Histone deacetylase inhibitors in adult patients with diffuse large b-cell lymphoma relapsed/refractory: a phase II study with Panobinostat

Backgrounds:Treatment of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) not eligible to high dose therapy represents an unmet medical need. Panobinostat showed encouraging therapeutic activity in studies conducted in lymphoma cell lines and in vivo in patients with advanced hematologic malignancies.Purpose:FIL-PanAL10 (NCT01523834) is a phase II, prospective multicenter trial of the Fondazione Italiana Linfomi (FIL) to evaluate safety and efficacy of single agent Panobinostat as salvage therapy for R/R DLBCL patients and to evaluate a possible relationships between response and any biological features. Patients and Methods:Patients with R/R DLBCL were included. The treatment plan included 6 induction courses with Panobinostat monotherapy followed by other 6 courses of consolidation. The primary objective was to evaluate Panobinostat activity in terms of overall response (OR); secondary objectives were: CR rate, time to response (TTR), progression-free survival (PFS), safety and feasibility of Panobinostat. We included evaluation of the impact of pharmacogenetics, immunohistochemical patterns and patient’s specific gene expression and mutations as potential predictors of response to Panobinostat as explorative objectives. To this aim a pre-enrollment new tissue biopsy was mandatory. ResultsThirty-five patients, 21 males (60%), were enrolled between June 2011 and March 2014. At the end of induction phase, 7 responses (20%) were observed, including 4 CR (11%), while 28 patients (80%) discontinued treatment due to progressive disease (PD) in 21 (60%) or adverse events in 7 (20%). Median TTR in 9 responders was 2.6 months (range 1.8-12). With a median follow up of 6 months (range 1-34), the estimated 12 months PFS and OS were 27% and 30.5%, respectively. Grade 3-4 thrombocytopenia and neutropenia were the most common toxicities (in 29 (83%) and 12 (34%) patients, respectively. Conclusions The results of this study indicate that Panobinostat might be remarkably active in some patients with R/R DLBCL, showing durable CR

Development of strategies for vascular damage repair in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive and rare disease with so far unclear pathogenesis, limited treatment options and poor prognosis. Unbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is an important hallmark of PAH. In this research Sodium butyrate (BU) has been evaluated in vitro and in vivo models of PAH. This histone deacetylase inhibitor (HDACi) counteracted platelet-derived growth factor (PDGF)-induced ki67 expression in PASMCs, and arrested cell cycle mainly at G0/G1 phases. Furthermore, BU reduced the transcription of PDGFRbeta, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing and pulmonary artery ring assays indicated that BU inhibited PDGF-induced PASMC migration. BU strongly inhibited PDGF-induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculinA. In vivo, BU showed efficacy in monocrotaline-induced PAH in rats. Indeed, the HDACi reduced both thickness of distal pulmonary arteries and right ventricular hypertrophy. Besides these studies, Serial Analysis of Gene Expression (SAGE) has be used to obtain complete transcriptional profiles of peripheral blood mononuclear cells (PBMCs) isolated from PAH and Healthy subjects. SAGE allows quantitative analysis of thousands transcripts, relying on the principle that a short oligonucleotide (tag) can uniquely identify mRNA transcripts. Tag frequency reflects transcript abundance. We enrolled patients naïve for a specific PAH therapy (4 IPAH non-responder, 3 IPAH responder, 6 HeritablePAH), and 8 healthy subjects. Comparative analysis revealed that significant differential expression was only restricted to a hundred of down- or up-regulated genes. Interestingly, these genes can be clustered into functional networks, sharing a number of crucial features in cellular homeostasis and signaling. SAGE can provide affordable analysis of genes amenable for molecular dissection of PAH using PBMCs as a sentinel, surrogate tissue. Altogether, these findings may disclose novel perspectives in the use of HDACi in PAH and potential biomarkers.

