In chronic lymphocytic leukaemia (CLL), mutation/deletion of TP53 is strongly associated with early disease progression, resistance to chemotherapy and short patient survival. Consequently, there is ...a pressing need to develop novel treatment protocols for this high-risk patient group. The present study was performed to evaluate Hsp90 inhibition as a possible therapeutic approach for such patients. Primary CLL cells of defined ataxia telangiectasia mutated (ATM)/p53 status were incubated with the Hsp90 inhibitor geldanamycin (GA) and analysed by western blotting for the expression of p53, p21, MDM2 and Akt. GA downregulated overexpressed mutant p53 protein (an oncogene) and upregulated wild-type (wt) p53 (a tumour suppressor). The upregulation of wt p53 by GA was independent of ATM and was accompanied by downregulation of Akt and the active form of MDM2, indicating a possible mechanism. GA also produced a p53/ATM-independent increase in the levels of p21-a potent inducer of cell-cycle arrest. In-vitro cytotoxicity studies showed that GA killed cultured CLL cells in a dose- and time-dependent fashion irrespective of their p53/ATM status and more effectively than normal blood mononuclear cells. In summary, our findings reveal important consequences of inhibiting Hsp90 in CLL cells and strongly support the therapeutic evaluation of Hsp90 inhibitors in poor-prognosis patients with p53 defects.
We have utilized a novel application of human genetics, illuminating the important role that rare genetic disorders can play in the development of novel drugs that may be of relevance for the ...treatment of both rare and common diseases. By studying a very rare Mendelian disorder of absent pain perception, congenital indifference to pain, we have defined Nav1.7 (endocded by SCN9A) as a critical and novel target for analgesic development. Strong human validation has emerged with SCN9A gain‐of‐function mutations causing inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder, both Mendelian disorder of spontaneous or easily evoked pain. Furthermore, variations in the Nav1.7 channel also modulate pain perception in healthy subjects as well as in painful conditions such as osteoarthritis and Parkinson disease. On the basis of this, we have developed a novel compound (XEN402) that exhibits potent, voltage‐dependent block of Nav1.7. In a small pilot study, we showed that XEN402 blocks Nav1.7 mediated pain associated with IEM thereby demonstrating the use of rare genetic disorders with mutant target channels as a novel approach to rapid proof‐of‐concept. Our approach underscores the critical role that human genetics can play by illuminating novel and critical pathways pertinent for drug discovery.
Summary
In chronic lymphocytic leukaemia (CLL) cells, functional impairment of the p53 pathway is detectable by Western blotting as impaired up‐regulation of p21 (a transcriptional target of p53) in ...response to ionizing radiation (IR). The type A defect is characterized by baseline p53 overexpression and is associated with TP53 mutation. The type B defect is characterized by impaired IR‐induced p53 up‐regulation and is associated with inactivation of the ataxia telangiectasia‐mutated gene (ATM). Both abnormalities are strongly associated with adverse clinical outcome. In the present study, flow cytometry was found to be an effective alternative to Western blotting in the detection of p53 dysfunction in CLL.
Inhibitors of voltage-gated sodium channels (NaVs) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We ...aimed to create novel, isoform selective inhibitors of Nav channels as a means of informing the development of improved antiseizure drugs.BACKGROUND AND PURPOSEInhibitors of voltage-gated sodium channels (NaVs) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We aimed to create novel, isoform selective inhibitors of Nav channels as a means of informing the development of improved antiseizure drugs.We created a series of compounds with diverse selectivity profiles enabling block of NaV1.6 alone or together with NaV1.2. These novel NaV inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding NaV1.6) and in wild-type mice.EXPERIMENTAL APPROACHWe created a series of compounds with diverse selectivity profiles enabling block of NaV1.6 alone or together with NaV1.2. These novel NaV inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding NaV1.6) and in wild-type mice.Pharmacologic inhibition of NaV1.6 in Scn8aN1768D/+ mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. NaV1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS NaV isoforms.KEY RESULTSPharmacologic inhibition of NaV1.6 in Scn8aN1768D/+ mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. NaV1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS NaV isoforms.Our data suggest NaV1.6 inhibition is a driver of efficacy for NaV inhibitor anti-seizure medicines. Sparing the NaV1.1 channels of inhibitory interneurons did not compromise efficacy. Selective NaV1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies.CONCLUSIONS AND IMPLICATIONSOur data suggest NaV1.6 inhibition is a driver of efficacy for NaV inhibitor anti-seizure medicines. Sparing the NaV1.1 channels of inhibitory interneurons did not compromise efficacy. Selective NaV1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies.
