The transforming proteins of acute promyelocytic leukaemias (APL) are fusions of the promyelocytic leukaemia (PML) and the promyelocytic leukaemia zinc-finger (PLZF) proteins with retinoic acid receptor-alpha (RARalpha). These proteins retain the RARalpha DNA- and retinoic acid (RA)-binding domains, and their ability to block haematopoietic differentiation depends on the RARalpha DNA-binding domain. Thus RA-target genes are downstream effectors. However, treatment with RA induces differentiation of leukaemic blast cells and disease remission in PML-RARalpha APLs, whereas PLZF-RARa APLs are resistant to RA. Transcriptional regulation by RARs involves modifications of chromatin by histone deacetylases, which are recruited to RA-target genes by nuclear co-repressors. Here we show that both PML-RARalpha and PLZF-RARalpha fusion proteins recruit the nuclear co-repressor (N-CoR)-histone deacetylase complex through the RARalpha CoR box. PLZF-RARalpha contains a second, RA-resistant binding site in the PLZF amino-terminal region. High doses of RA release histone deacetylase activity from PML-RARalpha, but not from PLZF-RARalpha. Mutation of the N-CoR binding site abolishes the ability of PML-RARalpha to block differentiation, whereas inhibition of histone deacetylase activity switches the transcriptional and biological effects of PLZF-RARalpha from being an inhibitor to an activator of the RA signalling pathway. Therefore, recruitment of histone deacetylase is crucial to the transforming potential of APL fusion proteins, and the different effects of RA on the stability of the PML-RARalpha and PLZF-RARalpha co-repressor complexes determines the differential response of APLs to RA.

Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphiapositive leukemias. To assess how BcrAbl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for IntroductionResistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) in chronic myeloid leukemia (CML) and Philadelphiapositive (Ph ϩ ) acute lymphoblastic leukemia (ALL) is often caused by selection of mutations in the Bcr-Abl kinase domain (KD), [1][2][3][4][5][6][7][8][9] altering residues that are directly or indirectly critical for IM binding. To overcome this problem, novel TKIs have been developed. Dasatinib and nilotinib [10][11][12][13] have been the first ones to enter clinical evaluation and to receive marketing approval in IMresistant patients. Preclinical experience with these agents showed that they are active against several IM-resistant Bcr-Abl mutants, with the exception of T315I. 10,11,14 However, both TKIs have been hypothesized to retain their own "Achilles heels." Recent studies have tried to profile mutations that will probably emerge under dasatinib and nilotinib, inducing random mutagenesis and then selecting for cell clones retaining viability when cultured in the presence of the inhibitors. [15][16][17][18] Results suggested that, besides T315I, other mutants might be critical. To assess how Bcr-Abl KD mutation status evolves under the selective pressure of sequential therapy with novel TKIs and which mutations among those predicted by in vitro studies may indeed develop in patients who relapse on dasatinib or nilotinib, we have monitored the mutation status of 95 IM-resistant patients before and during sequential treatment with one or both of these agents. Methods Patients and definitionsThis report focuses on 95 patients (Table 1) who were referred to our laboratory for mutation analysis at the time of IM failure and who received up to 2 subsequent TKIs (dasatinib and/or nilotinib). Thirty-eight patients had chronic-phase (CP) CML, 46 patients had advanced-phase CML (accelerated-phase [AP], n ϭ 11; myeloid blast crisis [BC], n ϭ 18; lymphoid BC, n ϭ 17), and 11 patients had Ph ϩ ALL. For CML patients, IM failure was defined according to European LeukemiaNet recommendations. 19 Similar criteria were applied to define IM failure in Ph ϩ ALL. All 95 patients received a second TKI (dasatinib, n ϭ 55; nilotinib, n ϭ 40). Twenty-six of 95 patients received a third TKI after relapse on second TKI (dasatinib, n ϭ 16; nilotinib, n ϭ 10). For the purpose of this analysis, response to dasat...

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