Asim Khwaja

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3‐OH kinase (PI 3‐kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3‐kinase or PKB/Akt block anoikis, while inhibition of PI 3‐kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3‐kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3‐kinase lipid products and PKB/Akt activity, both of which remain high in Ras‐transformed cells even in suspension. PI 3‐kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras‐transformed epithelial cells in the absence of attachment, and mediates matrix‐induced survival of normal epithelial cells.

Role of Phosphoinositide 3-OH Kinase in Cell Transformation and Control of the Actin Cytoskeleton by Ras

The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells and regulates survival and chemoresistance via NF-kB, MAPkinase and p53 pathways

The phosphoinositide 3-kinase (PI3-kinase) signalling pathway plays a key role in the regulation of cell survival and proliferation. We show that the PI3-kinase/Akt pathway is constitutively active in primary acute myeloid leukaemia (AML) cells and that blockade by the selective inhibitor LY294002 reduces survival of the total blast population (mean 52%). The ERK/MAPK module is also constitutively active and treatment with the MAPKK inhibitor U0126 reduces cell survival by 22%. In 10 of 18 samples, PI3-kinase contributes to MAPK activation as incubation with LY294002 leads to a marked reduction in its phosphorylation. PI3-kinase inhibition reduces survival of the CD34+38− AML progenitor subset by 44%, whereas MAPKK inhibition has little effect. Reporter assays in primary AML cells show that blocking PI3-kinase leads to a marked reduction of constitutive NF-kB activity and promotes p53-mediated transcription. This is associated with a synergistic interaction between LY294002 and Ara-C. An inducible activated form of Akt protects normal myeloid cells from Ara-C and etoposide-mediated apoptosis. These results show that blocking PI3-kinase has direct antileukaemic effects and potentiates the response to conventional cytotoxics via a number of targets including NF-kB, p53 and MAPK. Inhibitors of PI3-kinase and Akt may be useful in the treatment of AML.

Allogeneic Stem-Cell Transplantation Using a Reduced-Intensity Conditioning Regimen Has the Capacity to Produce Durable Remissions and Long-Term Disease-Free Survival in Patients With High-Risk Acute Myeloid Leukemia and Myelodysplasia

PURPOSE The toxicity of allogeneic stem-cell transplantation can be substantially reduced using a reduced-intensity conditioning (RIC) regimen. This has increased the proportion of patients with myeloid malignancies eligible for allogeneic transplantation. However, the capacity of RIC allografts to produce durable remissions in patients with acute myeloid leukemia (AML) and myelodysplasia (MDS) has not yet been defined, and consequently, the role of RIC allografts in the management of these diseases remains conjectural. PATIENTS AND METHODS Seventy-six patients with high-risk AML or MDS received an allograft using a fludarabine/melphalan RIC regimen incorporating alemtuzumab. The median age of the cohort was 52 years (range, 18 to 71 years). RESULTS The 100-day transplantation-related mortality rate was 9%, and no patient developed greater than grade 2 graft-versus-host disease. With a median follow-up of 36 months (range, 13 to 70 months), 27 patients were alive and in remission, with 3-year actuarial overall survival (OS) and disease-free survival (DFS) rates of 41% and 37%, respectively. The 3-year OS and DFS rates of patients with AML in complete remission at the time of transplantation were 48% and 42%, respectively. Disease relapse was the most common cause of treatment failure and occurred at a median time of 6 months after transplantation. All but one patient destined to relapse did so within 24 months of transplantation. CONCLUSION The extended follow-up in this series identifies a high risk of early disease relapse but provides evidence that RIC allografts can produce sustained DFS in a significant number of patients with AML who would be ineligible for allogeneic transplantation with myeloablative conditioning.

Constitutive activation of the Wnt/ β -catenin signalling pathway in acute myeloid leukaemia

The β-catenin protein is at the core of the canonical Wnt signalling pathway. Wnt stimulation leads to β-catenin accumulation, nuclear translocation and interaction with T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors to regulate genes important for embryonic development and proliferation. Wnt/β-catenin can promote stem cell self-renewal and is dysregulated in colon carcinoma. We have examined the role of the Wnt pathway in the development of acute myeloid leukaemia (AML) and find that the β-catenin protein is readily detected in primary AML samples. Using transfection of a TCF/LEF reporter construct into primary AML cells and normal human progenitors, we find increased reporter activity in 16/25 leukaemia samples. Retrovirally mediated expression of a mutant active β-catenin in normal progenitors preserves CD34 expression and impairs myelomonocytic differentiation. Activation of TCF/LEF signalling decreases factor withdrawal-induced apoptosis of normal progenitors. A significant proportion of AML cases show aberrant expression of components of the Wnt pathway including Wnt-1, Wnt-2b and LEF-1. These results provide evidence for the involvement of the Wnt/β-catenin pathway in the pathogenesis of AML.

