage have been reported to contribute to the inflammatory process in MDS through impaired phagocytosis of the apoptotic hemopoietic cells and abnormal production of cytokines. In the present study we assessed the number of peripheral blood (PB) monocyte subsets, namely the classical CD14bright/CD16-, intermediate CD14bright/CD16+ and non-classical CD14dim/CD16+ cells, in patients with lower risk (low/intermediate-I) MDS (n = 32). We also assessed the production of tumor necrosis factor (TNF)α by patient PB monocytes in response to immune stimulus as well as their transcriptome profile. Compared to age- and sex-matched healthy individuals (n = 19), MDS patients had significantly lower number of classical and increased number of intermediate monocytes. Patient intermediate monocytes displayed increased production of TNFα following stimulation with lipopolysaccharide, compared to healthy individuals. Transcriptional profiling comparison of CD16+ monocytes from patients and controls revealed 43 differentially expressed genes mostly associated with biological pathways/processes relevant to hemopoiesis, immune signaling and cell adhesion. These data provide evidence for the first-time that distinct monocyte subsets display abnormal quantitative and functional characteristics in lower risk MDS substantiating their role in the immune deregulation associated with the disease.

Aspartic Aminopeptidase Is a Novel Biomarker of Aggressive Chronic Lymphocytic Leukemia.

Treatment of chronic lymphocytic leukemia has advanced substantially as our understanding of the kinase signal transduction pathways driven by the B cell receptor (BcR) has developed. Particularly, understanding the role of Bruton tyrosine kinase and phosphatidyl inositol 3 kinase delta in driving prosurvival signal transduction in chronic lymphocytic leukemia (CLL) cells and their targeting with pharmacological inhibitors (ibrutinib and idelalisib, respectively) has improved patient outcomes significantly. The kinase signaling pathway induced by the BcR is highly complex and has multiple interconnecting branches mediated by tyrosine and serine/threonine kinases activated downstream of the BcR. There is a high level of redundancy in the biological responses, with several BcR-signaling kinases driving nuclear factor kappa B activation or inducing antiapoptotic Bcl-2 genes. Accordingly, common gene targets of BcR-signaling kinases may serve as biomarkers indicating enhanced BCR-signaling and aggressive disease progression. This study used a gene expression correlation analysis of malignant B cell lines and primary CLL cells to identify genes whose expression correlated with BCR-signaling kinases overexpressed and/or overactivated in CLL, namely: AKT1, AKT2, BTK, MAPK1, MAPK3, PI3KCD and ZAP70. The analysis identified a 32-gene signature with a strong prognostic potential and DNPEP, the gene coding for aspartic aminopeptidase, as a predictor of aggressive CLL. DNPEP gene expression correlated with MAPK3, PI3KCD, and ZAP70 expression and, in the primary CLL test dataset, showed a strong prognostic potential. The inhibition of DNPEP with a pharmacological inhibitor enhanced the cytotoxic potential of idelalisib and ibrutinib, indicating a biological functionality of DNPEP in CLL. DNPEP, as an aminopeptidase, contributes to the maintenance of the free amino acid pool in CLL cells found to be an essential process for the survival of many cancer cell types, and thus, these results warrant further research into the exploitation of aminopeptidase inhibitors in the treatment of drug-resistant CLL.

MeinteR: A framework to prioritize DNA methylation aberrations based on conformational and cis-regulatory element enrichment.

DNA methylation studies have been reformed with the advent of single-base resolution arrays and bisulfite sequencing methods, enabling deeper investigation of methylation-mediated mechanisms. In addition to these advancements, numerous bioinformatics tools address important computational challenges, covering DNA methylation calling up to multi-modal interpretative analyses. However, contrary to the analytical frameworks that detect driver mutational signatures, the identification of putatively actionable epigenetic events remains an unmet need. The present work describes a novel computational framework, called MeinteR, that prioritizes critical DNA methylation events based on the following hypothesis: critical aberrations of DNA methylation more likely occur on a genomic substrate that is enriched in cis-acting regulatory elements with distinct structural characteristics, rather than in genomic "deserts". In this context, the framework incorporates functional cis-elements, e.g. transcription factor binding sites, tentative splice sites, as well as conformational features, such as G-quadruplexes and palindromes, to identify critical epigenetic aberrations with potential implications on transcriptional regulation. The evaluation on multiple, public cancer datasets revealed significant associations between the highest-ranking loci with gene expression and known driver genes, enabling for the first time the computational identification of high impact epigenetic changes based on high-throughput DNA methylation data.

DNA methylation profiles in chronic lymphocytic leukemia patients treated with chemoimmunotherapy.

