UNDERSTANDING CHEMOREFRACTORINESS IN PERIPHERAL T CELL LYMPHOMA TO DEVELOP NOVEL TREATMENTS

UNDERSTANDING THE BIOLOGICAL BASIS OF CHEMOREFRACTORINESS IN PERIPHERAL T-CELL LYMPHOMA TO DEVELOP NOVEL TREATMENTS Tutors: Prof. Paolo Corradini, Dott.ssa Cristiana Carniti Sara Rizzitano, Matr: R10429 Peripheral T cell lymphoma (PTCL) is a mature T-cell lymphoma, considered as a rare and very heterogeneous disease. The current World Health Organization (WHO) classification recognizes several distinct PTCL subtypes classified by morphology, immunophenotype and genetic characteristics. Due to its rarity and to its complexity, the treatment approach of PTCL has traditionally been similar to the one used for B-cell lymphomas and the standard first-line therapy consists of CHOP or CHOP-like regimens (CHOEP). Therapeutic responses to this approach have been neither adequate nor durable. Because of the lack of appropriate successful treatments, the aim of this project was to study the differences in response to CHOP-like regimens using in vitro and in vivo models to better understand the biological basis of chemorefractoriness and to develop novel drug combinations for PTCLs We first demonstrated that treating T cell lines with CHOEP resulted in heterogeneous responses. Then, we investigated the activity of romidepsin (Ro), a histone deacetylase inhibitor, in combination with CHOEP in preclinical models of PTCL. The combination of Ro+CHOEP was effective in reducing tumor cell proliferation (median reduction: Ro 45.4%, range 44-46%; CHOEP 51.3%, range: 48-54%; Ro+CHOEP 76.4%, range 62-71%) determining an increase of apoptotic and necrotic cells and inducing a cytostatic effect when compared to Ro or CHOEP alone. Because of the promising results of Ro+CHOEP obtained in vitro, we tested the combination in vivo on a recently developed mouse model of PTCL characterized by the translocation t(5; 9) (q33; q22) (Pechloff et al, 2010) that generates the ITK-SYK fusion transcript together with a reporter gene (GFP). Unfortunately we found that monitoring disease status in this mouse model was not possible then we decide to quit this in vivo model. Gene expression profiling (GEP) studies were conducted in order to characterize the biological mechanisms responsible for the different responses induced by Ro+CHOEP treatment. GEP highlighted that cells exposed to CHOEP upregulated signalling pathways such as those prompted by Src family kinases, probably in order to escape death. This provides the basis for assessing the activity of a tyrosine kinase inhibitor (Dasatinib, Da), that if added to CHOEP might potentiate the antitumor efficacy of the treatment abrogating those survival pathways induced by chemotherapy. The addition of dasatinib to CHOEP showed interesting and promising antitumor results: the antiproliferative effect of the Da+CHOEP combination was related to a significant increase in cell death (median increase: Da 18%, range 16-22%; CHOEP 22%, range:17%-26%; Da+CHOEP 48%, range 35-48%) associated with a severe mitochondrial depolarization and cell cycle perturbations. We then set up two xenograft subcutaneous mouse models in which the combination of Da and CHOEP strongly reduced tumor weights when compared to Da and CHOEP used alone (Da+CHOEP tumor growth inhibition, TGI= 98%; Da TGI=38.9%; CHOEP TGI=77.4% for HDMAR cell line and Da+CHOEP TGI: 74.5%; Da TGI 27.5%; CHOEP TGI: 11.5% for OCI-Ly12 cell line). Dasatinib was efficacious in controlling tumor growth and potentiated chemotherapy. Although from these results Da+CHOEP represents a promising combination to be rapidly tested for the treatment of newly diagnosed T cell lymphomas, there are some open issues that hamper its introduction in the clinical setting. In fact, to date immunohistochemistry analysis failed to prove the upregulation of phosphorylated proteins in histological samples of patients relapsing after CHOP-based therapy, avoiding the upfront identification of those patients who could benefit from the combination therapy. Another important and open issue relates to refractory and relapsing patients. Iqbal and colleagues (Iqbal et al, 2014) identified that high expression of GATA3 in PTCL-NOS was associated with poor survival and showed enrichment of gene signatures related to proliferation driven by c-MYC expression (Iqbal et al, 2014)(Manso et al, 2016). We then targeted c-MYC using BET inhibitors, OTX-015 and JQ1, assessing their antitumor activity as single agents and in combination with agents used in lymphoma practice. They similarly induced cell cycle arrest with G1-phase accumulation suggesting that JQ1 and OTX-015 mainly exerted a cytostatic effect through the downregulation of Myc protein. OTX-015 was then tested in combination with bendamustine, dasatinib, gemcitabine, ibrutinib, idelalisib and venetoclax. OTX-015 was capable of synergizing with these important anticancer molecules and additional studies are ongoing in order to elucidate the mechanisms of action of OTX-015 as single agent and in combination in vitro and in vivo. In conclusion, in this study we have demonstrated that the antitumor efficacy of the conventionally used anthracycline-based therapeutic approach (CHOP or CHOP like regimens), can be potentiated by the addition of a histone deacetylase inhibitor such as romidepsin but also by the addition of a tyrosine kinase inhibitor such as dasatinib. Targeting tyrosine kinases that might be responsible for sustained cell proliferation in T cell lymphomas appears a promising strategy to enhance CHOP-like regimen activity for newly diagnosed patients. In the relapsed setting, we suggest that novel agents, such as BET inhibitors, might be combined with known antilymphoma drugs.