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Imatinib mesilate (STI-571, Gleevec(TM)), an inhibitior of bcr-abl tyrosine kinases (TK) which was primarily designed to treat chronic myeloid leukaemia, is also an inhibitor of c-kit receptor TK, and is currently the drug of choice for the therapy of metastatic gastrointestinal stromal tumors (GISTs), which frequently express constitutively activated forms of the c-kit-receptor. Despite the fact that the majority of patients receiving imatinib respond to treatment, early relapse and drug resistance occur in a large percentage of them [1]. The present study is focused on the ability of a new histone deacetylase (HDAC) inhibitor, 9-hydroxystearic acid (9-HSA) [2] to enhance the cytotoxicity of imatinib in imatinib-resistant cell lines. We chose as cellular model HT29, a colon adenocarcinoma cell line expressing the c-kit receptor and which is p53 mutated. Our results demonstrated that HT-29 exposed for 24 hr to a high dose (50 M) of imatinib and maintained in drug-free medium for up to 7 days, developed resistance characterized by loss of apoptotic response to the drug. Pretreating resistant cell line (IR-HT-29) with 9-HSA led to a growth-inhibitory and apoptotic effect of imatinib similar to that in imatinib-sensitive HT-29 cell line. More precisely, 9-HSA treatment is able per se to trigger apoptosis and subsequent imatinib treatment amplifies the process. In order to understand the mechanisms of this effect, we analyzed the degree of acetylation, methylation and phosphorylation of histones in IR-HT-29, and in 9-HSA treated IR-HT-29. Previous studies from our laboratory showed that diacetyl-dimethyl H4 content increased in a highly specific manner in 9-HSA treated HT-29. The results presented in this work clearly show that histone code in IR-HT-29 was completely changed with respect to control sensitive HT-29, and that 9-HSA treatment induced not only the increase of the diacetyl-dimethyl H4 content, but also several modification on H3-1 histone. In conclusion our data indicate that the use of HDAC inhibitor 9-HSA may be a powerful strategy to enhance cytotoxic effects of imatinib in resistant cell lines. BIBLIOGRAFIA 1. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002; 347:472-80 2. Calonghi N, Cappadone C, Pagnotta E, Boga C, Bertucci C, Fiori J, et al. Histone deacetylase 1: a target of 9-hydroxystearic acid in the inhibition of cell growth in human colon cancer. J Lipid Res 2005; 46:1596-1603

La costitutiva attività tirosin-cinasica della proteina chimerica p210bcr-abl ha un ruolo centrale nella patogenesi e nella progressione della Leucemia Mieloide Cronica (LMC). Interagendo con molteplici circuiti di trasduzione del segnale mitogenico essa inibisce l'adesione delle cellule leucemiche allo stroma midollare, abroga i checkpoints del ciclo cellulare e induce resistenza all'apoptosi. Ciò che si osserva è un'abnorme espansione dell'ematopoiesi leucemica sulla controparte normale e l'instabilità genomica che ne condiziona l'evoluzione da un fenotipo normale, caratterizzante la fase cronica, ad un fenotipo trasformato tipico della crisi blastica. I risultati dello studio qui presentato indicano che la costitutiva attività tirosin-cinasica di p210bcr-abl ha effetti su molteplici segnali coinvolti nella via intrinseca e nella via estrinseca della apoptosi e convergenti sulla integrità della membrana mitocondriale. L'espressione di un costrutto sensibile in temperatura di Bcr-Abl in progenitori ematopoietici clonali (32D) e l'esposizione ad un inibitore specifico dell'attività tirosin-cinasica di p210bcr-abl (STI571) ci ha permesso di individuare il ruolo della proteina chimerica nella fase decisionale dell'apoptosi. P210bcr-abl causa l'overespressione dei segnali anti-apoptotici Bcl-2 e Bcl-xL, previene l'attivazione dei segnali pro-apoptotici Bid, Bad e Bax, impedendo la formazione di omodimeri ed eterodimeri tra gli stessi membri della famiglia BcI-2 a livello della membrana mitocondriale. Tale condizione blocca il rilascio di citocromo-c e di altri fattori apoptogeni dal mitocondrio. La formazione dell'apoptosoma risulta pertanto inibita, così come la cascata caspasica mitocondrio-dipendente. La definizione dei circuiti biomolecolari coinvolti nella resistenza alla morte apoptotica ha un impatto importante nel trattamento della LMC: può infatti aiutare ad identificare bersagli farmacologici rilevanti per la terapia della malattia in associazione all'inibizione dell'attività tirosin-cinasica di p210bcr-abl.

