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.     

A new genetic abnormality leading to TP53 gene deletion in chronic lymphocytic leukaemia

The analysis of chromosomal abnormalities provides significant prognostic information in patients with chronic lymphocytic leukaemia (CLL), a disease with a highly heterogeneous clinical course. Chromosomal abnormalities commonly found are trisomy 12, del(13)(q14), del(11)(q22-23), del(17)(p13) and del(6)(q21). Translocations are present in some patients and affect regions recurrently involved in CLL. This report describes the clinical and pathological characteristics of four CLL patients showing a new recurrent chromosomal abnormality dic(8;17)(p11;p11), that implied loss of the TP53 gene in all cases. In addition, TP53 gene was mutated in three out of four patients. Mechanically, Low Copy Repeats (LCR) in 17p12 and 8p11 may explain the origin of the translocation by non-allelic homologous recombination (NAHR). Isolated dic(8;17)(p11;p11) in patients with mutated IGHV genes status may not have the same prognostic impact as other mutations or deletions affecting the TP53 gene. Larger series are needed to better evaluate the clinical impact of this chromosomal aberration during the course of the disease.     

Recent advances in therapy of chronic lymphocytic leukaemia

The last 5 to 10 years have been marked by considerable advances in both our understanding of the biology and treatment of chronic lymphocytic leukaemia (CLL). Fludarabine‐based immuno‐chemotherapy is the current standard of care for first line therapy in younger fit patients and although this can be highly effective its use in older co‐morbid patients is limited by toxicity, and the prognosis for patients with high risk or fludarabine‐refractory disease is poor. The introduction of new antibodies has however, facilitated the use of immuno‐chemotherapy in co‐morbid patients. Beyond this, the recognition that CLL cells are critically dependent on B‐cell receptor (BCR) signalling and interactions with the cellular micro‐environment for proliferation and survival has led to the investigation of BCR inhibitors in CLL treatment. These have been shown to be highly effective although a number of questions remain about how they should be optimally used in clinical practice.     

Jumping translocations,a novel finding in chronic lymphocytic leukaemia

A jumping translocation (JT) is a rare cytogenetic aberration that can occur in haematological malignancy. It involves the translocation of the same fragment of donor chromosome onto two or more recipient chromosomes, typically in different cells. In this study, we describe the first series of chronic lymphocytic leukaemia (CLL) patients with JTs reported to date. Following a review of 878 CLL patient karyotypes, we identified 26 patients (3%) with 97 JTs. The most commonly occurring breakpoint in these translocations was 17p11.2. Loss of TP53 was identified prior to or at the same time as JT in 23 of 26 patients (88%). All patients eventually developed a complex karyotype. All but one patient has required treatment for CLL, with estimated median time to treatment of 11·5 months. This study establishes JTs as a recurrent abnormality found in CLL patients with aggressive disease. JTs contribute to complex karyotypes and, in many cases, are involved in chromosomal rearrangements that result in loss of the tumour suppressor gene TP53.     

Chronic lymphocytic leukaemia with 17p deletion: a retrospective analysis of prognostic factors and therapy results

Patients with chronic lymphocytic leukaemia (CLL) whose tumour cells harbour a 17p deletion (17p‐) are universally considered to have a poor prognosis. The deletion can be detected at diagnosis or during the evolution of the disease, particularly in patients who have received chemotherapy. We sought to evaluate the natural history of patients with 17p‐ CLL, identify predictive factors within this prognostic subgroup, and evaluate the results of different therapeutic approaches. Data from 294 patients with 17p‐ CLL followed up at 20 different institutions was retrospectively collected and analysed. Median age was 68 (range 27–98) years at the time of fluorescence in situ hybridization analysis. After 17p‐ documentation, 52% received treatment, achieving an overall response rate of 50%. Median overall survival was 41 months, and was significantly shorter in patients with elevated beta₂‐microglobulin concentration (P < 0·001), B symptoms (P = 0·016), higher percentage of cells with deletion (P < 0·001), and acquired deletions (P = 0·012). These findings suggest that patients with 17p‐ CLL have a variable prognosis that can be refined using simple clinical and laboratory features, including 17p‐ clone size, beta2‐microglobulin concentration, presence of B symptoms and type of deletion (de novo versus acquired).     

B-cell chronic lymphocytic leukaemia with aberrant expression of CD8 antigen

The monoclonal antibodies against CD8 (T suppressor/cytotoxic) antigen (Leu2/OKT8) were found to bind to leukaemic lymphocytes from a patient with chronic lymphocytic leukaemia. The cells also had an unusual type of rod-like cytoplasmic immunoglobulin of IgM/lambda type as seen by light, fluorescence and electron microscopy, and displayed several antigens characteristic for B lymphocytes. Gene rearrangement analysis showed rearrangement of mu heavy chain gene. In spite of an expression of CD8 antigen, T-cell receptor genes were not rearranged.     

Breaking good: the inexorable rise of BTK inhibitors in the treatment of chronic lymphocytic leukaemia

Although expressed in several haematological lineages and involved in multiple different signalling pathways, Bruton tyrosine kinase (BTK) plays an indispensible role in B cells in signalling from the B cell receptor (BCR) for antigen. Many B cell malignancies remain dependent on constitutive BCR signalling, making BTK a functional therapeutic target. Several BTK inhibitors (BTKi) with different kinomes and modes of action are being assessed clinically. This review documents the efficacy and toxicity of BTKi in chronic lymphocytic leukaemia (CLL). Clinically, the furthest in development is ibrutinib (trade name, Imbruvica), an irreversible BTKi, which has shown spectacular preliminary efficacy, with rapid reductions in lymph nodes accompanied by peripheral blood lymphocytosis. The lymphocytosis resolves slowly and most patients do not enter a complete remission. Nevertheless, it is possible to maintain many CLL patients, even those with adverse cytogenetic features, on drug for many months with minimal toxicities, thus potentially transforming the therapeutic paradigms for CLL. The efficacy, lack of toxicity and oral administration of BTKi will ensure their adoption in a wide range of B cell malignancies. An outstanding challenge is to incorporate BTKi with other precision medicines in a mechanism‐based manner in order to dispense with conventional chemotherapy.     

