Key Points• In human aggressive B-NHLs, HSPH1 favors c-Myc and Bcl-6 expression, and its inhibition provides significant antilymphoma activity.• HSPH1 is expressed in function of Bcl-6 and c-Myc and constitutes a valuable alternative lymphoma therapeutic target of aggressive B-NHLs.We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10 4 cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs. (Blood. 2015;125(11):1768-1771 IntroductionWe have recently demonstrated that heat shock protein (HSP)H1/ 105 is a novel antigen and potential therapeutic target of B-cell nonHodgkin lymphomas (B-NHLs). We showed that it is expressed in function of B-NHL aggressiveness, and its targeting by a specific antibody (Ab) provides significant therapeutic activity against human aggressive B-NHLs in vivo. 1 We have now set out to clarify its role as a potential molecular target in these diseases.High-grade B-NHLs-including diffuse large B-cell lymphoma (DLBCL) with its numerous subtypes, and Burkitt lymphoma (BL)-account for ;60% of NHL cases.2 Despite their high response rate to anti-CD20 rituximab-based chemoimmunotherapy, alternative strategies are required to manage relapse and resistance, which still pose a very high risk of death in approximately one-third of cases.3 Sustained expression of Bcl-6 or c-Myc oncoprotein is the best-recognized trait of DLBCL 4 or BL, 5 respectively, whereas their concurrent overexpression defines a subset of aggressive B-NHLs with an extremely unfavorable prognosis.6 Although these transcription factors (TFs) have long been regarded as reliable lymphoma targets, 7,8 their selective inhibition has proven challenging. To ensure constitutive high expression of key oncoproteins, NHLs have shown to upregulate HSP90 and HSP70, 9,10 which assist protein folding and prevent protein degradation.11 Investigation of HSP90 inhibitors has thus been extended to lymphoma patients. 12Here we provide evidence that HSPH1 constitutes a viable alternative therapeutic target of aggressive B-NHLs insofar as the in vitro and in vivo antilymphoma activity determined by its knockdown is strongly associated with both Bcl-6 and c-Myc downmodulation...

Histone deacetylases (HDAC) extensively contribute to the c-Myc oncogenic program, pointing to their inhibition as an effective strategy against c-Myc-overexpressing cancers. We, thus, studied the therapeutic activity of the new-generation pan-HDAC inhibitor ITF2357 (GivinostatV R ) against c-Myc-overexpressing human B-cell non-Hodgkin lymphomas (B-NHLs). ITF2357 antiproliferative and pro-apoptotic effects were analyzed in B-NHL cell lines with c-Myc translocations (Namalwa, Raji and DOHH-2), stabilizing mutations (Raji) or post-transcriptional alterations (SU-DHL-4) in relationship to c-Myc modulation. ITF2357 significantly delayed the in vitro growth of all B-NHL cell lines by inducing G1 cell-cycle arrest, eventually followed by cell death. These events correlated with the extent of c-Myc protein, but not mRNA, downregulation, indicating the involvement of posttranscriptional mechanisms. Accordingly, c-Myc-targeting microRNAs let-7a and miR-26a were induced in all treated lymphomas and the cap-dependent translation machinery components 4E-BP1, eIF4E and eIF4G, as well as their upstream regulators, Akt and PIM kinases, were inhibited in function of the cell sensitivity to ITF2357, and, in turn, c-Myc downregulation. In vivo, ITF2357 significantly hampered the growth of Namalwa and Raji xenografts in immunodeficient mice. Noteworthy, its combination with suboptimal cyclophosphamide, achieved complete remissions in most animals and equaled or even exceeded the activity of optimal cyclophosphamide. Collectively, our findings provide the rationale for testing the clinical advantages of adding ITF2357 to current therapies for the still very ominous c-Myc-overexpressing lymphomas. They equally provide the proof-ofconcept for its clinical evaluation in rational combination with the promising inhibitors of B-cell receptor and PI3K/Akt/mTOR axis currently in the process of development.