Ruolo dell'Enantiomero (R)-9-HSA nel controllo della proliferazione in una linea di Adenocarcinoma del Colon umano

9-hydroxystearic acid (9-HSA) is an endogenous lipoperoxidation product and its administration to HT29, a colon adenocarcinoma cell line, induced a proliferative arrest in G0/G1 phase mediated by a direct activation of the p21WAF1 gene, bypassing p53. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity, showing interesting features as a new anticancer drug. The interaction of 9-HSA with the catalytic site of the 3D model has been tested with a docking procedure: noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one. Thus, in this study, (R)- and (S)-9-HSA were synthesized and their biological activity tested in HT29 cells. At the concentration of 50 M (R)-9-HSA showed a stronger antiproliferative effect than the (S) isomer, as indicated by the growth arrest in G0/G1. The inhibitory effect of (S)-9-HSA on HDAC1, HDAC2 and HDAC3 activity was less effective than that of the (R)-9-HSA in vitro, and the inhibitory activity of both the (R)- and the (S)-9-HSA isomer, was higher on HDAC1 compared to HDAC2 and HDAC3, thus demonstrating the stereospecific and selective interaction of 9-HSA with HDAC1. In addition, histone hyperacetylation caused by 9-HSA treatment was examined by an innovative HPLC/ESI/MS method. Analysis on histones isolated from control and treated HT29 confirmed the higher potency of (R)-9-HSA compared to (S)-9-HSA, severely affecting H2A-2 and H4 acetylation. On the other side, it seemed of interest to determine whether the G0/G1 arrest of HT29 cell proliferation could be bypassed by the stimulation with the growth factor EGF. Our results showed that 9-HSA-treated cells were not only prevented from proliferating, but also showed a decreased [3H]thymidine incorporation after EGF stimulation. In this condition, HT29 cells expressed very low levels of cyclin D1, that didn’t colocalize with HDAC1. These results suggested that the cyclin D1/HDAC1 complex is required for proliferation. Furthermore, in the effort of understanding the possible mechanisms of this effect, we have analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. EGF/EGFR/HDAC1 complex quantitatively increases in 9-HSA-treated cells but not in serum starved cells after EGF stimulation. Our data suggested that 9-HSA interaction with the catalytic site of the HDAC1 disrupts the HDAC1/cyclin D1 complex and favors EGF/EGFR recruitment by HDAC1, thus enhancing 9-HSA antiproliferative effects. In conclusion 9-HSA is a promising HDAC inhibitor with high selectivity and specificity, capable of inducing cell cycle arrest and histone hyperacetylation, but also able to modulate HDAC1 protein interaction. All these aspects may contribute to the potency of this new antitumor agent.

Role of (R)-9-hydroxystearic acid in zebrafish development

9-hydroxystearic acid (9-HSA) belongs to a class of lipid peroxidation products identified in several human and murine cell lines. These products are greatly diminished in tumors compared to normal tissues and their amount is inversely correlated with the malignancy of the tumor. 9-HSA activity has been tested in cancer cell lines, where it showed to act as a histone deacetylase 1 (HDAC1) inhibitor. In particular, in a colon cancer cell line (HT29), its administration resulted in an inhibition of proliferation together with an induction of differentiation. In this thesis the effect of (R)-9-hydroxystearic acid has been tested in vivo on cell proliferation and differentiation processes, in the early stages of zebrafish development. The final aim of this work was to elucidate the role of (R)-9-HSA in the control of cell differentiation and proliferation during normal development, in order to better understand its molecular control of cancerogenesis. The molecule has been administered via injection in the yolk of zebrafish embryos. The analysis of the histone acetylation pattern showed a hyperacetilation of histone H4 after treatment with the molecule, as detectable in HDAC1 mutants. (R)-9-HSA was also demonstrated to interfere with the signaling pathways that regulate proliferation and differentiation in zebrafish retina and hindbrain. This resulted in a reduction of proliferation in the hindbrain at 24 hours post injection (hpi), and in a hyperproliferation at 48 and 72 hpi in the retina, with a concomitant inhibition of differentiation. Finally, (R)-9-HSA effects were evident on proliferation of stem cell located in the ciliary marginal zone (CMZ) of the retina. The presence of ROS and 4-hydroxynoneal in the CMZ of wild-type embryos supports the hypothesis that oxidative stress could regulate stem cells fate in zebrafish retina.

