Maria Todaro

Convergent mutations and kinase fusions lead to oncogenic STAT3 activation in anaplastic large cell lymphoma.

A systematic characterization of the genetic alterations driving ALCLs has not been performed. By integrating massive sequencing strategies, we provide a comprehensive characterization of driver genetic alterations (somatic point mutations, copy number alterations, and gene fusions) in ALK(-) ALCLs. We identified activating mutations of JAK1 and/or STAT3 genes in ∼20% of 88 [corrected] ALK(-) ALCLs and demonstrated that 38% of systemic ALK(-) ALCLs displayed double lesions. Recurrent chimeras combining a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were also discovered in WT JAK1/STAT3 ALK(-) ALCL. All these aberrations lead to the constitutive activation of the JAK/STAT3 pathway, which was proved oncogenic. Consistently, JAK/STAT3 pathway inhibition impaired cell growth in vitro and in vivo.

PDGFR blockade is a rational and effective therapy for NPM-ALK-driven lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkins lymphoma found in children and young adults. ALCLs frequently carry a chromosomal translocation that results in expression of the oncoprotein nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). The key molecular downstream events required for NPM-ALK–triggered lymphoma growth have been only partly unveiled. Here we show that the activator protein 1 family members JUN and JUNB promote lymphoma development and tumor dissemination through transcriptional regulation of platelet-derived growth factor receptor-β (PDGFRB) in a mouse model of NPM-ALK–triggered lymphomagenesis. Therapeutic inhibition of PDGFRB markedly prolonged survival of NPM-ALK transgenic mice and increased the efficacy of an ALK-specific inhibitor in transplanted NPM-ALK tumors. Notably, inhibition of PDGFRA and PDGFRB in a patient with refractory late-stage NPM-ALK+ ALCL resulted in rapid, complete and sustained remission. Together, our data identify PDGFRB as a previously unknown JUN and JUNB target that could be a highly effective therapy for ALCL.

PRDM1/BLIMP1 is commonly inactivated in anaplastic large T-cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a mature T-cell lymphoma that can present as a systemic or primary cutaneous disease. Systemic ALCL represents 2% to 5% of adult lymphoma but up to 30% of all pediatric cases. Two subtypes of systemic ALCL are currently recognized on the basis of the presence of a translocation involving the anaplastic lymphoma kinase ALK gene. Despite considerable progress, several questions remain open regarding the pathogenesis of both ALCL subtypes. To investigate the molecular pathogenesis and to assess the relationship between the ALK(+) and ALK(-) ALCL subtypes, we performed a genome-wide DNA profiling using high-density, single nucleotide polymorphism arrays on a series of 64 cases and 7 cell lines. The commonest lesions were losses at 17p13 and at 6q21, encompassing the TP53 and PRDM1 genes, respectively. The latter gene, coding for BLIMP1, was inactivated by multiple mechanisms, more frequently, but not exclusively, in ALK(-)ALCL. In vitro and in vivo experiments showed that that PRDM1 is a tumor suppressor gene in ALCL models, likely acting as an antiapoptotic agent. Losses of TP53 and/or PRDM1 were present in 52% of ALK(-)ALCL, and in 29% of all ALCL cases with a clinical implication.

A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

The EGFR family members sustain the neoplastic phenotype of ALK+ lung adenocarcinoma via EGR1

In non-small cell lung cancer (NSCLC), receptor tyrosine kinases (RTKs) stand out among causal dominant oncogenes, and the ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nonetheless, long-term RTK inhibition leads invariably to acquired resistance, tumor recurrence and metastatic dissemination. In ALK+ cell lines, inhibition of ALK signaling was associated with coactivation of several RTKs, whose pharmacological suppression reverted the partial resistance to ALK blockade. Remarkably, ERBB2 signaling synergized with ALK and contributed to the neoplastic phenotype. Moreover, the engagement of wild-type epidermal growth factor receptor or MET receptors could sustain cell viability through early growth response 1 (EGR1) and/or Erk1/2; Akt activation and EGR1 overexpression prevented cell death induced by combined ALK/RTK inhibition. Membrane expression of ERBB2 in a subset of primary naive ALK+ NSCLC could be relevant in the clinical arena. Our data demonstrate that the neoplastic phenotype of ALK-driven NSCLC relays ‘ab initio’ on the concomitant activation of multiple RTK signals via autocrine/paracrine regulatory loops. These findings suggest that molecular and functional signatures are required in de novo lung cancer patients for the design of efficacious and multi-targeted ‘patient-specific’ therapies.

