Marcello Gaudiano

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.

Lenalidomide plus R-CHOP21 in elderly patients with untreated diffuse large B-cell lymphoma: results of the REAL07 open-label, multicentre, phase 2 trial

BACKGROUND Up to 40% of elderly patients with untreated diffuse large B-cell lymphoma (DLBCL) given a regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone every 21 days (R-CHOP21) relapse or develop refractory disease. Lenalidomide has high activity in relapsed or refractory aggressive B-cell lymphomas. In phase 2 of the REAL07 trial, we aimed to establish the safety and efficacy of the combination of lenalidomide and R-CHOP21 in elderly patients with untreated DLBCL. METHODS REAL07 was an open-label, multicentre trial that was done in 13 centres in Italy and one in Germany. Eligible patients were aged 60-80 years; had newly diagnosed, untreated, CD20-positive, Ann Arbor stage II-IV DLBCL or grade 3b follicular lymphoma; had an Eastern Cooperative Oncology Group performance status of 0-2; had an International Prognostic Index (IPI) risk of low-intermediate, intermediate-high, or high; and were fit according to comprehensive geriatric assessment. Participants were to receive 15 mg oral lenalidomide on days 1-14 of six 21-day cycles, and standard doses of R-CHOP21 chemotherapy (375 mg/m(2) intravenous rituximab, 750 mg/m(2) intravenous cyclophosphamide, 50 mg/m(2) intravenous doxorubicin, and 1·4 mg/m(2) intravenous vincristine on day 1, and 40 mg/m(2) oral prednisone on days 1-5). The primary endpoint was frequency of overall response (complete response [CR] and partial response [PR]), which was assessed by (18)F-fluorodeoxyglucose ((18)F-FDG) PET at the end of the treatment. Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00907348. FINDINGS 49 patients were included in phase 2: nine had been enrolled into phase 1 between Oct 23, 2008, and June 4, 2009, and had received the maximum tolerated dose of 15 mg lenalidomide; and 40 were enrolled into phase 2 between April 28, 2010, and June 3, 2011. 45 patients (92%, 95% CI 81-97) achieved a response (42 [86%] CR; three [6%] PR). Three patients (6%) did not respond and one (2%) died for reasons unrelated to treatment or disease. 277 (94%) of 294 planned cycles of lenalidomide and R-CHOP21 were completed. Grade 3-4 neutropenia was reported in 87 cycles (31%), grade 3-4 leukopenia in 77 (28%), and grade 3-4 thrombocytopenia in 35 (13%). No grade 4 non-haematological adverse events were reported. No patients died during the study as a result of toxic effects. INTERPRETATION Lenalidomide with R-CHOP21 is effective and safe in elderly patients with untreated DLBCL. FUNDING Fondazione Italiana Linfomi and Celgene.

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors

Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients.

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.

Abstract 2885: Somatic mutation of STAT3 leads to the preferential Th17 differentiation in human naïve CD4-positive cells and favor TCR-mediated proliferation

Intro: Mature Th17 lymphocytes play a key role in the host defenses against bacteria and fungi although unchecked Th17 activation can lead to autoimmune disorders, inflammation and cancer. The engagement of CD4+ naive T-cells via IL6 and TGFβ favors a Th17 differentiation program, which requires the presence of IL21 and IL23 to reach a complete maturation and the maintenance of Th17 phenotype. The Th17 differentiation requires multiple lineage defining transcription factors (i.e. RORα, RORγt, and STAT3) and the activation of STAT3 signaling, which regulates a plethora of factors (i.e. RORγt, IL23R), is required for the full execution of the Th17 program. We have recently shown that JAK1/STAT3 activating mutations in Anaplastic Large cell Lymphomas (ALCL) are oncogenic, but it is unknown their protumorigenic contribution in modulating/derailing the host microenviroment and the host responses. Methods: 50 FFPE ALCLs were investigated with pSTAT3, GATA3, RORγ and T-Bet antibodies using a dual color approach. CD4+ CD62L+ CD25- CD45RAhi CD4+ naive cells of healthy donor were sorted and cultured (7 days) in KBM (IL23 (25ng/ml), IL6 (25ng/ml), IL21 (25ng/ml), IL1β (12,5ng/ml), TGFβ (5ng/ml) + 1% FBS and anti-CD3/CD28 beads (1:50). Forced expressions of WT or mutated Y640F (YF) STAT3 were achieved via lentiviral transduction. STAT3 qRTPCR, luciferase assay, ATPlite, and ELISA were used as readouts on samples collected at day 7 post-transduction. Results: IHC stains showed that pSTAT3+ ALCL preferentially co-expressed nuclear RORγ (p Conclusions: Our data demonstrated that the ectopic expression of STAT3 leads to a robust Th17 differentiation of CD4 naive T-cells and YF STAT3 sustains their growth overtime in the presence of TCR signaling. The preferential expression of RORγ in pSTAT3+ ALCL (ALK+ and mutated STAT3 ALK-ALCL) suggest that the deregulated activation of STAT3 can regulate the plasticity of the neoplastic cells favoring the recruitment of inflammatory host cells within the neoplastic lesions. Additional studies are underway to define the lymphoma-host interactions and the role of JAK TKI in controlling the lymphoma growth and in modulating the pro-tumorigenic inflammatory phenotype of Patient Derived Tumor Xenograft Citation Format: Ramona Crescenzo, Valentina Fragliasso, Marcello Gaudiano, Marco Pizzi, Giorgio Inghirami. Somatic mutation of STAT3 leads to the preferential Th17 differentiation in human naive CD4-positive cells and favor TCR-mediated proliferation. [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 2885.

