Integration of Nanostring Profiling and Functional Characterization of Oxidative and Replicative Stress Biomarkers Identifies Poor Prognosis MYC/BCL-2 Positive Diffuse Large B-Cell Lymphoma Subsets, Providing Opportunities for Precision Therapies
Authors: Derenzini Enrico , Rossi Alessandra , Agostinelli Claudio , Rossi Maura , Melle Federica , Motta Giovanna , Fabbri Marco , Radice Tommaso , Ramadan Safaa , Vanazzi Anna , Tabanelli Valentina , Fiori Stefano , Chiappella Annalisa , Agazzi Alberto , Zinzani Pier Luigi , Vitolo Umberto , Tarella Corrado , Pileri Stefano A. 
Published: October 2019

Abstract
Diffuse large B-cell lymphoma (DLBCL) overexpressing MYC and BCL-2 have a poor outcome with standard therapies irrespective of the cell of origin (COO), representing an unmet medical need. Furthermore, precision therapy approaches specifically designed for MYC/BCL-2 positive DLBCL are lacking. Recently we reported that MYC positive DLBCL harbor high levels of inherent DNA damage and display constitutive activation of the DNA damage response (DDR) pathways, these alterations being correlated with poor outcome (Derenzini et al. Oncotarget 2015). In an effort to design specific therapies for MYC/BCL-2 positive DLBCL, we integrated nanostring-based gene expression profiling and functional characterization of replicative and oxidative stress biomarkers in 2 independent DLBCL cohorts treated with chemoimmunotherapy: 69 patients from the DLC04 study (Chiappella et al. Lancet Oncol 2017), and 66 patients treated in real life clinical practice (total 135 patients). We used a digital multiplex gene expression profiling (Nanostring technology) panel containing 28 genes: 20 genes used to assign COO subtype according to LST algorithm (Scott et al. Blood 2014), and 8 additional genes employed to assess the MYC and BCL-2 status and to detect potential therapeutic targets (STAT3, TP53, NFKBIA, PTEN, PIK3CA, CD68). Immunohistochemistry (IHC) profiling for MYC, BCL-2, the phosphorylated form of H2AX at S139 (γH2AX: a biomarker of DNA damage and DDR activation) and for 8-hydroxy-2'-deoxyguanosine (8-OHdG) (an oxidative DNA damage marker) was available in all patients.
MYC/BCL-2 status evaluated by nanostring outperformed IHC for prognostic stratification in both cohorts (patients were stratified in high and low MYC or BCL-2 expressors based on the median normalized MYC and BCL-2 mRNA levels in the respective cohorts). The 2 cohorts displayed similar patterns of γH2AX and 8-OHdG expression (cut off value 50% of positive cells). 8-OHdG negative cases showed very low levels of γH2AX whereas high 8-OHDG expressors displayed significantly higher levels of γH2AX phosphorylation. In line with this, γH2AX positivity was significantly associated with increased 8-OHdG levels in both cohorts (Figure 1A). These data suggest that oxidative stress could be a major source of inherent DNA damage contributing to constitutive DDR activation in DLBCL. Dual positivity for γH2AX and 8-OhDG was significantly associated with adverse outcome in both cohorts. However, this difference was even more pronounced when focusing the analysis on MYC/BCL-2 positive DLBCL (evaluated by nanostring): in fact dual positivity for H2AX and 8-OHdG identified a subgroup of MYC/BCL-2 positive cases (MYC/BCL-2ox) characterized by a dismal outcome. Interestingly MYC/BCL-2 positive cases without oxidative DNA damage had a very favorable outcome, with similar overall survival (OS) compared to MYC/BCL-2 negative cases. Similar trends were observed in both cohorts and a cumulative analysis confirmed a significantly worse OS for the MYC/BCL-2ox subgroup, compared to all other subgroups (Figure 1B).
We found an increased proportion of low NFKBIA expressors in the MYC/BCL-2ox subgroup (with no differences in the COO subtyping and in the fraction of double hit cases). Since NFKBIA is an endogenous inhibitor of NF-kB signaling, these data could indicate a compensatory NF-kB activation in MYC/BCL-2ox patients, in line with the role of NF-kB signaling in driving antioxidant responses. These findings suggest a model of oncogenic cooperation where constitutive NF-kB activation and BCL-2 overexpression could counteract the deleterious effects of inherent oxidative DNA damage driven by MYC.
To test this hypothesis and to determine the therapeutic implications of these findings we run a combinatorial high-throughput drug screening (HTS) investigating combinations based on 2 different inhibitors of the DDR (the CHK1/2 inhibitors AZD7762 and MK8776) in a large panel of DLBCL cell lines of ABC and GCB origin (n=10) (Figure 1C). Both CHK1/2 inhibitors showed synergistic interactions with BCL-2 inhibitors (venetoclax) and with drugs inhibiting NF-KB pathway such as PI3K, HDAC, and BET inhibitors. Taken together these data suggest synthetic lethal interactions between MYC, BCL-2, NF-kB and the DDR in DLBCL, providing the rationale for targeted therapeutic interventions in poor prognosis MYC/BCL-2 positive subgroups.

Disclosures
Chiappella: Nanostring: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Roche: Other: lecture fees; Teva: Other: lecture fees; Amgen: Other: lecture fees. Zinzani:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.