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Background Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL1 fusion protein. Accordingly, TK inhibitors have drastically changed the disease prognosis. However, persistence of the transformed hematopoiesis even in patients who achieved a complete response to TK inhibitors and the disease relapse upon therapy discontinuation represent a major obstacle to CML cure. Methods Thiostrepton, Danusertib and Volasertib were used to investigate the effects of FOXM1, AKA and Plk1 inhibition in K562-S and K562-R cells. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Quantitative reverse transcription (RT)-PCR was used to assess BCR-ABL1, FOXM1, PLK1 and AURKA expression. Protein expression and activation was assessed by Western Blotting (WB). Clonogenic assay were performed to confirm K562-R resistance to Imatinib and to evaluate cells sensitivity to the different drugs. Results Here we proved that BCR-ABL1 TK-dependent hyper-activation of Aurora kinase A (AURKA)-Polo-like kinase 1 (PLK1)-FOXM1 axis is associated with the outcome of Imatinib (IM) resistance in an experimental model (K562 cell line) and bone marrow hematopoietic cells. Notably, such a biomolecular trait was detected in the putative leukemic stem cell (LSC) compartment characterized by a CD34+ phenotype. Constitutive phosphorylation of FOXM1 associated with BCR-ABL1 TK lets FOXM1 binding with β-catenin enables β-catenin nuclear import and recruitment to T cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription complex, hence supporting leukemic cell proliferation and survival. Lastly, the inhibition of single components of AURKA-PLK1-FOXM1 axis in response to specific drugs raises the expression of growth factor/DNA damage-inducible gene a (GADD45a), a strong inhibitor of AURKA and, as so, a critical component whose induction may mediate the eradication of leukemic clone. Conclusions Our conclusion is that AURKA, PLK1 and FOXM1 inhibition may be considered as a promising therapeutic approach to cure CML. Electronic supplementary material The online version of this article (10.1186/s13046-019-1197-9) contains supplementary material, which is available to authorized users.

An in silico structure-based ligand design approach resulted in the identification of the first non-peptidic small molecule able to inhibit protein-protein interactions between 14-3-3 and c-Abl. This compound shows an anti-proliferative effect on human leukemia cells either sensitive or resistant to Imatinib, in consequence of the T315I mutation. It also mediates c-Abl release from 14-3-3 in a way similar to that found in response to Imatinib treatment.

To investigate the impact of TP53 (tumor protein 53, p53) on genomic stability of osteosarcoma (OS).In first instance, we expressed in OS cell line SAOS-2 (lacking p53) a wild type (wt) p53 construct, whose protein undergoes nuclear import and activation in response to ionizing radiations (IR). Thereafter, we investigated genomic imbalances (amplifications and deletions at genes or DNA regions most frequently altered in human cancers) associated with radio-resistance relative to p53 expression by mean of an array-based comparative genomic hybridization (aCGH) strategy. Finally we investigated a putative marker of radio-induced oxidative stress, a 4,977 bp deletion at mitochondrial (mt) DNA usually referred to as 'common' deletion, by mean of a polimerase chain reaction (PCR) strategy.In radio-resistant subclones generated from wt p53-transfected SAOS-2 cells DNA deletions were remarkably reduced and the accumulation of 'common' deletion at mtDNA (that may let the persistence of oxidative damage by precluding detoxification from reactive oxygen species [ROS]) completely abrogated.The results of our study confirm that wt p53 has a role in protection of OS cell DNA integrity. Multiple mechanisms involved in p53 safeguard of genomic integrity and prevention of deletion outcome are discussed.

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.

