Hematology in the United Kingdom

This article is the first in a series of features comparing and contrasting aspects of oncology care delivery in non-US settings. The Journal of Oncology Practice will occasionally publish similar pieces in anticipation of discovering best practices from international health care systems. Dr Prentice's contribution is based on his presentation to the Committee on Practice of the American Society of Hematology at their December 2005 meeting. </jats:p

Pifithrin α, a Selective Inhibitor of p53-Mediated Transcription, Augments Apoptotic Killing of Chronic Lymphocytic Leukemia Cells by Nutlin 3a and Cytotoxic Drugs.

Abstract The p53 protein plays a key role in triggering DNA damage-induced apoptosis. The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins Puma and Noxa. However, recent studies identified a non-transcriptional mechanism, involving direct association of p53 with mitochondrial anti-apoptotic proteins Bcl-2 or Bcl-XL. We studied the relative contributions of transcription-dependent and -independent mechanisms of p53-mediated apoptosis in CLL cells in vitro. The cytotoxic drugs chlorambucil (chl) and fludarabine (flu) were used to induce p53, as was nutlin 3a, which augments p53 levels by inhibiting interaction with its negative regulator MDM2. Using differential detergent fractionation to isolate cytosolic (cyt), mitochondrial (mt) and nuclear (nuc) fractions, we found that a significant proportion (&amp;gt;50%) of the induced p53 was stably associated with mitochondria in cells treated with nutlin 3a (Fig 1), chl or flu. Co-immunoprecipitation studies established that p53 bound to Bcl-2. PFTα, an inhibitor of p53-mediated transcription, blocked upregulation of the known p53 targets p21CIP1, MDM2 and Puma. Surprisingly, apoptosis induction by nutlin 3a (Figure 2), chl or flu, quantified by western blot analysis of cleavage of poly(ADP ribose) polymerase (PARP), was augmented by PFTα. This observation was confirmed by morphological analysis of apoptosis. Cytochrome c release from mitochondria following p53 induction was also enhanced by PFTα. PFTα did not augment nutlin-, chl- or flu-induced killing of CLL cells lacking functional p53. Furthermore, PFTα did not augment killing by the p53-independent drug parthenolide and failed to increase apoptosis of normal T lymphocytes treated with chl or nutlin 3a. Our observations suggest that p53 induces apoptosis of CLL cells principally via its transcription-independent function, involving direct association with mitochondrial Bcl-2. p53’s transcriptional function apparently blocks apoptosis at a point between p53 association with Bcl-2 and subsequent release of cytochrome c. Therefore, therapeutic strategies aimed at blocking p53-mediated transcription may be of value in enhancing the action of agents which induce apoptosis of CLL cells via p53 upregulation. Figure 1 Figure 1. Figure 2 Figure 2.</jats:p

2-Phenylacetylenesulfonamide Upregulates Noxa and Induces p53-Independent Apoptosis of CLL Cells.