Constitutive tyrosine kinase (TK) activity of p210 bcr-abl fusion protein is the causative event of Chronic Myeloid Leukemia (CML). Its inhibition abrogates the proliferative advantage and apoptosis resistance of clonal leukemic progenitors and induces the disease remission and the emergence of normal hematopoiesis. Imatinib mesylate (IM) let hope to have a cure for Ph1-(bcr-abl)-positive leukemias. Unfortunaley, the IM resistance apparent from longer clinical follow-up emerged as the drug major limitation. It imposes the development of combined therapies capable of targeting genes located downstream of bcr-abl and endowed with a bcr-abl-independent role in CML progression towards the fully transformed phenotype of blast crisis. PI3 kinase inhibitors could be considered as very promising drugs as they target AKT pathway. We investigated the in vitro effects of Wortmannin alone or in combination with IM on proliferation and survival of bcr-abl-transducing 32D cell clones. Here we demonstrate that Wortmannin and IM combined treatment is more effective than IM alone in inhibiting the enzymatic activity of Cdk2, the cyclin-dependent kinase mostly responsible for p210 TK-induced acceleration of cell cycle progression. Cdk2 activity was prevented by the enhanced effects of drug combination on Chk2-Cdc25A axis and PI3K/Akt pathway (leading to increased nuclear import of Cdk inhibitors p21 and p27 and to cyclin D downmodulation). In conclusion, the combined use of TK and PI3K inhibitors may prevent IM resistance and, therefore, improve the prognosis of CML at clinical diagnosis, but its advantage in the treatment of IM-resistant CML is likely marginal and conditional upon the mechanisms responsible for IM resistance.

Extended survival of clonal hematopoietic progenitors associated with the bcr-abl expression has a key role in the pathogenesis of Chronic Myeloid Leukemia (CML) and may concur to promote the disease progression towards the fully transformed, drug-resistant phenotype of blast crisis. Here we demonstrated that p210 bcr-abl protein tyrosine kinase (TK) activity protects bcr-abl-transducing 32D cell clones from apoptotic death through multiple pathways preserving mitochondrial membrane integrity. P210 TK prevents Trail/DR4, Bim and Bax induction and caspase 9 and Bid activation and upregulates the expression of anti-apoptotic BclxL in the cytoplasm. Moreover, it precludes the integration and aggregation of pro-apoptotic signals Bid, Bax and Bak at mitochondrial membranes letting, in turn, pore opening and apoptogenic molecule leakage from intermembrane spaces. Our results underscore the central role of Akt serine/threonine kinase, activated by p210 TK downstream of phosphatidyl-inositol 3 kinase (PI3K), in leukemic progenitor failure to undergo apoptotic death and support that pharmacological targeting of its enzymatic activity may implement the effects of TK inhibitor Imatinib mesylate (IM).