Hodgkin’s lymphoma (HL) is a heterogeneous lymphoid malignancy curable in more than 80% of cases. Its morbility and mortality are mostly contingent upon chemo- and radio-therapy side effects including cardiovascular or pulmonary diseases, infections and second malignancies. Indeed, the high incidence of secondary cancers and leukemias in potentially cured LH patients supports that genomic instability and defective DNA repair system have a crucial role in secondary cancerogenesis/leukemogenesis processes as they let the emergence of genomic aberrations and clonal cell evolution towards a transformed phenotype. The aim of our study was to define the genomic profile associated with HL evolution towards secondary leukemia. To the purpose, formalin-fixed/paraffin-embedded biopsies from 4 LH patients who developed a secondary acute myeloid leukemia (AML) were analyzed by mean of array-based comparative genomic hybridization (aCGH). This method allows the identification of changes in DNA sequence copy number (amplifications or deletions) at high resolution. The microarrays used in our study contain 287 genomic targets involved in most human cancers, including oncogenes, tumor-suppressor genes and DNA sequences localized within chromosomal regions most frequently rearranged. DNAs from lymphonode biopsies of LH patient who developed AML were compared with pooled DNAs from reactive and LH lymphonodes (comparable for histotype and follow-up) from cured patients. CGH profiles of single LH patients were extensively altered. However, they shared a common pattern of structural genomic alterations. Bioinformatic analysis of CGH results performed with dedicated softwares let distinguish statistatically significant (ratio >1,2) amplifications relative to 3 DNA sequences (AFM137XA11, FGFR1, PPABP) and statistatically significant (ratio< 0,83) deletions in 4 (AFM217YD10, FGR(SRC2), GATA3, TOP1, WT1). CGH data relative to the above mentioned sequences were further validated by FISH and immunohistochemistry. In spite of the low number of LH patients our results suggest that the evolution to AML in LH patients may be genetically determined. The role of single genes in leukemic transformation requires further investigation.

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

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.

Nuclear accumulation of c-Abl protein in response to genotoxic damage addresses apoptotic cell death. The process, driven by the disruption of its binding to 14-3-3 scaffolding proteins, is independent from c-Abl catalytic state and conditional upon 14-3-3 phosphorylation by the c-jun N-terminal kinase (JNK) (Yoshida et al Nat Cell Biol 7,278,2005). Our work moved from the observation that the constitutive activation of Abl tyrosine kinase (TK) by juxtaposed Bcr sequences in p210 fusion protein Bcr-Abl precludes c-Abl nuclear import in response to gamma irradiation, advancing a role for residual c-Abl protein “loss of function” in the apoptosis-resistant phenotype of Chronic Myeloid Leukemia (CML) progenitors. In 32D cell clones transducing a temperature-conditional Bcr-Abl mutant, active p210 TK is associated with the overexpression of 14-3-3sigma mostly driven by transcriptional events involving histone H4 acetylation (but not methylation) status of the gene promoter. P210 TK inhibition in response to 24 hour exposure to imatinib mesylate (IM) is associated with an early downregulation of 14-3-3sigma followed by c-Abl nuclear import and cell commitment to apoptotic death. The two last events are further enhanced by complementary inhibition of the 14-3-3 binding site by R18 peptide, supporting that the negative impact of p210 TK on pro-apoptotic function of residual normal c-Abl arises from its effects on 14-3-3sigma. Previous studies proved that the activation of p38 MAP kinase contributes to IM effects in CML (Parmar et al, J Biol Chem279,25345,2004). Interestingly, p38 MAP kinase is also involved in tuberous sclerosis 2 gene protein (TSC2, also known as tuberin) phosphorylation and enhanced binding to 14-3-3 possibly promoting the activation of IM-compensatory signals, including m-Tor (Li et al, J Biol Chem278,13663,2003). Here we show that the m-Tor inhibitor RAD001 (everolimus; kindly provided by Novartis Pharma AG) at 100 nM concentration induces c-Abl nuclear import and apoptotic cell death in 32D cell clones expressing active p210 TK. Both events are further and significantly enhanced by combination with IM. C-Abl nuclear shuttling in response to the RAD001 and IM combination procedes from Akt and p38 MAP kinase inactivating dephosphorylation and 14-3-3 phosphorylation at serine residues critical for client protein binding including the apoptotic death effectors Bad, Bax and Ask1. In conclusion, our work supports the advantage of TK and m-Tor inhibitor association for CML treatment.