Abstract 3098: Epigenetic therapy to target neuroendocrine prostate cancer using precision medicine models

Background Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer that may either arise de novo or much more commonly after hormonal therapy for prostate adenocarcinoma. Patients diagnosed with NEPC are often treated with platinum chemotherapy able to produce only short duration responses underling the urgent need of identifying novel potential therapeutic targets for this lethal disease. In the context of our Englander Institute for Precision Medicine we developed patient derived 3D NEPC tumor organoids and patient derived PDXs to test specific inhibitors on molecular targets identified by genomic analysis of native tumors. Emerging data from an integrative molecular analysis of metastatic tumors from a large cohort of castration resistant prostate cancer (CRPC) patients, including NEPC, points to a key role of the Polycomb gene EZH2 and the epigenome in the pathogenesis of NEPC. Methods Tumor organoids were developed according to protocols developed by our Englander Institute for Precision Medicine and other Institutes. Briefly the tissue biopsies (liver and bone biopsy) were washed, enzymatically digested and then seeded in a Matrigel (BD) droplet. Organoids were then characterized at both genomic (WES) and protein level (IHC) to confirm the expression of specific markers. Organoids were also subcutaneously injected in NSG mice to generate PDX for drug treatment in vivo. Results Based on the significant EZH2 overexpression in NEPC tumors by RNA-Seq and tissue microarray, we checked the expression of EZH2 and H3K273M, the readout of its activity, in NEPC organoids and we found out that both EZH2 and H3K273M were high expressed in NEPC organoids. Therefore we evaluated the effects of the EZH2 inhibitor, GSK343, in NEPC versus CRPC organoids and in the castration resistant line DU145 versus the NEPC cell line NCI-H660. We found out that GSK343 effectively inhibited H3K27me3 and resulted in a significant reduction of NEPC organoids and H660 viability while DU145 as well as CRPC organoids were insensitive to the drug. We extended our studies generating PDXs by subcutaneously injecting NEPC tumor organoids in NSG mouse. The tumor extracted from the PDXs showed a high proliferative phenotype with molecular features characteristic of NEPC as chromogranin A expression and no androgen receptor expression. NEPC PDXs were treated with the EZH2 inhibitor, GSK126, and we observed a significant reduction of tumor size along with the treatment suggesting that EZH2 is a potential therapeutic target for this highly aggressive disease. Conclusions In the Englander Institute for Precision Medicine we are generating NEPC patient tumor organoids and PDXs to unveil new targets to facilitate therapeutic decision at this late stage disease. Among the possible hits, EZH2 represents a promising drug target and a potential modulator of the NEPC phenotype. Citation Format: Loredana Puca, Wouter R. Karthaus, Dong Gao, John Wongvipat, Andrea Sboner, Marcello Gaudiano, Chantal Pauli, Rema A. Rao, Juan Miguel Mosquera, Joanna Cyrta, Theresa Y. MacDonald, Giorgio Ga Inghirami, Yu Chen, Mark A. Rubin, Himisha Beltran. Epigenetic therapy to target neuroendocrine prostate cancer using precision medicine models. [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 3098.