N S B Thomas

CDKN2B methylation status and isolated chromosome 7 abnormalities predict responses to treatment with 5-azacytidine

5-Azacytidine, a DNA methyl transferase inhibitor, is effective in patients with myelodysplastic syndromes (MDS). Whether responses to 5-Azacytidine are achieved by demethylation of key genes or by cytotoxicity is unclear. Of 34 patients with MDS or acute myeloid leukaemia (AML) treated with 5-Azacytidine, 7 achieved complete remissions (CR) (21%) and 6 achieved haematological improvement. All six had less than 5% bone marrow (BM) blasts at the time of haematological improvements (HI) (2 had pre-existing refractory anaemia (RA), 4 had refractory anaemia with excess blasts (RAEB)). A further patient with RAEB had blast reduction to less than 5% without HI. Five of the seven (71%) complete responders had chromosome 7 abnormalities. BM CR predicted longer overall survival (OS) (median 23 versus 9 months, P=0.015). Bisulphite genomic sequencing (BGS) of the CDKN2B (p15INK4b) promoter showed low level, heterogeneous pretreatment methylation (mean 12.2%) in 14/17 (82%) patients analysed. Lower baseline methylation occurred in responders (9.8% versus 16.2% in non-responders P=0.07). No response was seen in patients with >24% methylation, in whom p15INK4b mRNA was not expressed. 5-Azacytidine reduced CDKN2B methylation by mean 6.8% in 8/17 (47%) patients, but this did not correlate with response. At 75 mg/m2, cell death (reduced BM cellularity (P=0.001) and increased apoptosis (P=0.02)) rather than demethylation of CDKN2B correlates with response. Patients with >24% methylation may benefit from alternative dosing or combination strategies.

Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.

JAK inhibition induces silencing of T Helper cytokine secretion and a profound reduction in T regulatory cells

CD4+ T cells maintain cancer surveillance and immune tolerance. Chronic inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN), suggesting that T cells play an important role in their pathogenesis. Treatment with JAK inhibitors (JAKi) results in improvements in MPN‐associated constitutional symptoms as well as reductions in splenomegaly. However, effects of JAKi on T cells in MPN are not well established and the baseline immune signature remains unclear. We investigated the frequency and function of CD4+ T cell subsets in 50 MPN patients at baseline as well as during treatment with either ruxolitinib or fedratinib in a subset. We show that CD4+ CD127low CD25high FOXP3+ T regulatory cells are reduced in MPN patients compared to healthy controls and that this decrease is even more pronounced following JAKi therapy. Moreover, we show that after 6 months of treatment the number of T helper (Th)‐17 cells increased. We also describe a functional ‘silencing’ of T helper cells both in vivo and in vitro and a blockade of pro‐inflammatory cytokines from these cells. This profound effect of JAKi on T cell function may underlay augmented rates of atypical infections that have been reported with use of these drugs.

Reducing MCM levels in human primary T cells during the G(0)-->G(1) transition causes genomic instability during the first cell cycle.

DNA replication is tightly regulated, but paradoxically there is reported to be an excess of MCM DNA replication proteins over the number of replication origins. Here, we show that MCM levels in primary human T cells are induced during the G0→G1 transition and are not in excess in proliferating cells. The level of induction is critical as we show that a 50% reduction leads to increased centromere separation, premature chromatid separation (PCS) and gross chromosomal abnormalities typical of genomic instability syndromes. We investigated the mechanisms involved and show that a reduction in MCM levels causes dose-dependent DNA damage involving activation of ATR & ATM and Chk1 & Chk2. There is increased DNA mis-repair by non-homologous end joining (NHEJ) and both NHEJ and homologous recombination are necessary for Mcm7-depleted cells to progress to metaphase. Therefore, a simple reduction in MCM loading onto DNA, which occurs in cancers as a result of aberrant cell cycle control, is sufficient to cause PCS and gross genomic instability within one cell cycle.

Tumor-derived IL-6 may contribute to the immunological defect in CLL

Chronic lymphocytic leukemia (CLL) is an indolent disorder with a highly variable course whose clinical features arise through the accumulation of tumor cells in the bone marrow, blood and secondary lymphoid tissue. While hematopoietic failure may occur in those with advanced disease, for the majority it is immune dysfunction, manifested as susceptibility to infection or autoimmunity, which dominates the clinical picture. Both the disease and its treatment may affect the number of normal cells in the innate and adaptive immune systems, however, it is clear that there are also more subtle qualitative defects which presumably arise either through contact with the expanded neoplastic B cell compartment or because of the secretion of immunomodulatory cytokines. A variety of functional defects in T cells from patients with CLL have been reported including, reduction in activation-induced CD40L expression1 and abnormalities of gene expression affecting the differentiation of CD4 cells and cytoskeletal function and vesicle transport in CD8 cells.2 Although these abnormalities were shown to arise through contact with the malignant clone it is clear that soluble mediators may also play a role, as we have previously shown in myeloid malignancies.3 In the present study, we sought to characterize and identify soluble immunomodulatory factors in CLL since these might lend themselves to therapeutic intervention.

Tumor supernatant from myeloid malignancies inhibits T-cell apoptosis and cell cycle entry independently

Tumor supernatant from myeloid malignancies inhibits T-cell apoptosis and cell cycle entry independently

Analysis of ultrasound pulse-wave Doppler systems

The aim of the investigation reported here is to clarify the way in which spectral-modifying artefacts, such as tissue attenuation, compromise pulse-wave Doppler measurements, and to accurately measure the magnitude of the corrupting influence of attenuation under controlled laboratory conditions. A theoretical description of the structure of the pulse-echo sequence from a moving scatterer field is constructed from first principles by utilizing a time-domain description of the Doppler process. It is demonstrated that the essential features of the echo signal may be rather more accurately described by a wavelet-, rather than by a Fourier-, transform, and that the power spectrum of the Doppler signal does not necessarily encode the range of the scatterer velocities present in the (pulse-echo) sampling volumes. The analysis provides a better understanding of the origins of the significant levels of noise present in pulse-wave Doppler signals, and allows a novel approach towards noise-reduction -- by zero manipulation in complex frequency space -- to be developed.