The well-established association between TP53 mutations and adverse clinical outcome in a range of human cancers reflects the importance of p53 protein in regulating tumor-cell growth and survival. ...Although it is theoretically possible for p53 dysfunction to arise through mechanisms that do not involve TP53 mutation, such a phenomenon has not previously been demonstrated in a sporadic tumor. Here, we show that p53 dysfunction in B-cell chronic lymphocytic leukemia (CLL) can occur in the absence of TP53 mutation and that such dysfunction is associated with mutation of the gene encoding ATM, a kinase implicated in p53 activation. Forty-three patients with CLL were examined for p53 dysfunction, as detected by impaired up-regulation of p53 and of the p53-dependent protein p21(CIP1/WAF1) after exposure to ionizing radiation (IR). Thirty (70%) patients had normal p53 responses and underwent progressive IR-induced apoptosis. In 13 (30%) patients, p21 up-regulation was markedly impaired, indicating p53 dysfunction. Six (14%) of these patients with p53 dysfunction had increased baseline levels of p53, were found to have TP53 mutations, and were completely resistant to IR-induced apoptosis. In the other 7 (16%) patients with p53 dysfunction, IR-induced p53 up-regulation and apoptosis were markedly impaired, but baseline levels of p53 were not increased, and no TP53 mutations were detected. Each of these patients was found to have at least one ATM mutation, and a variable reduction in ATM protein was detected in all 4 patients examined. This is the first study to provide a direct demonstration that p53 dysfunction can arise in a sporadic tumor by a mechanism that does not involve TP53 mutation. (Blood. 2001;98:814-822)
Although the nucleoside analogues fludarabine and chlorodeoxyadenosine have become important therapeutic agents in chronic lymphocytic leukemia (CLL), their effectiveness is limited by drug ...resistance. Because such resistance is likely to result from impaired drug-induced apoptosis, it is clearly important to understand the mechanisms involved in this process. Whereas p53 can contribute to the nucleoside-induced killing of CLL cells, recent work from this laboratory and elsewhere has shown that such killing can also occur by p53-independent mechanisms. Because poly(ADP-ribose) polymerase (PARP)-mediated NAD+/ATP depletion has been implicated in the nucleoside-induced killing of normal resting lymphocytes, we postulated that this mechanism might account for the p53-independent component of nucleoside cytotoxicity in CLL. To address this question, we used 3-aminobenzamide (3AB) at a concentration (200 microM) known to produce selective inhibition of poly(ADP-ribosyl)ation in intact cells and examined nucleoside-induced killing using a number of different end points (cell membrane disruption, cell shrinkage, mitochondrial depolarization, exposure of phosphatidyl serine, morphological changes, DNA fragmentation, and PARP-1 cleavage). In 27 of the 30 cases of CLL examined, 3AB delayed nucleoside-induced cell membrane disruption without inhibiting other manifestations of cytotoxicity. This indicates that PARP activity, rather than contributing to the induction of cell killing, was accelerating cell membrane disruption during the late stages of apoptosis. This novel observation has important implications for previous studies of PARP-mediated cytotoxicity. However, in cells from one CLL patient, 3AB inhibited all manifestations of nucleoside cytotoxicity; this was the only case in the study known to have a p53 gene defect affecting both alleles. This indicates that PARP activity can occasionally be central to nucleoside-induced killing and that such PARP-mediated killing is p53 independent.
To clarify the role of p53 in the killing of chronic lymphocytic leukaemia (CLL) cells by purine analogues, we examined the cytotoxic effects of chlorodeoxyadenosine and fludarabine on CLL cells that ...had been characterized according to their p53 functional status.
Cases of CLL with p53 dysfunction (n = 7) displayed slight, but significant, resistance to nucleoside‐induced cell killing when compared with cases with functionally intact p53 (n = 12). The small difference between the two groups indicated that p53 plays a minor role in such killing.
These findings suggest that the poor therapeutic response to purine analogues observed in patients with p53 defects is likely to be caused by the emergence, on a background of genomic instability, of CLL‐cell clones that are resistant to nucleoside‐induced killing for reasons unrelated to p53.
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