IL-10 production by CLL cells is enhanced in the anergic IGHV mutated subset and associates with reduced DNA methylation of the IL10 locus.

Chronic lymphocytic leukemias (CLLs) with unmutated (U-CLL) or mutated (M-CLL) IGHV have variable features of immunosuppression, possibly influenced by those CLL cells activated to produce interleukin 10 (IL-10). The two subsets differ in their levels of anergy, defined by low surface immunoglobulin M levels/signaling capacity, and in their DNA methylation profile, particularly variable in M-CLL. We have now found that levels of IL-10 produced by activated CLL cells were highly variable. Levels were higher in M-CLL than in U-CLL and correlated with anergy. DNA methylation analysis of IL10 locus revealed two previously uncharacterized 'variably methylated regions' (CLL-VMRs1/2) in the gene body, but similarly low methylation in the promoter of both U-CLL and M-CLL. CLL-VMR1/2 methylation was lower in M-CLL than in U-CLL and inversely correlated with IL-10 induction. A functional signal transducer and activator of transcription 3 (STAT3) binding site in CLL-VMR2 was confirmed by proximity ligation and luciferase assays, whereas inhibition of SYK-mediated STAT3 activation resulted in suppression of IL10. The data suggest epigenetic control of IL-10 production. Higher tumor load may compensate the reduced IL-10 production in U-CLL, accounting for clinical immunosuppression in both subsets. The observation that SYK inhibition also suppresses IL-10 provides a potential new rationale for therapeutic targeting and immunological rescue by SYK inhibitors in CLL.

Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.

Longitudinal copy number, whole exome and targeted deep sequencing of 'good risk' IGHV-mutated CLL patients with progressive disease.

The biological features of IGHV-M chronic lymphocytic leukemia responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in 13 patients presenting with cMBL or Stage A disease and good-risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom 10 have required therapy. Using cytogenetics, fluorescence in situ hybridisation, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing at diagnosis, we identified mutations in established chronic lymphocytic leukemia driver genes in nine patients (69%), non-coding mutations (PAX5 enhancer region) in three patients and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition before therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low-risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good-risk disease.

The SF3B1 inhibitor spliceostatin A (SSA) elicits apoptosis in chronic lymphocytic leukaemia cells through downregulation of Mcl-1.

The pro-survival Bcl-2 family member Mcl-1 is expressed in chronic lymphocytic leukaemia (CLL), with high expression correlated with progressive disease. The spliceosome inhibitor spliceostatin A (SSA) is known to regulate Mcl-1 and so here we assessed the ability of SSA to elicit apoptosis in CLL. SSA induced apoptosis of CLL cells at low nanomolar concentrations in a dose- and time-dependent manner, but independently of SF3B1 mutational status, IGHV status and CD38 or ZAP70 expression. However, normal B and T cells were less sensitive than CLL cells (P=0.006 and P<0.001, respectively). SSA altered the splicing of anti-apoptotic MCL-1(L) to MCL-1(s) in CLL cells coincident with induction of apoptosis. Overexpression studies in Ramos cells suggested that Mcl-1 was important for SSA-induced killing since its expression inversely correlated with apoptosis (P=0.001). IL4 and CD40L, present in patient lymph nodes, are known to protect tumour cells from apoptosis and significantly inhibited SSA, ABT-263 and ABT-199 induced killing following administration to CLL cells (P=0.008). However, by combining SSA with the Bcl-2/Bcl-x(L) antagonists ABT-263 or ABT-199, we were able to overcome this pro-survival effect. We conclude that SSA combined with Bcl-2/Bcl-x(L) antagonists may have therapeutic utility for CLL.

p53 and Notch signaling in chronic lymphocytic leukemia: clues to identifying novel therapeutic strategies.

The p53 tumor suppressor protein has a key role in the induction of apoptosis of chronic lymphocytic leukemia (CLL) cells. Abnormalities within the p53 pathway identify a subset of patients with a poor prognosis. This review describes recent advances in understanding the mechanisms that regulate p53 levels and the role of p53 in the control of the cell cycle and of apoptosis. The classical model of p53-mediated apoptosis emphasizes the transcriptional activation of proapoptotic genes. In contrast, a novel model emphasizes p53's non-transcriptional actions as the major route of apoptosis induction, whereas its transcriptional arm predominantly upregulates antiapoptotic genes, thus providing a negative feedback mechanism that limits apoptosis. Further studies have identified the Notch pathway as a candidate p53-induced antiapoptotic mechanism. In contrast to the classical model, the novel model predicts that pharmacological inhibition of p53's transcriptional function or of the Notch signaling pathway will augment apoptosis induction by cytotoxic agents. Therapeutic strategies based on the novel model, which we review here for the first time, may significantly augment the antitumor actions of cytotoxic agents in CLL and in other malignancies.

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD(50) for PTL was 6.2 muM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1-3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34(+) haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IkappaB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

Detection of serum reverse transcriptase activity in patients with ALS and unaffected blood relatives.

BACKGROUND: Retroviral involvement in the etiology of sporadic ALS has been suspected for several years since the recognition that both murine and human retroviruses can cause motor neuron disease-like syndromes. In a pilot study, an increased prevalence of a retroviral marker (reverse transcriptase [RT] activity) was demonstrated in the serum of British patients with ALS. The current investigation was designed to confirm and extend these findings in a geographically distinct patient cohort under blinded testing conditions. METHODS: A highly sensitive product-enhanced RT assay was employed to test coded sera obtained from 30 American patients with sporadic ALS and from 14 of their blood relatives, 16 of their spouses, and 28 nonrelated, nonspousal control subjects. RESULTS: Serum RT activity was detected in a higher proportion of ALS patients (47%) than in non-blood-related controls (18%; p = 0.008). The prevalence of RT activity in the serum of spousal controls (13%) was similar to that in other non-blood-related controls. Unexpectedly, the prevalence of serum RT activity in blood relatives of ALS patients (43%) approached that in the ALS patients themselves. CONCLUSIONS: These results confirm that patients with ALS have a significantly higher prevalence of serum reverse transcriptase (RT) activity than that seen in unrelated control subjects. The finding of a similarly increased prevalence in blood relatives of ALS patients raises the possibility that the observed RT activity might be due to an inherited endogenous retrovirus.