The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank

Introduction Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs). Methods One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted. Results RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively). Conclusion RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.

Therapeutic efficacy of the bromodomain inhibitor OTX015/MK-8628 in ALK-positive anaplastic large cell lymphoma: an alternative modality to overcome resistant phenotypes

Anaplastic large cell lymphomas (ALCL) represent a peripheral T-cell lymphoma subgroup, stratified based on the presence or absence of anaplastic lymphoma kinase (ALK) chimeras. Although ALK-positive ALCLs have a more favorable outcome than ALK-negative ALCL, refractory and/or relapsed forms are common and novel treatments are needed. Here we investigated the therapeutic potential of a novel bromodomain inhibitor, OTX015/MK-8628 in ALK-positive ALCLs. The effects of OTX015 on a panel of ALK+ ALCL cell lines was evaluated in terms of proliferation, cell cycle and downstream signaling, including gene expression profiling analyses. Synergy was tested with combination targeted therapies. Bromodomain inhibition with OTX015 led primarily to ALCL cell cycle arrest in a dose-dependent manner, along with downregulation of MYC and its downstream regulated genes. MYC overexpression did not compensate this OTX015-mediated phenotype. Transcriptomic analysis of OTX015-treated ALCL cells identified a gene signature common to various hematologic malignancies treated with bromodomain inhibitors, notably large cell lymphoma. OTX015-modulated genes included transcription factors (E2F2, NFKBIZ, FOS, JUNB, ID1, HOXA5 and HOXC6), members of multiple signaling pathways (ITK, PRKCH, and MKNK2), and histones (clusters 1-3). Combination of OTX015 with the Brutons tyrosine kinase (BTK) inhibitor ibrutinib led to cell cycle arrest then cell death, and combination with suboptimal doses of the ALK inhibitor CEP28122 caused cell cycle arrest. When OTX015 was associated with GANT61, a selective GLI1/2 inhibitor, C1156Y-resistant ALK ALCL growth was impaired. These findings support OTX015 clinical trials in refractory ALCL in combination with inhibitors of interleukin-2-inducible kinase or SHH/GLI1.

Magic pills: new oral drugs to treat chronic lymphocytic leukemia

ABSTRACT Introduction: A deeper understanding of chronic lymphocytic leukemia (CLL) biology has led to the identification of new promising therapeutic targets. Different classes of molecules are currently under investigation and novel oral drugs have recently been approved or are in a late stage of clinical development. Areas covered: We present biological data illustrating the heterogeneous mechanisms of action of new oral drugs in CLL. Moreover, we provide clinical data from phase I to III studies, and discuss efficacy and side effects profile of these new therapies. Data are derived from peer-reviewed articles indexed in PubMed and from abstracts presented at major international meetings. Expert opinion: Novel oral drugs represent a valuable alternative to chemo-immunotherapy for patients with CLL, especially when high-risk disease features are present and when age or comorbidities preclude the use of standard treatments. Based on data from ongoing clinical trials, the indications of already approved agents will most likely be expanded and new options will soon be available. Moreover, treatment combinations will broaden the therapeutic armamentarium of physicians treating CLL. The availability of multiple choices is of benefit for patients with CLL, but also represents a challenge for the need of choosing the right drug for each patient.

Abstract A219: OTX015, a bromodomain and extraterminal inhibitor, represents a novel agent for ALK positive anaplastic large cell lymphoma.