BACKGROUND: In order to gain insight into the contribution of DNA methylation to disease progression of chronic lymphocytic leukemia (CLL), using 450K Illumina arrays, we determined the DNA methylation profiles in paired pre-treatment/relapse samples from 34 CLL patients treated with chemoimmunotherapy, mostly (n = 31) with the fludarabine-cyclophosphamide-rituximab (FCR) regimen. RESULTS: The extent of identified changes in CLL cells versus memory B cells from healthy donors was termed "epigenetic burden" (EB) whereas the number of changes between the pre-treatment versus the relapse sample was termed "relapse changes" (RC). Significant (p < 0.05) associations were identified between (i) high EB and short time-to-first-treatment (TTFT); and, (ii) few RCs and short time-to-relapse. Both the EB and the RC clustered in specific genomic regions and chromatin states, including regulatory regions containing binding sites of transcription factors implicated in B cell and CLL biology. CONCLUSIONS: Overall, we show that DNA methylation in CLL follows different dynamics in response to chemoimmunotherapy. These epigenetic alterations were linked with specific clinical and biological features.

EZH2 upregulates the PI3K/AKT pathway through IGF1R and MYC in clinically aggressive chronic lymphocytic leukaemia.

EZH2 is overexpressed in poor-prognostic chronic lymphocytic leukaemia (CLL) cases, acting as an oncogene; however, thus far, the EZH2 target genes in CLL have not been disclosed. In this study, using ChIP-sequencing, we identified EZH2 and H3K27me3 target genes in two prognostic subgroups of CLL with distinct prognosis and outcome, i.e., cases with unmutated (U-CLL, n = 6) or mutated IGHV genes (M-CLL, n = 6). While the majority of oncogenic pathways were equally enriched for EZH2 target genes in both prognostic subgroups, PI3K pathway genes were differentially bound by EZH2 in U-CLL versus M-CLL. The occupancy of EZH2 for selected PI3K pathway target genes was validated in additional CLL samples (n = 16) and CLL cell lines using siRNA-mediated EZH2 downregulation and ChIP assays. Intriguingly, we found that EZH2 directly binds to the IGF1R promoter along with MYC and upregulates IGF1R expression in U-CLL, leading to downstream PI3K activation. By investigating an independent CLL cohort (n = 96), a positive correlation was observed between EZH2 and IGF1R expression with higher levels in U-CLL compared to M-CLL. Accordingly, siRNA-mediated downregulation of either EZH2, MYC or IGF1R and treatment with EZH2 and MYC pharmacological inhibitors in the HG3 CLL cell line induced a significant reduction in PI3K pathway activation. In conclusion, we characterize for the first time EZH2 target genes in CLL revealing a hitherto unknown implication of EZH2 in modulating the PI3K pathway in a non-canonical, PRC2-independent way, with potential therapeutic implications considering that PI3K inhibitors are effective therapeutic agents for CLL.

Study of gene expressions' correlation structures in subgroups of Chronic Lymphocytic Leukemia Patients.

In chronic lymphocytic leukemia (CLL) the interaction of leukemic cells with the microenvironment ultimately affects patient outcome. CLL cases can be divided in two subgroups with different clinical course based on the mutational status of the immunoglobulin heavy variable (IGHV) genes: mutated CLL (M-CLL) and unmutated CLL (U-CLL). Since in CLL, the differentiated relation of genes between the two subgroups is of greater importance than the individual gene behavior, this paper investigates the differences between the groups' gene interactions, by comparing their correlation structures. Fisher's test and Zou's confidence intervals are employed to detect differences of correlation coefficients. Afterwards, networks created by the genes participating in most differences are estimated with the use of structural equation models (SEM). The analysis is enhanced with graph modeling in order to visualize the between group differences in the gene structures of the two subgroups. The applied methodology revealed stronger correlations between genes in U-CLL patients, a finding in line with related biomedical literature. Using SEM for multigroup analysis, different gene structures between the two groups of patients were confirmed. The study of correlation structures can facilitate the detection of differential gene expression profiles in CLL subgroups, with potential applications in other diseases. Comparison of correlations can be very useful in understanding the complex internal structural differences which signify the variations of a disease.

Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness.

The inhibitory receptor toll interleukin-1R 8 (TIR8/IL-1R8/SIGIRR) is downregulated in chronic lymphocytic leukemia.

Toll interleukin-1 receptor 8 (also known as TIR8, SIGIRR, or IL1R8) is a transmembrane receptor that inhibits inflammation. Accordingly, genetic inactivation of this protein exacerbates chronic inflammation and inflammation-associated tumors in mice. In particular, lack of TIR8 triggers leukemia progression in a mouse model of chronic lymphocytic leukemia (CLL), supporting its role as a novel tumor restrainer. The aim of this study was to measure the amount of TIR8 mRNA and protein in CLL cells, and to analyze its regulation of expression. Circulating leukemic cells expressed lower levels of TIR8 compared to normal B-lymphocytes. Treatment of CLL cells with Azacytidine restored higher levels of TIR8 suggesting that DNA methylation may be involved in modulating TIR8 expression, with implications for novel therapeutic strategies.

NF-κB activation in chronic lymphocytic leukemia: A point of convergence of external triggers and intrinsic lesions.