The Src-selective Kinase Inhibitor PP1 Also Inhibits Kit and Bcr-Abl Tyrosine Kinases

4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases, including events downstream of the stem cell factor (SCF) receptor c-Kit. While investigating the role of Src kinases in SCF signaling, we found that PP1 completely abrogated the proliferation of M07e cells in response to SCF. PP1 inhibited SCF-induced c-Kit autophosphorylation in intact cells and blocked the activation of mitogen-activated protein kinase and Akt. In vitro kinase assays using immunoprecipitated c-Kit confirmed direct inhibition by PP1. SCF-induced c-Kit phosphorylation was also inhibited by the related inhibitor 4-amino-5- (4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) and by STI571 but not by the Src inhibitor SU6656. PP1 inhibited the activity of mutant constitutively active forms of c-Kit (D814V and D814Y) found in mast cell disorders, and triggered apoptosis in the rat basophilic leukemia cell line RBL-2H3 that expresses mutant c-Kit. In addition, PP1 (and PP2) inhibited the in vitrokinase activity and autophosphorylation in whole cells of p210 Bcr-Abl. PP1 reduced the constitutive activation of signal transducer and activators of transcription 5 and mitogen-activated protein kinase and triggered apoptosis in FDCP1 cells expressing Bcr-Abl. These results have implications for the use of PP1 in investigating intracellular signaling and suggest that PP1 or related compounds may be useful in the treatment of malignant diseases associated with dysregulated c-Kit or Abl tyrosine kinase activity.

A selective inhibitor of the p110δ isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16

Current therapy for acute myeloid leukaemia (AML) is suboptimal with a high incidence of relapse. There is strong evidence that constitutive phosphoinositide 3-kinase (PI3K) activity plays a significant role in the pathophysiology of AML. PI3K products are derived from the activity of a number of PI3K catalytic isoforms (class I, II and III) but the relative contribution of these enzymes in AML remains unknown. As non-isoform-selective inhibitors of PI3K such as LY294002 may produce unwanted toxicity to normal tissues, we have investigated the role of the leukocyte-restricted p110δ PI3K isoform in 14 cases of AML. p110δ was detected in all cases whereas the expression levels of the other class I PI3Ks varied more widely, and were often undetectable. The p110δ-selective compound IC87114 inhibited constitutive phosphorylation of the PI3K target Akt/PKB and reduced cell number to a mean of 66±5% (range 14–88%). In eight cases, the combination of IC87114 and VP16 (a topoisomerase II inhibitor) was synergistic in reducing viable cell number, and was associated with a reduction in constitutive NF-κB activity. IC87114 did not have direct adverse effects or enhance the activity of VP16 on the proliferation and survival of normal haemopoietic progenitors. Overall, our results identify the p110δ isoform as a potential therapeutic target in AML and support a clinical approach to use isoform-selective over broad-spectrum PI3K inhibitors.

Arsenic trioxide and all-trans retinoic acid treatment for acute promyelocytic leukaemia in all risk groups (AML17): results of a randomised, controlled, phase 3 trial

