Andrea Ghelli Luserna Di Rorà

Targeting the p53-MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph−) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph− ALL.

Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions

Pixantrone is a novel aza-anthracenedione active against aggressive lymphoma and is being evaluated for use against various hematologic and solid tumors. The drug is an analog of mitoxantrone, but displays less cardiotoxicity than mitoxantrone or the more commonly used doxorubicin. Although pixantrone is purported to inhibit topoisomerase II activity and intercalate with DNA, exact mechanisms of how it induces cell death remain obscure. Here we evaluated the effect of pixantrone on a panel of solid tumor cell lines to understand its mechanism of cell killing. Initial experiments with pixantrone showed an apparent discrepancy between its anti-proliferative effects in MTS assays (short-term) compared with clonogenic assays (long-term). Using live cell videomicroscopy to track the fates of cells, we found that cells treated with pixantrone underwent multiple rounds of aberrant cell division before eventually dying after approximately 5 d post-treatment. Cells underwent abnormal mitosis in which chromosome segregation was impaired, generating chromatin bridges between cells or within cells containing micronuclei. While pixantrone-treated cells did not display γH2AX foci, a marker of DNA damage, in the main nuclei, such foci were often detected in the micronuclei. Using DNA content analysis, we found that pixantrone concentrations that induced cell death in a clonogenic assay did not impede cell cycle progression, further supporting the lack of canonical DNA damage signaling. These findings suggest pixantrone induces a latent type of DNA damage that impairs the fidelity of mitosis, without triggering DNA damage response or mitotic checkpoint activation, but is lethal after successive rounds of aberrant division.

The Combination of the PARP Inhibitor Rucaparib and 5FU Is an Effective Strategy for Treating Acute Leukemias

The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known. In this study we test, for the first time on leukemic cells, a combined therapy that associates the conventional chemotherapeutic agent fluorouracil (5FU), used as a source of DNA damage, and a PARP inhibitor, rucaparib. We demonstrate the efficacy and the specificity of this combined therapy in killing both acute myeloid leukemia and acute lymphoid leukemia cells in vitro and in vivo. We clearly show that the inhibition of DNA repair induced by rucaparib is synthetic lethal with the DNA damage caused by 5FU in leukemic cells. Therefore, we propose a new therapeutic strategy able to enhance the cytotoxic effect of DNA-damaging agents in leukemia cells via inhibiting the repair of damaged DNA. Mol Cancer Ther; 14(4); 889–98. ©2015 AACR.

Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Abstract 2723: The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia

Different Checkpoint kinase inhibitors (Chk-i) have been developed to increase the cytotoxic effect of genotoxic agents inhibiting the key elements of the DNA damage response (DDR) pathways. Our group has already showed the efficacy of this class of compounds in single agent in different in vitro/ex vivo/in vivo studies for the treatment of acute lymphoblastic leukemia (ALL). The aim of the study was to evaluate the efficacy of a Chk1/Chk2 inhibitor in combination with the topoisomerase II inhibitor doxorubicin for the treatment of ALL. Firstly we evaluate the efficacy of doxorubicin on human B (NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathways. Cells were treated with doxorubicin (0.25-2.5 uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. The induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was also evaluated in all the cell lines. Due to the inhibitory effect of the compound on the topoisomerase II enzyme and due to the activation of the cell cycle checkpoint, cells were arrested in G2/M phase. Then the effectiveness of the Chk-i as a chemo-sensitizer agent was evaluated. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk-i (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a synergistic effect in term of reduction of the cell viability and induction of apoptosis. The effect of the combination was also analyzed using western blot looking for specific marker of activation of the DDR pathway showing the same synergistic effect. Moreover the effect of the combination on cell cycle profile was evaluated using a double staining Pi/Anti-phospho-Histone H3 ser10 (marker of mitosis). Cell lines were pre-treated for 18 hours with doxorubicin and then with the Chk-i for different time points (1, 2, 3, 6 and 9 hours). The treatment with Chk-i removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells positive for the mitotic marker p-HH3 (ser10) and increasing the percentage of cells in sub-G1 phase. In our opinion the combination between the Chk1 inhibitor,LY2606368, and the topoisomerase II inhibitor, doxorubicin, could be a promising strategy for the treatment of B/T-ALL. Supported by ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni Martinelli. The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2723.

