Nicholas Lea

TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17–89 years) and median follow‐up was 45 months [95% confidence interval (CI) 27–62 months]. TP53 mutations occurred in 30 (9·4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with ‐5/5q‐(72%), correlated with International Prognostic Scoring System intermediate‐2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild‐type (9 versus 66 months, P < 0·001) and it retained significance in multivariable model (Hazard Ratio 3·8, 95%CI 2·3–6·3,P < 0·001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5‐azacitidine, however clones increased in non‐responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q‐ and CK with ‐5/5q‐, possibly implies two different mechanistic roles for TP53 protein.

Novel TET2 Mutations Associated With UPD4q24 in Myelodysplastic Syndrome

PURPOSE Cryptic chromosomal aberrations, such as regions of uniparental disomy (UPD), have been shown to harbor homozygous mutations and are a common feature in myelodysplastic syndrome (MDS). We investigated the sequence integrity of 4q24 candidate tumor suppressor gene TET2 in MDS patients with UPD on chromosome 4. PATIENTS AND METHODS The coding exons of TET2 were analyzed by 454 deep sequencing and Sanger sequencing in nine patients with UPD on 4q. Four patients had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) and UPD4q24, and five patients (refractory anemia with excess blasts-II, n = 1; 5q- syndrome, n = 1; RCMD-RS, n = 1; refractory anemia, n = 1; refractory cytopenia with multilineage dysplasia, n = 1) had no UPD4q24. RESULTS Mutations on TET2 were identified in all four patients with UPD4q24. These were localized to exons 3, 6, and 9 and resulted in two premature stop codons, one frameshift mutation, and one cysteine to glycine amino acid change. Mutant clone size varied between 30% and 85%. One patient with UPD outside of q24 (UPD4q28.3) displayed additional TET2 mutations, but these were at low clonal levels (13%, 4%, and 4% for a silent mutation, a 180-base pair deletion in exon 3, and a lysine to phenylalanine substitution in exon 11, respectively). The other patients who did not have UPD4q24 did not have verifiable TET2 mutations. CONCLUSION Our data identify novel TET2 mutations in a dominant clone in patients with UPD4q24. The presence of UPD4q24 and mutations in RCMD-RS patients may suggest specificity to this subtype. Our preliminary results need to be confirmed in a large cohort of all MDS subtypes.

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.

Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-κB, c-Myc, and pRb Pathways

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-β, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-κB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G0 to G1. Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.

BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression.

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27Kip1 to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27Kip1 expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27Kip1 on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27Kip1 have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27Kip1 can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27Kip1, cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.

Commitment Point during G0→G1 That Controls Entry into the Cell Cycle

ABSTRACT Initiation of T-lymphocyte-mediated immune responses involves two cellular processes: entry into the cell cycle (G0→G1) for clonal proliferation and coordinated changes in surface and secreted molecules that mediate effector functions. However, a point during G0→G1 beyond which T cells are committed to enter the cell cycle has not been defined. We define here a G0→G1 commitment point that occurs 3 to 5 h after CD3 and CD28 stimulation of human CD4 or CD8 T cells. Transition through this point requires cdk6/4-cyclin D, since inhibition with TAT-p16INK4A during the first 3 to 5 h prevents cell cycle entry and maintains both naive and memory T cells in G0. Transition through the G0→G1 commitment point is also necessary for T cells to increase in size, i.e., to enter the cellular growth cycle. However, transition through this point is not required for the induction of effector functions. These can be initiated while cells are maintained in G0 with TAT-p16INK4A. We have termed this quiescent, activated state G0(A). Our data provide proof of the principle that entry of T cells into the cell cycle and cellular growth cycles are coupled at the G0→G1 commitment point but that these processes can be uncoupled from the early expression of molecules of effector functions.

The JAK2 V617F mutation identifies a subgroup of MDS patients with isolated deletion 5q and a proliferative bone marrow

The JAK2 V617F mutation identifies a subgroup of MDS patients with isolated deletion 5q and a proliferative bone marrow

Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes

Hematopoietic cells from patients with acute myeloid leukemia and myelodysplastic syndrome often have defects in DNA repair processes. This study shows that these defects make the cells susceptible to induction of apoptosis by poly ADP-ribose polymerase (PARP) inhibitors. Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors. Design and Methods Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5’ aza-2’-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents. Results PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5’ aza-2’-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.

BCR signals target p27 Kip1 and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27Kip1, which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27Kip1 by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27Kip1 have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27Kip1 and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27Kip1. Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27Kip1 expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27Kip1 expression induced by BCR-engagement. These results indicate that BCR activation targets p27Kip1 and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.

Spliceosome mutations exhibit specific associations with epigenetic modifiers and proto-oncogenes mutated in myelodysplastic syndrome

The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P<0.03). Furthermore, mutations in SF3B1 and SRSF2 were mutually exclusive to TP53 mutations both at diagnosis and at the time of disease transformation. Moreover, mutations in FLT3, NRAS, RUNX1, CCBL and C-KIT were more likely to co-occur with splicing factor mutations generally (P<0.02), and SRSF2 mutants in particular (P<0.003) and were significantly associated with disease transformation (P<0.02). SF3B1 and TP53 mutations had varying impacts on overall survival with hazard ratios of 0.2 (P<0.03, 95% CI, 0.1–0.8) and 2.1 (P<0.04, 95% CI, 1.1–4.4), respectively. Moreover, patients with splicing factor mutations alone had a better overall survival than those with epigenetic modifier mutations, or cell signaling/transcription regulator mutations with and without coexisting mutations of splicing factor genes, with worsening prognosis (P<0.001). These findings suggest that splicing factor mutations are maintained throughout disease evolution with emerging oncogenic mutations adversely affecting patients’ outcome, implicating spliceosome mutations as founder mutations in myelodysplastic syndromes.