Complexity of N-Myc transcriptional function in childhood neuroblastoma

Myc is a transcription factor that can activate transcription of several hundreds genes by direct binding to their promoters at specific DNA sequences (E-box). However, recent studies have also shown that it can exert its biological role by repressing transcription. Such studies collectively support a model in which c-Myc-mediated repression occurs through interactions with transcription factors bound to promoter DNA regions but not through direct recognition of typical E-box sequences. Here, we investigated whether N-Myc can also repress gene transcription, and how this is mechanistically achieved. We used human neuroblastoma cells as a model system in that N-MYC amplification/over-expression represents a key prognostic marker of this tumour. By means of transcription profile analyses we could identify at least 5 genes (TRKA, p75NTR, ABCC3, TG2, p21) that are specifically repressed by N-Myc. Through a dual-step-ChIP assay and genetic dissection of gene promoters, we found that N-Myc is physically associated with gene promoters in vivo, in proximity of the transcription start site. N-Myc association with promoters requires interaction with other proteins, such as Sp1 and Miz1 transcription factors. Furthermore, we found that N-Myc may repress gene expression by interfering directly with Sp1 and/or with Miz1 activity (i.e. TRKA, p75NTR, ABCC3, p21) or by recruiting Histone Deacetylase 1 (Hdac1) (i.e. TG2). In vitro analyses show that distinct N-Myc domains can interact with Sp1, Miz1 and Hdac1, supporting the idea that Myc may participate in distinct repression complexes by interacting specifically with diverse proteins. Finally, results show that N-Myc, through repressed genes, affects important cellular functions, such as apoptosis, growth, differentiation and motility. Overall, our results support a model in which N-Myc, like c-Myc, can repress gene transcription by direct interaction with Sp1 and/or Miz1, and provide further lines of evidence on the importance of transcriptional repression by Myc factors in tumour biology.

Progettazione e sintesi di nuovi derivati azetidinonici biologicamente attivi

In this PhD-thesis new synthetic approaches towards new azetidinone derivatives are described. In particular, 4-alkyliden-β-lactams were used as starting materials for the preparation of new biologically active compounds. The carbapenem Thienamycin has got a broad spectrum of activity as antibiotic. It has got 3 stereocenters and apart of one epimer, all isomers have been synthesized. Using the 4-alkyliden-β-lactam benzilyc ester as precursor, we developed a synthesis for this missing epimer, which is described in chapter II. Biological tests in order to establish its biological activity are under way. The Hunsdiecker-Borodine reaction was extensively studied for the preparation of the mono halogenated and – surprisingly – the dihalogenated derivative from the 4-alkyliden-azetidinone carboxylic acid. The herein described synthetic procedures allowed the preparation of chloro-, bromo- and iodo derivatives in good to excellent yield. Furthermore, the reaction mechanism was investigated by NMR-experiments and is described in detail in chapter III. In chapter IV, synthetic approaches towards new β-lactam derivatives for inhibition of the histone deacetylase enzymes (HDACs) are reported. In collaboration with the company Sigma-Tau (Rome), 14 new β-lactams were synthesized. The new β-lactams were evaluated for the activity showing a promising activityparticulary, 10 of the β-lactams synthesized were evaluated for the in vitro inhibitory activity against the 11 human HDACs isoforms and they showed a selective inhibition of HDAC6 or HDAC8 in micromolar range. Finally, preliminary studies were conducted for the employment of 4-alkyliden-β-lactams as precursors for the synthesis of chiral β-amino acids by an opening of the β-lactam ring. In chapter V is described the ring opening reaction catalyzed by the enzyme lipase Cal-B. Preliminary results have shown that the enzyme not only catalyzes the ring opening of the β-lactam precursor, moreover, it leads to the formation of a cyclic dimer by the reaction of two molecules of β-amino acid obtained.