Therapeutic implications of intratumor heterogeneity for TP53 mutational status in Burkitt lymphoma

Therapeutic implications of intra-tumor heterogeneity are still undefined. In this study we report a genetic and functional analysis aimed at defining the mechanisms of chemoresistance in a 43-year old woman affected by stage IVB Burkitt lymphoma with bulky abdominal masses and peritoneal effusion. The patient, despite a transient initial response to chemotherapy with reduction of the bulky masses, rapidly progressed and died of her disease. Targeted TP53 sequencing found that the bulky mass was wild-type whereas peritoneal fluid cells harbored a R282W mutation. Functional studies on TP53 mutant cells demonstrated an impaired p53-mediated response, resistance to ex vivo doxorubicin administration, overexpression of DNA damage response (DDR) activation markers and high sensitivity to pharmacologic DDR inhibition. These findings suggest that intra-tumor heterogeneity for TP53 mutational status may occur in MYC-driven cancers, and that DDR inhibitors could be effective in targeting hidden TP53 mutant clones in tumors characterized by genomic instability and prone to intra-tumor heterogeneity.

Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients

BackgroundIn chronic lymphocytic leukemia (CLL) disease onset and progression are influenced by the behavior of specific CD4+ T cell subsets, such as T regulatory cells (Tregs). Here, we focused on the phenotypic and functional characterization of Tregs in CLL patients to improve our understanding of the putative mechanism by which these cells combine immunosuppressive and effector-like properties.MethodsPeripheral blood mononuclear cells were isolated from newly diagnosed CLL patients (n = 25) and healthy volunteers (n = 25). The phenotypic and functional characterization of Tregs and their subsets was assessed by flow cytometry. In vitro analysis of TH1, TH2, TH17 and Tregs cytokines was evaluated by IFN-γ, IL-4, IL-17A and IL-10 secretion assays. The transcriptional profiling of 84 genes panel was evaluated by RT2 Profiler PCR Array. Statistical analysis was carried out using exact non parametric Mann–Whitney U test.ResultsIn all CLL samples, we found a significant increase in the frequency of IL-10-secreting Tregs and Tregs subsets, a significant rise of TH2 IL-4+ and TH17 IL-17A+ cells, and a higher percentage of IFN-γ/IL-10 and IL-4/IL-10 double-releasing CD4+ T cells. In addition, we also observed the up-regulation of innate immunity genes and the down-regulation of adaptive immunity ones.ConclusionsOur data show that Tregs switch towards an effector-like phenotype in CLL patients. This multifaceted behavior is accompanied by an altered cytokine profiling and transcriptional program of immune genes, leading to a dysfunction in immune response in the peripheral blood environment of CLL patients.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia

BackgroundDespite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study.MethodsThe effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts.ResultsWe showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation.ConclusionsOur data suggest that WEE1 plays a role in ALL blast’s survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

Abstract 294: Override the doxorubicin-induced G2/M checkpoint using cell-cycle checkpoint inhibitors on acute lymphoblastic leukemia

The topoisomerase 2 inhibitor, doxorubicin, has been showed by different groups to induce cell cycle arrest in various kind of tumor cells. Specifically doxorubicin-treated cells activate the G2/M cell cycle checkpoint as a consequence of the induction of DNA damages. During the last years many studies have been showed the efficacy of different cell cycle checkpoint inhibitors in single agent or in combination with various DNA damaging agents. These studies showed that the inhibition of key proteins of the cell cycle, like Chk1 and Wee1, deeply sensitize tumor cells to the treatment with genotoxic agent. On these bases, the aim of the study was to evaluate the efficacy of a selective Chk1/Chk2 inhibitor and a Wee1 inhibitor in combination with doxorubicin for the treatment of acute lymphoblastic leukemia. Firstly we evaluate the effect of doxorubicin treatment on a panel of human B and T ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DNA damage response. For this reason the cells were treated with doxorubicin (0.25, 0.5 and 1uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. Then the induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was evaluated in all the cell lines. In line with the literature the treatment with doxorubicin arrested the cells in G2/M phase. Then the effect of the combinations between doxorubicin and the two checkpoint kinase inhibitor was assessed in all the cell lines. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk1/Chk2 inhibitor (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a additively effect in term of reduction of the cell viability and induction of apoptosis. Different cell lines were pre-treated for 18 hours with doxorubicin and then with Chk1/Chk2 inhibitor for different time points. Interestingly the inhibition of both Chk1/Chk2 proteins removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells in sub-G1 phase. Similar results were seen combining a Wee1 inhibitor with doxorubicin on several ALL cell lines. In our opinion the combination between the cell cycle checkpoint inhibitors and doxorubicin could be a promising strategy for the treatment of B/T-ALL. Supported by ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Anna Ferrari, Valentina Robustelli, Cristina Papayannidis, Maria Chiara Abbenante, Antonella Padella, Giovanni Marconi, Sandro Grilli, Giovanni Martinelli. Override the doxorubicin-induced G2/M checkpoint using cell-cycle checkpoint inhibitors on acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 294. doi:10.1158/1538-7445.AM2017-294