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Background - Treatment of ovarian cancer remains very challenging, with 80-85% of the cases still dying after relapse to standard chemotherapy, and alternative treatments are urgently needed. Expansion of regulatory T cells (Tregs) is considered the major factor limiting spontaneous immune responses to ovarian cancer. Agonist antibodies against the co-stimulatory receptor OX40 have recently demonstrated to abrogate Treg functions and are under clinical evaluation. We thus studied whether OX40 constituted a valid target of ovarian cancer-associated Tregs. Methods -Treg immunophenotypic analyses were performed by flow cytometry in ascites and ovarian cancer specimens and studied in association with patients' outcome. Results - CD4+CD25+Foxp3+ Treg% and OX40 expression were measured in 50 diagnostic ovarian cancer cases (36, high grade serous carcinomas; 14, other histotypes). Treg% was significantly higher in ovary (47 evaluable cases) relative to ascites (39 evaluable cases) (p<0.0001). Treg % in ascites and peripheral blood from ovarian cancer patients or healthy donors were not significantly different. The fraction of Tregs expressing OX40, instead, dramatically increased from peripheral blood to ascites (p<0.01), and was even higher within the tumor (p<0.001). In 24 patients (stage IIC-IV; 19, high grade serous carcinoma; 5, other histotypes) with ≥12-month follow-up after surgery and carboplatin-paclitaxel, we analyzed OX40 expression on intratumor Tregs in function of the outcome. Patients with Treg% or OX40 MFI above the mean had a significantly reduced OS (p=0.004, p=0.02). Interestingly, having both high Treg% and OX40 MFI trended toward the worst OS, followed by high Treg% or OX40 MFI alone, whereas the lack of these parameters was associated with a significantly improved survival (mean: 28, 36, 41 and 53 months). OX40+ Tregs in both ascites and ovarian cancer tissue included CD45RA-, Foxp3hi, ICOS+ IFNγ- effector Tregs, thus indicating that OX40 may define a subset of highly suppressive Tregs in ovarian cancer. Conclusions - Our study underscores a negative prognostic impact of OX40 expression on ovarian cancer-infiltrating Tregs and indicates that OX40 may constitute a rational target to counteract effector Treg functions and unleash potentially effective immune responses against ovarian cancer. Citation Format: Massimo Di Nicola, Roberta Zappasodi, Alessia Burocci, Giusi Ruggiero, Fabio Martinelli, Claudio Tripodo, Domenica Lorusso, Filippo De Braud, Francesco Raspagliesi, Mario P. Colombo. OX40 expression in tumor-associated Tregs as a potential prognostic biomarker and immunotherapeutic target in ovarian cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B157.

Background: The standard chemotherapy treatment for Non-Hodgkin lymphoma (NHL) consists in the combination of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). Because of the relevant treatment-related toxicity and the minor therapeutic effectiveness of CHOP therapy variants, the development of novel therapeutic approaches represents an urgent clinical need. Despite treatment with the anti-CD20 monoclonal antibody Rituximab and CHOP has led to favourable results for CD20+ lymphoma, many patients experienced drug resistance. Based on studies indicating that the malignant behaviour of tumors depends on the interactions between tumor and its microenvironment, we tested the effect of the novel heparanase inhibitor Roneparstat (sigma-tau Research Switzerland) in combination with various clinically relevant agents against B-cell lymphoma models, poorly responsive to conventional agents. Methods: We assessed the CD20 cell surface expression by flow cytometry, the heparanase (HPA-1) expression levels by immunoblotting, the VEGF spontaneous release in cell supernatant by proteome array in B-NHL models. The therapeutic activity was evaluated in SCID mice xenografted with CD20+ and VEGF+ B-NHL models. The efficacy of the drug treatment was estimated as tumor volume inhibition percentage (TVI%), log10 cell kill (LCK) and complete regression (CR). To gain insight the mechanisms underlying the anti-tumor activity of Roneparstat plus rituximab histopathological/immunoistochemical analyses were performed on treated tumors versus controls. Results: In a model of aggressive diffuse large B-cell NHL, except for doxorubicin, the combinations cyclophosphamide, dexamethasone and rituximab exhibited significantly superior efficacy over single-agent therapy. The most impressive enhancement of anti-tumor activity was observed with Roneparstat plus rituximab, with a high rate of complete tumor regressions and no evidence of disease at the end of the experiment in 50% of treated animals. Histological analysis revealed inflammatory cell infiltration, stromal destructuration, most pronounced apoptotic changes in Roneparstat plus rituximab-treated tumors versus controls; stromal architecture analysis showed signs of an altered, incomplete reticulin network suggestive of impaired stromal scaffolding that might have promoted the recruitment of complement components (C1q, C5) within tumors increasing the cytotoxic effect of rituximab. This interpretation was also supported by the favourable interaction of Roneparstat with bevacizumab in a Burkitt's lymphoma model characterized by high levels of VEGF.. Conclusion: Our results provide evidence that targeting the tumor microenvironment with Roneparstat may have therapeutic potential in combination with various agents against aggressive lymphoma subtypes. Citation Format: Massimo Di Nicola, Franco Zunino, Anna Rossini, Giusi Ruggiero, Micheleandrea De Cesare, Denis Cominetti, Monica Tortoreto, Cinzia Lanzi, Giuliana Cassinelli, Roberta Zappasodi, Claudio Tripodo, Nadia Zaffaroni. Microenvironment modulation and enhancement of cytotoxic therapy by the heparanase inhibitor Roneparstat against human B-non Hodgkin lymphomas. [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 3289.