DAP Kinase-Related Apoptosis-Inducing Protein Kinase 2 (DRAK2) Is a Key Regulator and Molecular Marker in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World and it is characterized by a marked degree of clinical heterogeneity. An impaired balance between pro- and anti-apoptotic stimuli determines chemorefractoriness and outcome. The low proliferation rate of CLL cells indicates that one of the primary mechanisms involved in disease development may be an apoptotic failure. Here, we study the clinical and functional significance of DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in CLL. We have analyzed CLL patient samples and showed that low expression levels of DRAK2 were significantly associated with unfavorable outcome in our CLL cohort. DRAK2 expression levels showed a positive correlation with the expression of DAPK1, and TGFBR1. Consistent with clinical data, the downregulation of DRAK2 in MEC-1 CLL cells strongly increased cell viability and proliferation. Further, our transcriptome data from MEC-1 cells highlighted MAPK, NF-κB, and Akt and as critical signaling hubs upon DRAK2 knockdown. Taken together, our results indicate DRAK2 as a novel marker of CLL survival that plays key regulatory roles in CLL prognosis

Low BACH2 Expression Predicts Adverse Outcome in Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a highly variable clinical outcome. There are well‐established CLL prognostic biomarkers that have transformed treatment and improved the understanding of CLL biology. Here, we have studied the clinical significance of two crucial B cell regulators, BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) and BCL6 (B‐cell CLL/lymphoma 6), in a cohort of 102 CLL patients and determined the protein interaction networks that they participate in using MEC‐1 CLL cells. We observed that CLL patients expressing low levels of BCL6 and BACH2 RNA had significantly shorter overall survival (OS) than high BCL6‐ and BACH2‐expressing cases. Notably, their low expression specifically decreased the OS of immunoglobulin heavy chain variable region‐mutated (IGHV‐M) CLL patients, as well as those with 11q and 13q deletions. Similar to the RNA data, a low BACH2 protein expression was associated with a significantly shorter OS than a high expression. There was no direct interaction observed between BACH2 and BCL6 in MEC‐1 CLL cells, but they shared protein networks that included fifty different proteins. Interestingly, a prognostic index (PI) model that we generated, using integrative risk score values of BACH2 RNA expression, age, and 17p deletion status, predicted patient outcomes in our cohort. Taken together, these data have shown for the first time a possible prognostic role for BACH2 in CLL and have revealed protein interaction networks shared by BCL6 and BACH2, indicating a significant role for BACH2 and BCL6 in key cellular processes, including ubiquitination mediated B‐cell receptor functions, nucleic acid metabolism, protein degradation, and homeostasis in CLL biology

Non-genotoxic MDM2 inhibition selectively induces a pro-apoptotic p53 gene signature in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous hematologic malignancy. In approximately 90% of cases the TP53 gene is in its wildtype state at diagnosis of this malignancy. As mouse double-minute-2 homolog (MDM2) is a primary repressor of p53, targeting this protein is an attractive therapeutic approach for non-genotoxic reactivation of p53. Since the discovery of the first MDM2 inhibitor, Nutlin-3a, newer potent and bioavailable compounds have been developed. In this study we tested the second-generation MDM2 inhibitor, RG7388, in patient-derived CLL cells and normal cells, examining its effect on the induction of p53-transcriptional targets. RG7388 potently decreased viability in p53-functional CLL cells, whereas p53-non-functional samples were more resistant to the drug. RG7388 induced a pro-apoptotic gene expression signature with upregulation of p53-target genes involved in the intrinsic (PUMA, BAX) and extrinsic (TNFRSF10B, FAS) pathways of apoptosis, as well as MDM2. Only a slight induction of CDKN1A was observed and upregulation of pro-apoptotic genes dominated, indicating that CLL cells are primed for p53-dependent apoptosis. Consequently, RG7388 led to a concentration-dependent increase in caspase-3/7 activity and cleaved poly (ADP-ribose) polymerase. Importantly, we observed a preferential pro-apoptotic signature in CLL cells but not in normal blood and bone marrow cells, including CD34+ hematopoietic cells. These data support the further evaluation of MDM2 inhibitors as a novel additional treatment option for patients with p53-functional CLL

Splicing Modulation Results in Aberrant Isoforms and Protein Products of p53 Pathway Genes and the Sensitization of B Cells to Non-Genotoxic MDM2 Inhibition

Several molecular subtypes of cancer are highly dependent on splicing for cell survival. There is a general interest in the therapeutic targeting of splicing by small molecules. E7107, a first-in-class spliceosome inhibitor, showed strong growth inhibitory activities against a large variety of human cancer xenografts. Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous hematologic malignancy, with approximately 90% of cases being TP53 wild-type at diagnosis. An increasing number of studies are evaluating alternative targeted agents in CLL, including MDM2–p53 binding antagonists. In this study, we report the effect of splicing modulation on key proteins in the p53 signalling pathway, an important cell death pathway in B cells. Splicing modulation by E7107 treatment reduced full-length MDM2 production due to exon skipping, generating a consequent reciprocal p53 increase in TP53WT cells. It was especially noteworthy that a novel p21WAF1 isoform with compromised cyclin-dependent kinase inhibitory activity was produced due to intron retention. E7107 synergized with the MDM2 inhibitor RG7388, via dual MDM2 inhibition; by E7107 at the transcript level and by RG7388 at the protein level, producing greater p53 stabilisation and apoptosis. This study provides evidence for a synergistic MDM2 and spliceosome inhibitor combination as a novel approach to treat CLL and potentially other haematological malignancies

Analysis of SF3B1 mutations in monoclonal B-cell lymphocytosis

[No abstract available

Inter- and intra-patient clonal and subclonal heterogeneity of chronic lymphocytic leukaemia: evidences from circulating and lymph nodal compartments

Whole exome sequencing and copy number aberration (CNA) analysis were performed on cells taken from peripheral blood (PB) and lymph nodes (LN) of patients with chronic lymphocytic leukaemia (CLL). Of 64 non-silent somatic mutations, 54 (84·4%) were clonal in both compartments, 3 (4·7%) were PB-specific and 7 (10·9%) were LN-specific. Most of the LN- or PB-specific mutations were subclonal in the other corresponding compartment (variant frequency 0·5-5·3%). Of 41 CNAs, 27 (65·8%) were shared by both compartments and 7 (17·1%) were LN- or PB-specific. Overall, 6 of 9 cases (66·7%) showed genomic differences between the compartments. At subsequent relapse, Case 10, with 6 LN-specific lesions, and Case 100, with 6 LN-specific and 8 PB-specific lesions, showed, in the PB, the clonal expansion of LN-derived lesions with an adverse impact: SF3B1 mutation, BIRC3 deletion, del8(p23·3-p11·1), del9(p24·3-p13·1) and gain 2(p25·3-p14). CLL shows an intra-patient clonal heterogeneity according to the disease compartment, with both LN and PB-specific mutations/CNAs. The LN microenvironment might contribute to the clonal selection of unfavourable lesions, as LN-derived mutations/CNAs can appear in the PB at relapse