Abstract 368: Specific chromosomic alterations confer therapy resistance in a cohort of 49 patients with newly diagnosed acute myeloid leukemia treated with intensive chemotherapy

Introduction. Intensive induction chemotherapy in non-M3 young Acute Myeloid Leukemia (AML) patients is represented by the association of an antracycline and Cytarabine. Some treatment regimens including fludarabine or the addition of Gemtuzumab Ozogamicin (GO) as a third or fourth drug, proved to give a benefit in terms of CR rates. Aims of the study. In a group of 49 patients treated with intensive chemotherapy, we evaluated chromosomal abnormalities with SNP 6.0 and Cytoscan HD (Affymetrix) in order to improve conventional cytogenetic analysis and discover novel chromosomic aberrations related to clinical data and therapy response. Patients and Methods. From 2001 to 2014, 489 patients were treated in our Institution. Among those, in 49 newly diagnosed AML patients (median age 54 (range 19-71)), SNP microarray based-genotyping were performed and then analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team. According to karyotype, FLT3 and NPM1 mutational status, 55.9% of the patients were considered at High Risk (HR) and 4.1% at low risk (LR). Ten patients had secondary AML. Patients were treated with induction schemes including MyFLAI, MyAIE, FLAI, FLAN, FLAG, 3+7 or DAE. Results. The CR rate after induction was 87.8% (43/49 patients). Deaths during induction (DDI), occurring in the first 50 days from 1st line therapy, were 1/49. The median OS was 135 months, the 5-years OS in our patients was 55,1%. Patients treated with GO showed a non-statistical trend toward a better OS than patients treated with other regimens (median OS not reached vs 133 months, respectively). We explored the alterations found by SNP array in our patients searching for novel markers of therapy resistance. We found a median of 192,5 total copy number aberrations (range 72- 1071): a median of 145,5 total copy number aberrations in responding patients group (RPG), and a median of 361 total copy number aberrations (p = ns) in non-responding patients group (NRPG). We compared the frequency of detected aberrations in RPG and in NRPG with Fisher9s exact test. We found that PIK3CA, Gain chr3:178,927,088-178,929,550 (p = 0,0016), SMAD4, Gain chr18:48,573,154-48,573,255 (p = 0,0166) and several other gene9s loci (CASC18, TCF12, UTY, GRB10, ZFY) are significant aberrations in NRPG compared with RPG. Conclusions. We identified a number of genes with significant aberrations in NRPG, particularly PIK3CA, a protein-coding gene involved in cell proliferation and metabolic pathway with interaction with HRAS/KRAS and EGF, and SMAD4, a transcription factor activated by TGF-beta. Those 2 genes were found overexpressed in other solid tumors. We suppose that those genes may be involved in a hyper-proliferative pathway that underlies a mechanism of chemo-resistance. Acknowledgments Work supported by ELN, AIL, AIRC, Progetto Regione-Universit⁁ 2010-12 (L.Bolondi), FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Maria Chiara Fontana, Giovanni Marconi, Viviana Guadagnuolo, Giorgia Simonetti, Antonella Padella, Simona Soverini, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Silvia Lo Monaco, Marco Manfrini, Elisa Zuffa, Eugenia Franchini, Claudia Venturi, Maria Teresa Bochicchio, Andrea Ghelli Luserna di Rora, Emanuela Ottaviani, Giovanni Martinelli. Specific chromosomic alterations confer therapy resistance in a cohort of 49 patients with newly diagnosed acute myeloid leukemia treated with intensive chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 368.