Design and Synthesis of Naphthalene Diimides Based Small Molecules as Anticancer Agents: Targeting the Polyamine Transporter, G-quadruplex Structures and HDAC

Cancer is a multifactorial disease characterized by a very complex etiology. Basing on its complex nature, a promising therapeutic strategy could be based by the “Multi-Target-Directed Ligand” (MTDL) approach, based on the assumption that a single molecule could hit several targets responsible for the pathology. Several agents acting on DNA are clinically used, but the severe deriving side effects limit their therapeutic application. G-quadruplex structures are DNA secondary structures located in key zones of human genome; targeting quadruplex structures could allow obtaining an anticancer therapy more free from side effects. In the last years it has been proved that epigenetic modulation can control the expression of human genes, playing a crucial role in carcinogenesis and, in particular, an abnormal expression of histone deacetylase enzymes are related to tumor onset and progression. This thesis deals with the design and synthesis of new naphthalene diimide (NDI) derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development, such as HDACs. NDI-polyamine and NDI-polyamine-hydroxamic acid conjugates have been designed with the aim to provide potential MTDLs, in order to create molecules able simultaneously to interact with different targets involved in this pathology, specifically the G-quadruplex structures and HDAC, and to exploit the polyamine transport system to get selectively into cancer cells. Macrocyclic NDIs have been designed with the aim to improve the quadruplex targeting profile of the disubstituted NDIs. These compounds proved the ability to induce a high and selective stabilization of the quadruplex structures, together with cytotoxic activities in the micromolar range. Finally, trisubstituted NDIs have been developed as G-quadruplex-binders, potentially effective against pancreatic adenocarcinoma. In conclusion, all these studies may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

Preclinical neuroblastoma models for a pharmacological study of a new MYCN oncogene inhibitor

Background. Neuroblastoma is the most deadly solid tumor of childhood. In the 25% of cases it is associated with MYCN amplification (MA), resulting in the disregulation of several genes involved in cancer progression, chemotherapy resistance and poor prognosis causing the disregulation of several genes involved in cancer progression and chemotherapy resistance and resulting in a poor prognosis. Moreover, in this contest, therapy-related p53 mutations are frequently found in relapsed cases conferring an even stronger aggressiveness. For this reason, the actual therapy requires new antitumor molecules. Therefore, rapid, accurate, and reproducible preclinical models are needed to evaluate the evolution of the different subtypes and the efficacy of new pharmacological strategies. Procedures. We report the real-time tumorigenesis of MA Neuroblastoma mouse models: transgenic TH-MYCN mice and orthotopic xenograft models with either p53wt or p53mut, by non-invasive micro PET and bioluminescent imaging, respectively. Characterization of MYCN amplification and expression was performed on every collected sample. We tested the efficacy of a new MYCN inhibitor in vitro and in vivo. Results. MicroPET in TH-MYCN mice permitted the identification of Neuroblastoma at an early stage and offered a sensitive method to follow metabolic progression of tumors. The MA orthotopic model harboring multitherapy-related p53 mutations showed a shorter latency and progression and a stronger aggressiveness respect to the p53wt model. The presence of MA and overexpression was confirmed in each model and we saw a better survival in the TH-MYCN homozigous mice treated with the inhibitor. Conclusions. The mouse models obtained show characteristics of non-invasiveness, rapidity and sensitivity that make them suitable for the in vivo preclinical study of MA-NB. In particular, our firstly reported p53mut BLI xenograft orthotopic mouse model offers the possibility to evaluate the role of multitherapy-related p53 mutations and to validate new p53 independent therapies for this highly aggressive Neuroblastoma subtype. Moreover, we have shown potential clinical suitability of an antigene strategy through its cellular and molecular activity, ability to specifically inhibit transcription and in vivo efficacy with no evidence of toxicity.