BACKGROUND Acute promyelocytic leukaemia is a chemotherapy-sensitive subgroup of acute myeloid leukaemia characterised by the presence of the PML-RARA fusion transcript. The present standard of care, chemotherapy and all-trans retinoic acid (ATRA), results in a high proportion of patients being cured. In this study, we compare a chemotherapy-free ATRA and arsenic trioxide treatment regimen with the standard chemotherapy-based regimen (ATRA and idarubicin) in both high-risk and low-risk patients with acute promyelocytic leukaemia. METHODS In the randomised, controlled, multicentre, AML17 trial, eligible patients (aged ≥16 years) with acute promyelocytic leukaemia, confirmed by the presence of the PML-RARA transcript and without significant cardiac or pulmonary comorbidities or active malignancy, and who were not pregnant or breastfeeding, were enrolled from 81 UK hospitals and randomised 1:1 to receive treatment with ATRA and arsenic trioxide or ATRA and idarubicin. ATRA was given to participants in both groups in a daily divided oral dose of 45 mg/m(2) until remission, or until day 60, and then in a 2 weeks on-2 weeks off schedule. In the ATRA and idarubicin group, idarubicin was given intravenously at 12 mg/m(2) on days 2, 4, 6, and 8 of course 1, and then at 5 mg/m(2) on days 1-4 of course 2; mitoxantrone at 10 mg/m(2) on days 1-4 of course 3, and idarubicin at 12 mg/m(2) on day 1 of the final (fourth) course. In the ATRA and arsenic trioxide group, arsenic trioxide was given intravenously at 0·3 mg/kg on days 1-5 of each course, and at 0·25 mg/kg twice weekly in weeks 2-8 of course 1 and weeks 2-4 of courses 2-5. High-risk patients (those presenting with a white blood cell count >10 × 10(9) cells per L) could receive an initial dose of the immunoconjugate gemtuzumab ozogamicin (6 mg/m(2) intravenously). Neither maintenance treatment nor CNS prophylaxis was given to patients in either group. All patients were monitored by real-time quantitative PCR. Allocation was by central computer minimisation, stratified by age, performance status, and de-novo versus secondary disease. The primary endpoint was quality of life on the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 global health status. All analyses are by intention to treat. This trial is registered with the ISRCTN registry, number ISRCTN55675535. FINDINGS Between May 8, 2009, and Oct 3, 2013, 235 patients were enrolled and randomly assigned to ATRA and idarubicin (n=119) or ATRA and arsenic trioxide (n=116). Participants had a median age of 47 years (range 16-77; IQR 33-58) and included 57 high-risk patients. Quality of life did not differ significantly between the treatment groups (EORTC QLQ-C30 global functioning effect size 2·17 [95% CI -2·79 to 7·12; p=0·39]). Overall, 57 patients in the ATRA and idarubicin group and 40 patients in the ATRA and arsenic trioxide group reported grade 3-4 toxicities. After course 1 of treatment, grade 3-4 alopecia was reported in 23 (23%) of 98 patients in the ATRA and idarubicin group versus 5 (5%) of 95 in the ATRA and arsenic trioxide group, raised liver alanine transaminase in 11 (10%) of 108 versus 27 (25%) of 109, oral toxicity in 22 (19%) of 115 versus one (1%) of 109. After course 2 of treatment, grade 3-4 alopecia was reported in 25 (28%) of 89 patients in the ATRA and idarubicin group versus 2 (3%) of 77 in the ATRA and arsenic trioxide group; no other toxicities reached the 10% level. Patients in the ATRA and arsenic trioxide group had significantly less requirement for most aspects of supportive care than did those in the ATRA and idarubicin group. INTERPRETATION ATRA and arsenic trioxide is a feasible treatment in low-risk and high-risk patients with acute promyelocytic leukaemia, with a high cure rate and less relapse than, and survival not different to, ATRA and idarubicin, with a low incidence of liver toxicity. However, no improvement in quality of life was seen.

Activity of any class IA PI3K isoform can sustain cell proliferation and survival

Small molecule inhibitors of PI3K for oncology mainly target the class I PI3Ks, comprising the p110α, β, γ, and δ isoforms, of which only p110α is mutated in cancer. To assess the roles of class I PI3K isoforms in cell proliferation and survival, we generated immortalized mouse leukocyte and fibroblast models in which class I PI3Ks were inactivated by genetic and pharmacological approaches. In IL3-dependent hemopoietic progenitor cells (which express all four class I PI3K isoforms), genetic inactivation of either p110α or p110δ did not affect cell proliferation or survival or sensitize to p110β or p110γ inactivation. Upon compound inactivation of p110α and p110δ, which removed >90% of p85-associated PI3K activity, remarkably, cells continued to proliferate effectively, with p110β assuming an essential role in signaling and cell survival. Furthermore, under these conditions of diminished class I PI3K activity, input from the ERK pathway became important for cell survival. Similar observations were made in mouse embryonic fibroblasts (which mainly express p110α and p110β) in which p110α or p110β could sustain cell proliferation as a single isoform. Taken together, these data demonstrate that a small fraction of total class I PI3K activity is sufficient to sustain cell survival and proliferation. Persistent inhibition of selected PI3K isoforms can allow the remaining isoform(s) to couple to upstream signaling pathways in which they are not normally engaged. Such functional redundancy of class IA PI3K isoforms upon sustained PI3K inhibition has implications for the development and use of PI3K inhibitors in cancer.

Resistance to the cytotoxic effects of tumor necrosis factor alpha can be overcome by inhibition of a FADD/caspase-dependent signaling pathway

Tumor necrosis factor (TNF) α initiates the activation of a pro-apoptotic pathway involving the recruitment of the death domain containing protein FADD and the subsequent activation of specific proteases (caspases). Many cells are resistant, however, to the cytotoxic effects of TNFα due to the concurrent activation of pro-survival pathways involving the transcription factor NFκB and TRAF2. Here we show that the TNFα-activated FADD/caspase pathway can also exert an unexpected pro-survival effect. Inhibition of this pathway in NIH3T3 fibroblasts or U937 leukemic cells by peptide caspase inhibitors or expression of dominant-negative FADD leads to rapid death following treatment with TNFα, whereas control cells are TNFα-resistant. FADD/caspase-inhibited cells die by a non-apoptotic mechanism caused by increased production of reactive oxygen species which precedes loss of the mitochondrial membrane potential. Cytotoxicity can be prevented by preincubation with antioxidants including reduced glutathione or by expression of a dominant-negative Rac GTP-binding protein. These results indicate that caspase activation in response to TNFα has anti-necrotic as well as pro-apoptotic effects and extend our understanding of the biological role of these proteases.