Constitutive tyrosine kinase (TK) activity of p210 bcr-abl fusion protein is the causative event of Chronic Myeloid Leukemia (CML). Accordingly, its inhibition abrogates the proliferative advantage and apoptosis resistance of clonal leukemic progenitors and induces the disease remission and the emergence of normal hematopoiesis. Imatinib mesylate (IM), the first TK inhibitor approved for clinical use in CML, let to hope to have a cure for Ph1-(bcr-abl)-positive leukemias. Unfortunaley, the IM resistance apparent from longer clinical follow-up emerged as the drug major limitation. It imposes the development of combined therapies capable of targeting genes located downstream of bcr-abl and endowed with a bcr-abl-independent role in CML progression towards the fully transformed phenotype of blast crisis. Src kinase inhibitors are now regarded as the most promising drugs as they target p210 constitutive phosphorylation at Tyr177. We investigated the in vitro effects of a new compound, a 4-anilino-3-quinolinecarbonitrile (SKI-606) alone or in combination with IM on proliferation and survival of bcr-abl-transducing 32D cell clones. Here we demonstrate that SKI606 and IM combined treatment is more effective than IM alone in inhibiting the enzymatic activity of Cdk2, the cyclin-dependent kinase mostly responsible for p210 TK-induced acceleration of cell cycle progression. Cdk2 activity was prevented by the enhanced effects of drug combination on Chk2-Cdc25A axis and PI3K/Akt pathway (leading to increased nuclear import of Cdk inhibitors p21 and p27 and to cyclin D downmodulation). Drug combination effects on Chk2-Cdc25A and PI3K/Akt were greater in IM-sensitive bcr-abl-expressing cells compared to IM-resistant cells and were abrogated by IL3. In conclusion, the combined use of TK and Src kinase inhibitors may prevent IM resistance and, therefore, improve the prognosis of CML at clinical diagnosis, but its advantage in the treatment of IM-resistant CML is likely marginal and conditional upon the mechanisms responsible for IM resistance

The bcr-abl fusion gene originated from the balanced (9;22) translocation is the molecular hallmark and the causative event of chronic myeloid leukemia (CML). Constitutive activation and improper localization to the cytosol of p210 protein of bcr-abl induce enlargement of clonal hematopoiesis over its normal counterpart and consequently genetic instability that drives leukemic progenitor progression towards the fully transformed phenotype of blast crisis. To define a role to tyrosine kinase p210bcr-abl, we considered its impact on histone H4 acetylation status, focusing on the proceeding of enzyme HDAC1 (histone deacetylase1) during progression of the pathology. We performed molecular analysis in vitro in 32D cell clone expressing a temperature-sensitive (ts) bcr-abl mutant and in vivo in CD34+ from patients affected by CML. We observed that HDAC1 has several effects on gene expression in different phases of CML. In particular p210bcr-abl tyrosine kinase binds HDAC1, resulting in its cytoplasmic compartimentalization and consequently loss of function of HDAC1 itself: this event may be prominent in early chronic phase of the disease, when bcr-abl is the only genetic lesion and its transcription is essential for clonal hematopoiesis to proliferate and survive. It may also contribute to the disease progression by promoting clonal progenitor genomic instability driving the accumulation of additional genetic aberrations. Deregulation of transcription of genes controlling differentiation, apoptosis and growth arrest resulting from HDAC1 compartimentalization in the cytoplasm may be protagonist of more advanced stages of CML, when a fully transformed, drug-resistant phenotype emerges.

Covalent modifications of the core histone tails, includind acetylation, methylation, phosphorylation, ADP-ribosylation and ubiquitination govern the chromatine accessibility and create binding surfaces for protein recognition (such as the bromodomain far acetylated lysines and the chromodomain for methylated lysines), therefore dictating the rate of gene transcription. We investigated the impact of p210 bcr-abl tyrosine kinase of chronic myeloid leukemia (CMLl) on the acetylation and methylation at individual lysine residues within the NH2-terminal tails of histone H4, an integral component of transcriptional competence at the onset of S phase. To the purpose, we used RP-HPLC coupled with electrospray mass spectrometry (LCMS) to resolve the single H4 species (non-acetylated and mono-, di- or three-acetylated) and enzymatic digestion coupled with MALDI mass spectrometry to define the individuai lysine residues involved in acetylation and methylation processes. In single cell clones generated from the murine myeloid 32D cell line transducing a temperature-sensitive (ts) p210 bcr-abl construct (that owns constitutive tyrosine kinase activity at the permissive temperature of 33°C, but lacks it at the non- permissive temperature of 39°C) the inhibition of p210 bcr-abl tyrosine kinase induced by 1 M STI571 was associated with a significant (p