Innovative Strategies for the Synthesis of Biologically Active Small Molecules

The post genomic era, set the challenge to develop drugs that target an ever-growing list of proteins associated with diseases. However, an increase in the number of drugs approved every year is nowadays still not observed. To overcome this gap, innovative approaches should be applied in drug discovery for target validation, and at the same time organic synthetic chemistry has to find new fruitful strategies to obtain biologically active small molecules not only as therapeutic agents, but also as diagnostic tools to identify possible cellular targets. In this context, in view of the multifactorial mechanistic nature of cancer, new chimeric molecules, which can be either antitumor lead candidates, or valuable chemical tools to study molecular pathways in cancer cells, were developed using a multitarget-directed drug design strategy. According to this approach, the desired hybrid compounds were obtained by combining in a single chemical entity SAHA analogues, targeting histone deacetylases (HDACs), with substituted stilbene or terphenyl derivatives able to block cell cycle, to induce apoptosis and cell differentiation and with Sorafenib derivative, a multikinase inhibitor. The new chimeric derivatives were characterized with respect to their cytotoxic activity and their effects on cell cycle progression on leukemia Bcr-Abl-expressing K562 cell lines, as well as their HDACs inhibition. Preliminary results confirmed that one of the hybrid compounds has the desired chimeric profile. A distinct project was developed in the laboratory of Dr Spring, regarding the synthesis of a diversity-oriented synthesis (DOS) library of macrocyclic peptidomimetics. From a biological point of view, this class of molecules is extremely interesting but underrepresented in drug discovery due to the poor synthetic accessibility. Therefore it represents a valid challenge for DOS to take on. A build/couple/pair (B/C/P) approach provided, in an efficient manner and in few steps, the structural diversity and complexity required for such compounds.

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.

Mechanisms Contributing to Tyrosin Kinase Inhibitor Resistance in GISTs: Toward a Personalized Therapy

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. This work considers the pharmacological response in GIST patients treated with imatinib by two different angles: the genetic and somatic point of view. We analyzed polymorphisms influence on treatment outcome, keeping in consideration SNPs in genes involved in drug transport and folate pathway. Naturally, all these intriguing results cannot be considered as the only main mechanism in imatinib response. GIST mainly depends by oncogenic gain of function mutations in tyrosin kinase receptor genes, KIT or PDGFRA, and the mutational status of these two genes or acquisition of secondary mutation is considered the main player in GIST development and progression. To this purpose we analyzed the secondary mutations to better understand how these are involved in imatinib resistance. In our analysis we considered both imatinib and the second line treatment, sunitinib, in a subset of progressive patients. KIT/PDGFRA mutation analysis is an important tool for physicians, as specific mutations may guide therapeutic choices. Currently, the only adaptations in treatment strategy include imatinib starting dose of 800 mg/daily in KIT exon-9-mutated GISTs. In the attempt to individualize treatment, genetic polymorphisms represent a novelty in the definition of biomarkers of imatinib response in addition to the use of tumor genotype. Accumulating data indicate a contributing role of pharmacokinetics in imatinib efficacy, as well as initial response, time to progression and acquired resistance. At the same time it is becoming evident that genetic host factors may contribute to the observed pharmacokinetic inter-patient variability. Genetic polymorphisms in transporters and metabolism may affect the activity or stability of the encoded enzymes. Thus, integrating pharmacogenetic data of imatinib transporters and metabolizing genes, whose interplay has yet to be fully unraveled, has the potential to provide further insight into imatinib response/resistance mechanisms.

The constitutive activation of the DNA damage response pathway is a novel therapeutic target in aggressive B-cell lymphoma

The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL. The second part of this work is the clinical, molecular and functional description of a paradigmatic case of primary refractory Burkitt lymphoma characterized by spatial intratumor heterogeneity for the TP53 mutational status, high expression levels of genomic instability and DDR activation markers, primary resistance to chemotherapy and exquisite sensitivity to DDR inhibitors.