Victoria Weston

We detected an interaction of the N-terminus of huntingtin (htt171) with the C-terminal region of the nuclear receptor co-repressor (N-CoR) using the yeast two-hybrid system. This interaction was repeat length dependent and specific to htt171; the co-repressor did not interact with the repeat carrying a section of atrophin 1 nor with the androgen receptor or polyglutamine alone. The interaction was confirmed using His-tagged Escherichia coli -expressed C-terminal human and rat co-repressor protein which pulled full-length huntingtin out of homogenized rat brain and in pull-down assays. The N-CoR represses transcription from sequence-specific ligand-activated receptors such as the retinoid X-thyroid hormone receptor dimers and other nuclear receptors including Mad-Max receptor dimers. The mechanism of this repression appears to be through the formation of a complex of repressor proteins including the N-CoR, mSin3 and histone deacetylases. We have used N-CoR and mSin3A antibodies in immunohistochemical studies and find that in Huntington's disease (HD) cortex and caudate, the cellular localization of these proteins is exclusively cytoplasmic whilst in control brain they are localized in the nucleus as well as the cytoplasm; mSin3A immunoreactivity also occurred in a subset of huntingtin positive intranuclear inclusions. The relocalization of repressor proteins in HD brain may alter transcription and be involved in the pathology of the disease.

The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.

B-cell chronic lymphocytic leukemia (B-CLL) is a clinically variable disease where mutations in DNA damage response genes ATM or TP53 affect the response to standard therapeutic agents. The in vitro cytotoxicity of a novel cyclin-dependent kinase inhibitor, CYC202, was evaluated in 26 B-CLLs, 11 with mutations in either the ATM or TP53 genes, and compared with that induced by ionizing radiation and fludarabine. CYC202 induced apoptosis within 24 hours of treatment in all 26 analyzed tumor samples independently of ATM and TP53 gene status, whereas 6 of 26 B-CLLs, mostly ATM mutant, showed marked in vitro resistance to fludarabine-induced apoptosis. Compared with B-CLLs, normal T and B lymphocytes treated with CYC202 displayed reduced and delayed apoptosis. Using global gene expression profiling, we found that CYC202 caused a significant down-regulation of genes involved in regulation of transcription, translation, survival, and DNA repair. Furthermore, induction of apoptosis by CYC202 was preceded by inhibition of RNA polymerase II phosphorylation, leading to down-regulation of several prosurvival proteins. We conclude that CYC202 is a potent inducer of apoptosis in B-CLL regardless of the functional status of the p53 pathway, and may be considered as a therapeutic agent to improve the outcome of resistant B-CLL tumors.

The ATM/p53-dependent DNA damage response pathway plays an important role in the progression of lymphoid tumors. Inactivation of the ATM or TP53 gene is frequent in B-cell lymphocytic leukemia (B-CLL) and leads to aggressive disease. Although the ATM and p53 pathways overlap, they are not congruent, and it is unclear how the mechanism of tumor progression differs between ATM- and p53-deficient tumors. Using microarray analysis of ATM-mutant, TP53-mutant, and ATM/TP53 wild-type B-CLLs, we show that after exposure to DNA damage transcriptional responses are entirely dependent on ATM function. The p53 proapoptotic responses comprise only a part of ATM-regulated transcription; additionally, ATM regulates prosurvival responses independently of p53. Consequently, the greater severity of the TP53-mutant B-CLLs compared with ATM-mutant B-CLLs is consistent with the additive effect of defective apoptotic and elevated survival responses after DNA damage in these tumors. We also show that transcription expression profiles of ATM-deficient, TP53-deficient, and wild-type B-CLLs are indistinguishable before irradiation. Therefore, damage-induced transcriptional fingerprinting can be used to stratify tumors according to their biologic differences and simultaneously identify potential targets for treating refractory tumors.

Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1, a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in paediatric ALL. We showed a potent in vitro cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic features but dependent on high MYC expression and coupled with transcriptional downregulation of multiple pro-survival pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone. Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.