The nuclear factor-κB (NF-κB) pathway is constitutively activated in chronic lymphocytic leukemia (CLL) patients, and hence plays a major role in disease development and evolution. In contrast to many other mature B-cell lymphomas, only a few recurrently mutated genes involved in canonical or non-canonical NF-κB activation have been identified in CLL (i.e. BIRC3, MYD88 and NFKBIE mutations) and often at a low frequency. On the other hand, CLL B cells seem 'addicted' to the tumor microenvironment for their survival and proliferation, which is primarily mediated by interaction through a number of cell surface receptors, e.g. the B-cell receptor (BcR), Toll-like receptors and CD40, that in turn activate downstream NF-κB. The importance of cell-extrinsic triggering for CLL pathophysiology was recently also highlighted by the clinical efficacy of novel drugs targeting microenvironmental interactions through the inhibition of BcR signaling. In other words, CLL can be considered a prototype disease for studying the intricate interplay between external triggers and intrinsic aberrations and their combined impact on disease evolution. In this review, we will discuss the current understanding of mechanisms underlying NF-κB deregulation in CLL, including micro-environmental, genetic and epigenetic events, and summarize data generated in murine models resembling human CLL. Finally, we will also discuss different strategies undertaken to intervene with the NF-κB pathway and its upstream mediators.

The histone methyltransferase EZH2 as a novel prosurvival factor in clinically aggressive chronic lymphocytic leukemia.

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 overexpression has been reported in many types of cancer and associated with poor prognosis. Here we investigated the expression and functionality of EZH2 in chronic lymphocytic leukemia (CLL). Aggressive cases with unmutated IGHV genes (U-CLL) displayed significantly higher EZH2 expression compared to indolent CLL cases with mutated IGHV genes (M-CLL); furthermore, in U-CLL EZH2 expression was upregulated with disease progression. Within U-CLL, EZH2high cases harbored significantly fewer (p = 0.033) TP53 gene abnormalities compared to EZH2low cases. EZH2high cases displayed high H3K27me3 levels and increased viability suggesting that EZH2 is functional and likely confers a survival advantage to CLL cells. This argument was further supported by siRNA-mediated downmodulation of EZH2 which resulted in increased apoptosis. Notably, at the intraclonal level, cell proliferation was significantly associated with EZH2 expression. Treatment of primary CLL cells with EZH2 inhibitors induced downregulation of H3K27me3 levels leading to increased cell apoptosis. In conclusion, EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Pharmacologic inhibition of EZH2 catalytic activity promotes apoptosis, highlighting EZH2 as a novel potential therapeutic target for specific subgroups of patients with CLL.

B Cell Anergy Modulated by TLR1/2 and the miR-17∼92 Cluster Underlies the Indolent Clinical Course of Chronic Lymphocytic Leukemia Stereotyped Subset #4.

Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BCR Ig) display a particularly indolent disease course. Immunogenetic studies of the clonotypic BCR Ig of CLL subset #4 suggested a resemblance with B cells rendered anergic through chronic autoantigenic stimulation. In this article, we provide experimental evidence that subset #4 CLL cells show low IgG levels, constitutive ERK1/2 activation, and fail to either release intracellular Ca(2+) or activate MAPK signaling after BCR cross-linking, thus displaying a signature of B cell anergy at both biochemical and functional levels. Interestingly, TLR1/2 triggering restored BCR functionality, likely breaching the anergic state, and this was accompanied by induction of the miR-17∼92 cluster, whose members target critical BCR-associated molecules, including MAPKs. In conclusion, we demonstrate BCR anergy in CLL subset #4 and implicate TLR signaling and the miR-17∼92 cluster in the regulation of the anergic state. This detailed signaling profiling of subset #4 has implications for advanced understanding of the complex regulation of intracellular signaling pathways in CLL, currently a major therapeutic target of the disease.

Excessive antigen reactivity may underlie the clinical aggressiveness of chronic lymphocytic leukemia stereotyped subset #8.

Subset #8 is a distinctive subset of patients with chronic lymphocytic leukemia (CLL) defined by the expression of stereotyped IGHV4-39/IGKV1(D)-39 B-cell receptors. Subset #8 patients experience aggressive disease and exhibit the highest risk for Richter transformation among all CLL. In order to obtain biological insight into this behavior, we profiled the antigen reactivity and signaling capacity of subset #8 vs other clinically aggressive stereotyped subsets, namely subsets #1 and #2. Twenty-seven monoclonal antibodies (mAbs) from subsets #1, #2, and #8 CLL clones were prepared as recombinant human immunoglobulin G1 and used as primary antibodies in enzyme-linked immunosorbent assays against representatives of the major classes of established antigenic targets for CLL. Subset #8 CLL mAbs exhibited broad polyreactivity as they bound to all antigens tested, in clear contrast with the mAbs from the other subsets. Antigen challenge of primary CLL cells indicated that the promiscuous antigen-binding activity of subset #8 mAbs could lead to significant cell activation, again in contrast to the less responsive CLL cells from subsets #1 and #2. These features constitute a distinctive profile for CLL subset #8, supporting the existence of distinct mechanisms of aggressiveness in different immunogenetic subsets of CLL.