Aberrantly activated anti-apoptotic signalling mechanisms in chronic lymphocytic leukaemia cells: clues to the identification of novel therapeutic targets

Chronic lymphocytic leukaemia (CLL) is the commonest haematological malignancy in the western world and is incurable by cytotoxic therapy. Considerable research effort has identified the signal transduction pathways in CLL cells that contribute to anti-apoptotic signalling. Some pathways are constitutively activated in CLL cells but upregulated in normal cells only when protein tyrosine kinases (PTKs) are activated by ligands. This review describes which PTKs are aberrantly activated in CLL cells and are potential targets for inhibition. Additional potential targets within pathways downstream of these PTKs include Mek/Erk, mTorc1, protein kinase C, PI-3 kinase/Akt, nuclear factor-κB and cyclin-dependent protein kinase. Numerous studies have identified chemical agents and antibodies that selectively kill CLL cells, irrespective of their genetic resistance to conventional chemotherapeutic agents, and which can overcome cytoprotective microenvironmental signalling. These studies have resulted in identification of novel therapies, some of which are currently undergoing clinical trials. In vitro and animal model studies and clinical trials could determine which inhibitors of which targets are the likely to be most effective and least toxic either singly or in combination.     

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c‐Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of Syk^Y348 phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol‐3‐kinase and mitogen‐activated protein kinase activation following BCR crosslinking, and blocked the Mcl‐1‐dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro‐apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)‐4 (CD154L/IL‐4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co‐culture to both fludarabine and chlorambucil, the addition of CD154 and IL‐4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17‐DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL‐4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen‐independent microenvironmental signals.     

A prognostic model for survival in chronic lymphocytic leukaemia based on p53 expression

As the abnormal expression of p53 protein is prognostically significant in some human cancers, its significance in patients with B-cell chronic lymphocytic leukaemia (CLL) was assessed. Two investigators evaluated the percentage of bone marrow mononuclear cells that stained for p53, using biopsies stained with anti-p53 monoclonal antibody (DO-7), and graded the degree of staining (0, +, ++, +++). Samples from a cohort of 90 patients with CLL were studied (median age 60 years, range 30-89 years; 57 patients were (63%) previously untreated, 22 patients (24%) had received one or two prior regimens, 11 patients had received (12%) three to seven regimens. The overall percentage of cells positive for p53 staining was a median of 43 (range 1-88). No investigator effect was detected either in overall percentage cells rated p53 positive or on the degree of staining (Pearson's correlation coefficient 0.980, P-value < 0.001). A Cox proportional hazards model showed that the percentage of ++ and +++ p53-positive cells correlated with various prognostic factors in CLL (P < 0.0001). A multivariate model incorporating prior therapy, Rai stage, beta2 microglobulin (beta2M) and p53 expression showed that only the percentage of p53-positive cells and beta2M were predictive of survival, and enabled the development of a highly predictive model of survival based on these two parameters.     

Differential control of G0 programme in chronic lymphocytic leukaemia: a novel prognostic factor

Chronic lymphocytic leukaemia (CLL) is a unique malignancy where quiescent B cells accumulate in the peripheral blood. Since clinical outcomes in CLL are very heterogeneous, it is of utmost importance to correctly assess the disease prognosis in each individual case. Recently, it has been shown that high ZAP-70 [Zeta-chain (T-cell receptor) associated protein kinase (70 kDa)] expression level strongly correlates with lack of IgV(H) mutations and poor prognosis in B-CLL. As CLL malignant cells are arrested in G(0), we investigated whether Dipeptidyl Peptidase 2 (DPP2), a serine protease that plays a key role in keeping cells in the quiescent state, is involved in cell-cycle control in CLL. We have previously shown that specific inhibition of DPP2 results in apoptosis of normal lymphocytes. In this study, cell apoptosis experiments were conducted in 38 patients with B-CLL. Two distinct subsets of B-CLL were identified, susceptible and resistant to DPP2-inhibition-induced apoptosis. If resistant to apoptosis (42.1%), the CLL cells have higher expression of ZAP-70 and exhibit a worse prognosis, such as shorter treatment-free time period. Thus, resistance vs. susceptibility to DPP2-inhibiton induced apoptosis can be employed as a novel prognostic factor in CLL.     

TP53 mutations and relevance of expression of TP53 pathway genes in paediatric acute myeloid leukaemia

Limited data are available on the incidence and impact of TP53 alterations and TP53 pathway deregulation in paediatric acute myeloid leukaemia (AML). We analysed TP53 alterations in bone marrow samples of 229 patients with de novo paediatric AML, and detected heterozygous missense exon mutations in two patients (1%) and 17p deletions of the TP53 gene in four patients (2%). These patients more frequently had complex karyotype (50% vs. 4%, P = 0·002) or adverse cytogenetic abnormalities, including complex karyotype (67% vs. 17%, P = 0·013), compared to TP53 wild-type. Differential expression of TP53 pathway genes was associated with poor survival, indicating a role for TP53 regulators and effector genes.