Abstract The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins including Puma and Noxa. However, recent studies have identified a novel non-transcriptional mechanism mediated by direct binding of p53 to Bcl-2 or Bcl-XL at the mitochondrial surface and consequent neutralization of their anti-apoptotic function (Chipuk and Green, Cell Cycle, 2004; 3: 429–431). Induction of apoptosis of chronic lymphocytic leukaemia (CLL) cells by cytotoxic agents is strongly dependent on a functional p53 system and genetic changes which compromise p53 function result in drug resistance. We are therefore attempting to identify novel therapeutic agents which induce selective apoptosis of CLL cells by mechanisms independent of p53. Here we present data suggesting that 2-phenylacetylenesulfonamide (PAS; trivial name pifithrin μ) is a potential therapeutic agent for CLL. PAS was toxic to CLL cells, with a median LC50 of 10.72 μM (SD 3.8 μM n=20) as measured by a dye reduction cytotoxicity assay. Killing of CLL cells was by apoptosis, documented by the ability of 10–20 μM PAS to induce cytochrome c release from mitochondria (Fn2) to the cytosol (Fn1; Figure 1, upper), conformational change of Bax to a pro-apoptotic conformation, translocation of Bax from the cytosol to mitochondria (Figure 1, lower) and cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP). PAS toxicity was p53-independent, since p53-deleted CLL isolates were susceptible to killing. Furthermore, PAS was toxic towards leukemic cell lines with either inducible (Daudi), non-functional (Raji) or deleted (K562) p53. PAS treatment of CLL cells resulted in upregulation of Noxa and the subsequent displacement by Noxa of the pro-apoptotic Bim long (L) and extra-long (EL) isoforms from Bcl-2, as determined by western blot analysis of Bcl-2 immunoprecipitates (Figure 2). These observations contrast with those of Strom et al (Nat Chem Biol, 2006; 2: 474–479), who reported that PAS caused the displacement of p53 from mitochondria to the cytosol and thereby protected the cells from p53-dependent apoptosis. These differences may result from ectopic viral expression of p53 in the latter study whereas this protein was under normal cellular regulation in CLL cells. Normal T lymphocytes were relatively resistant to PAS, with IC50 values four times greater than those observed for CLL cells. In conclusion, our data suggest that PAS represents a novel class of agent which is toxic towards CLL cells via a mechanism involving p53-independent upregulation of Noxa and the subsequent unleashing of pro-apoptotic Bim from anti-apoptotic Bcl-2. This p53-independent toxicity may be of particular value in the treatment of drug-resistant CLL patients with compromised p53 pathways. Figure 1. Figure 1. Figure 2. Figure 2.</jats:p

A Prospective, Randomised Trial of Chlorambucil, Mitoxantrone and Dexamethasone (CMD) Versus Fludarabine, Mitoxantrone and Dexamethasone (FMD) for Advanced Follicular Lymphoma (Real Grades I-III, Stages III/IV).

Abstract Fludarabine, alone and in combination, has activity in de novo and relapsed/refractory follicle centre cell lymphoma (FCCL), as do many other combinations of chemotherapy, including chlorambucil, mitoxantrone and steroids. In this multicentre study, between May 2000 and April 2006, 400 adults with FCCL (REAL grades I-III) were randomised to receive either CMD (chlorambucil 10mgs po, od, days 1–10, mitoxantrone 12mgs/m2 IV, day 1 and dexamethasone 20mgs po, od, days 1–5) or FMD (fludarabine 25mgs/m2, IV days 1–3, with M and D as for CMD) on physicians’ decision to treat advanced stage (III or IV) disease (B symptoms, bone marrow failure, bulk or progression and compression syndromes). Rituximab (R) was not available for this trial. Randomisation was prospectively stratified by IPI score. Patients received four courses before assessment of response, then had no further trial therapy with progression or inadequate response or had a maximum total of eight courses if responding. The primary end points were progression-free survival (PFS) and overall survival (OS) and the secondary end points were complete (C) and partial (P) remission rates (RR). This preliminary report is at median follow-up of 31 months (range 0–71). Clinical features and pathological diagnoses (reviewed centrally) were equivalent across both arms. The median age in years (range; number &gt;60 y) for CMD was 56 (32–72; 68) and for FMD 55 (29–75; 68) and 70% in both arms had stage IV FCCL. For CMD versus FMD, the CRR and PRR after course 4 were 18% v 21% and 72% v 68% and after course 8 or at the end of treatment were 44% v 45% and 47% v 47%. The hazard ratios were in favour of CMD, for OS at 1.65 (95%CI 0.97–2.8, p=0.063) and for PFS at 1.61 (95%CI 1.2–2.17; p=0.0018). The numbers of deaths due to lymphoma were equivalent (19 v 21) but there were fewer deaths in the first year (most due to lymphoma in both arms) with CMD (7 v 15; ns). The difference between those who received between 6 and 8 courses in either arm was not significant (152[80%] v 135[71%]). With CMD there were higher rates of grade 3/4 neutropenia (8 v 1) and infections (29 v 20) but these differences were not statistically significant; other toxicities were low grade and equivalent for both arms. Days between cycles (median 28 days in both arms) and dose adherence (96% and 97%) were equivalent; both regimens were well tolerated and relatively easy to deliver. The equivalent dose intensity, toxicities and RRs of the two arms indicate that the statistically significant superior PFS for CMD was not due to undertreatment in the FMD arm. The outcomes for CMD are at least as good as those reported for other combinations of chemotherapy without R and R-CVP.</jats:p