Unrestrained progression throughout the cell cycle, mostly resulting from abrogation of the G1/S checkpoint, has a key role in the pathogenesis and progression of CML. It permits, in fact, the illegitimate enlargement of clonal hematopoiesis over its normal counterpart and causes the genomic instability that drives further progression of CML towards the fully transformed phenotype of its terminal phase, the blast crisis. It is conditional upon the constitutive tyrosine kinase activity of the p210 protein of bcr-abl rearranged gene, the molecular marker of CML. Accordingly, the tyrosine kinase inhibitor STI571 (Imatinib), approved as the first-choice drug in the treatment of CML since 2001, significantly improved the prognosis of CML. More recently, the outcomes of large clinical trials uncovered the Achille heel of STI571, the drug resistance occurring at variable intervals from the beginning of therapy and likely arising from the selection of clonal myeloid progenitors bearing additional, drug-insensitive biomolecular traits. In our previous studies we proved that Cdk2 has a role in the early escape from STI571 of CML hematopoiesis (Mazzacurati et al, The Hematology Journal, 5, 168-177, 2004). Here, we confirmed that Cdk2 is constitutively activated in STI571-resistant clonal hematopoietic progenitors in spite of the drug pharmacological effects on its target, the p210 bcr-abl tyrosine kinase. Multiple pathways participate in sustaining Cdk2 enzymatic activity associated with resistance to STI571. They include: 1- the persistence of Cdc25A phosphatase (that activates Cdk2 by removing the inhibitory phosphorylation at Thr 14 and Tyr15), resulting, in turn, from the downmodulation of Chk2 serin-threonin kinase (that under adverse conditions addresses Cdc25A to the ubiquitin-dependent/proteasome mediated degradation) and of 14.3.3 chaperon protein (that binds Cdc25A and keeps it inactive within the cytoplasmatic compartment) as well as from the upmodulation of c-Myc (that transcriptionally regulates Cdc25A); 2- the down-modulation of Wee 1 (that provides the Cdk2 inhibitory phosphorylation at Tyr15). In conclusion, our results address to Cdk2 as an early molecular marker of responsiveness to STI571 of CML hematopoietic progenitors. Moreover and more importantly, they contribute to the identification of additional signals whose targeting, combined with the inhibition of p210 bcr-abl tyrosine kinase, may greatly help to improve the CML prognosis and circumvent the selection of drug-resistant clones.

The resistance to apoptotic death has a key role in the illegitimate enlargement of chronic myeloid leukemia (CML) hematopoiesis over its normal counterpart and in the genetic instability that drives clonal evolution of bcr-abl-rearranged myeloid progenitors towards the fully transformed phenotype of blast crisis. It results from multiple events preventing proapoptotic pathways (Trail-DR4, Fas-ligand and Bax induction, Bax and Bad translocation to the membranes of subcellular organelles such as mitochondria or endoplasmic reticulum, cytochrome-c release and caspase-3 activation) and/or enhancing pro-survival signals such as Bcl-2, Bcl-xL and survivin, and is mostly conditional upon the tyrosine kinase of p210 bcr-abl fusion protein. Our study addressed the matter of p210 bcr-abl tyrosine kinase effects on expression levels and subcellular locations of proteins that trigger apoptotic death in response to extrinsic or intrinsic signals by antagonizing anti-apoptotic Bcl- 2 and Bcl-xL at the mitochondrial membranes. To the purpose, in individuai cell clones generated from the murine myeloid 32D cell line transduced with a temperature-sensitive (ts) p210 bcr-abl construct (lacking the abl constitutive tyrosine kinase activity at the non-permissive temperature of 39°C) we investigated transcriptional induction and post-transcriptional modifications of pro-apoptotic signals in response to growth factor withdrawal, starvation, TNF-a and tyrosine kinase inhibitor STI571. Trail-DR4, Fas, Bax and Bim transcriptional induction, initiator caspase 8, 9 and 12 activation, Bad dephosphorylation, Bid cleavage and Bax and Bak aggregation at the mitochondrial membranes preceding citochrome-c release and executioner caspase activation are prevented by p210 bcr-abl tyrosine kinase through interactions with multiple trascription factors including Stat5, PI3K/Akt, Foxo3A and c-Myc. Our results may be helpful to design novel therapeutic strategies intended for targeting gene products relevant for CML progression in addition to p210 bcr-abl tyrosine kinase.