Introduction: Inhibitors of the bromodomain and extraterminal (BET) family (BRD2, BRD3, BRD4, and BRDT) are a promising new class of anticancer agents. Here, we assessed the activity and the mechanism of action of a novel molecule OTX015, a selective orally bioavailable BRD2/3/4 inhibitor, in a panel of anaplastic large cell lymphoma (ALCL) cell lines. Material and Methods: Human cell lines derived from ALKpos ALCL (SUPM2/TS, SU-DHL-1, L82, JB-6, Karpas 299) were treated with increasing doses of OTX015 (OncoEthix SA, Swtizerland). Cell proliferation was evaluated by ATPlite and MTT methods over time. For cell cycle analysis cells were treated and stained with citrate buffer and PI and analyzed for DNA content using a FACScan flow cytometer. RNA was extracted by TRIzol and reverse-transcribed using the Superscript First-Strand Synthesis System kit according to the manufacturer9s instructions. RT-qPCR was performed using SYBR Green Master Mix on a Bio-RAD Real-Time PCR System. For WB analysis, cell lysates were fractionated by 8% polyacrilamide gels and membranes incubated with specific antibodies. Results: ALKpos ALCL cell lines (5) treated with different doses of OTX015 (ranging from 100nM to 1µM) underwent to a proliferation arrest as compare to 0.1% DMSO-treated cells. This was already detectable after 24h of treatment, and more pronounced at 48h and 72h. Cell cycle analysis showed a rapid G1 cell cycle arrest of all OTX015 treated ALCLpos cells as early as 24hrs. In 2 cell lines (SUPM2/TS, and JB-6) an increment in cell death rate was observed. Dose-curve studies and kinetics experiments demonstrated that a single exposure of 250nM of OTX015, without any subsequent refills, could sustain the OTX015 mediated changes (cell cycle arrest and c-MYC down-regulation) over time (up to 72-96 hrs). A time and dose dependent decreased of c-MYC m-RNA and protein levels was observed after OTX015 exposure in all ALCL cell lines, even after an exposure of 24h. Loss of c-MYC expression was associated with a concomitant down-regulation of known c-MYC regulated genes (CAD, NUC). The treatment with OTX015 (250nM) and suboptimal concentration of selective ALK inhibitor (5nM of CEP28122) led to a rapid and more significant down-regulation of c-MYC expression than those seen after each individual drug treatment. Once OTX015 was compared with OTX015 analog and a bona fine BET-inhibitor, JQ1, similar activity were observed. Conclusion: OTX015 is a new potent BRD-inhibitor with cytostatic anti-proliferative activity in several ALCL cell lines. The rapid down-regulation of c-MYC expression, which precedes the cell cycle G1 arrest, may provide a model of action in this experimental context. However, the identification of other biomarkers may be relevant in determining the appropriate drug dose and schedule and to predict clinical responses. The synergistic effects of OTX015 with anti-ALK inhibitors provide the rational for more efficacious and possibly less toxic protocols. This could represent a desirable strategy in patients partially responsive or refractory to ALK therapy. Overall, these findings demonstrated that BET bromodomains inhibitors represent promising therapeutic agents for the treatment of ALKpos ALCL patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A219. Citation Format: Michela Boi, Maria Todaro, Valentina Vurchio, Esteban Cvitkovic, Eugenia Riveiro, Francesco Bertoni, Giorgio Inghirami. OTX015, a bromodomain and extraterminal inhibitor, represents a novel agent for ALK positive anaplastic large cell lymphoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A219.

Regulation of HIF-1 α in TP53 Disrupted Chronic Lymphocytic Leukemia Cells and Its Potential Role as a Therapeutic Target

S214 CLL-101 Regulation of HIF-1 a in TP53 Disrupted Chronic Lymphocytic Leukemia Cells and Its Potential Role as a Therapeutic Target Candida Vitale ,1,y Valentina Griggio,1,y Maria Todaro, Chiara Riganti, Joanna Kopecka, Chiara Salvetti, Riccardo Bomben, Michele Dal Bo, Davide Rossi, Gabriele Pozzato, Monia Marchetti, Paola Omedè, Lisa Bonello, Ahad Ahmed Kodipad, Luca Laurenti, Giovanni Del Poeta, Francesca Romana Mauro, Rosa Bernardi, Valter Gattei, Gianluca Gaidano, Robin Foà, Massimo Massaia, Mario Boccadoro, Marta Coscia Division of Hematology, University of Torino, A.O.U. Città della Salute e della Scienza di Torino, Torino, Italy; Department of Oncology, University of Torino, Torino, Italy; Clinical and Experimental OncoHematology Unit, CRO Aviano National Cancer Institute, Aviano, Italy; Department of Hematology, Oncology Institute of Southern Switzerland and Institute of Oncology Research, Bellinzona, Switzerland; Department of Internal Medicine and Hematology, Maggiore General Hospital, University of Trieste, Trieste, Italy; Hematology Day Service, Oncology SOC, Hospital Cardinal Massaia, Asti, Italy; Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy; Division of Hematology, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy; Department of Hematology, Catholic University of Sacred Heart, Roma, Italy; Division of Hematology, S. Eugenio Hospital and University of Tor Vergata, Roma, Italy; Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Roma, Italy; Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milano, Italy; Ematology Unit, ASO Santa Croce e Carle, Cuneo, Italy