Dual targeting of the DNA damage response pathway and BCL-2 in diffuse large B-cell lymphoma

Standard chemotherapies for diffuse large B-cell lymphoma (DLBCL), based on the induction of exogenous DNA damage and oxidative stress, are often less effective in the presence of increased MYC and BCL-2 levels, especially in the case of double hit (DH) lymphomas harboring rearrangements of the MYC and BCL-2 oncogenes, which enrich for a patient’s population characterized by refractoriness to anthracycline-based chemotherapy. Here we hypothesized that adaptive mechanisms to MYC-induced replicative and oxidative stress, consisting in DNA damage response (DDR) activation and BCL-2 overexpression, could represent the biologic basis of the poor prognosis and chemoresistance observed in MYC/BCL-2-positive lymphoma. We first integrated targeted gene expression profiling (T-GEP), fluorescence in situ hybridization (FISH) analysis, and characterization of replicative and oxidative stress biomarkers in two independent DLBCL cohorts. The presence of oxidative DNA damage biomarkers identified a poor prognosis double expresser (DE)-DLBCL subset, characterized by relatively higher BCL-2 gene expression levels and enrichment for DH lymphomas. Based on these findings, we tested therapeutic strategies based on combined DDR and BCL-2 inhibition, confirming efficacy and synergistic interactions in in vitro and in vivo DH-DLBCL models. These data provide the rationale for precision-therapy strategies based on combined DDR and BCL-2 inhibition in DH or DE-DLBCL.


INTRODUCTION
Diffuse Large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) subtype, and yet 40% of patients are resistant to current therapies [1][2][3], which are still based on the induction of exogenous DNA damage with anthracycline-based chemotherapy regimens representing the standard of care [1][2][3]. The cell of origin (COO) determined by gene expression profiling (GEP) is a well-established prognostic predictor in DLBCL [4][5][6][7]. In general, DLBCLs with a GEP signature related to activated B-cell cells (ABC subgroup) have a worse outcome compared to their germinal center B-cell (GCB) counterparts [4][5][6][7], and display higher expression levels of MYC and BCL2 [8] and oncogenic addiction to nuclear factor kappa-B (NF-kB) signaling [9]. However, despite its prognostic relevance, COO-based precision therapy has not yet translated into meaningful clinical benefits [10][11][12]. Besides the COO, overexpression or genomic rearrangements of the MYC and BCL-2 oncogenes are powerful negative prognostic factors in DLBCL [13,14]. Due to the fact that concurrent MYC and BCL-2 rearrangements enrich for a patient's population characterized by refractoriness to standard anthracycline-based chemotherapy, these lymphomas are now classified as a separate disease entity (HG-BCL w/DH) [15] and currently treated with more intensive chemotherapy regimens, representing a major unmet need in lymphoma therapy [16,17]. On the other hand, the observation that a fraction of HG-BCL w/DH can be cured with standard therapies underlines the concept that the mechanisms underlying chemoresistance in MYC/BCL-2 positive DLBCL are still poorly defined. Recent evidence suggests that MYC-positive tumors are characterized by replicative and oxidative stress leading to inherent DNA damage and genomic instability [18][19][20]. Constitu-tive activation of the DNA damage response (DDR) pathway is one of the main mechanisms by which cancer cells cope with replicative stress, avoiding intolerable levels of endogenous DNA damage [21][22][23]. On the other hand, it is well known that BCL-2 overexpression synergizes with MYC in driving B-cell lymphomagenesis, by counteracting MYC-related proapoptotic effects and oxidative stress [24][25][26][27][28]. Of note, constitutive DDR activation correlates with MYC levels predicting poor prognosis [29], and therapeutic approaches targeting DDR through inhibition of the Ataxia teleangiectasia and Rad3 related (ATR)-checkpoint kinase 1/ 2 (CHK1/2) axis showed efficacy in preclinical models of MYCpositive DLBCL, including those with TP53 mutations/deletions and CDKN2A loss which are mechanistically linked to anthracycline resistance [30][31][32][33].
Since chemotherapy exerts its cytotoxic effects through exogenous DNA damage and induction of reactive oxygen species (ROS), adaptive mechanisms to replicative and oxidative stress (consisting in DDR activation and upregulation of antioxidant capacity), could represent the biologic basis of the poor prognosis and chemoresistance observed in MYC/BCL-2-double expresser DLBCL and HG-BCL w/DH.
In an effort to design specific therapies for MYC/BCL-2 positive DLBCL, we first integrated targeted-GEP (T-GEP), fluorescence in situ hybridization (FISH) analysis, and functional characterization of replicative and oxidative stress biomarkers in two independent DLBCL cohorts. Since the presence of oxidative DNA damage biomarkers identified a poor prognosis DE-DLBCL subset, which was characterized by relatively higher BCL-2 gene expression levels and enrichment in HG-BCL w/DH, we then tested therapeutic strategies based on combined DDR and BCL-2 inhibition, confirming efficacy and synergistic interactions in in vitro and in vivo HG-BCL w/DH models. These data provide the rationale for novel precision therapy strategies based on combined DDR and BCL-2 inhibition in double-hit DLBCL.

METHODS Patients
In the present study, we analyzed two independent patients cohorts: 69 patients from the DLCL04 study [34], a prospective randomized phase 3 clinical trial investigating the role of first-line autologous stem cell transplant (ASCT) consolidation after chemoimmunotherapy in CD20 + DLBCL, and 66 patients from a real-life cohort treated with R-CHOP/CHOPlike regimens at S. Orsola-Malpighi Hospital, Bologna (Italy), from 2007 to 2012. Patients characteristics are summarized in Table 1.
The study flowchart is depicted in Fig. 1 and additional details are provided in supplement.
This study was approved by the Institutional Review Boards of the participating centers, in accordance with the Declaration of Helsinki.

Targeted gene expression profiling (T-GEP) panel
Gene expression was measured on the NanoString nCounter Analysis System (NanoString Technologies, Seattle, WA, USA). The original T-GEP panel contains 22 genes: 15 genes used to assign COO subtype [6], 5 housekeeping genes (UBXN4, ISY1, R3HDM1, WDR55, TRIM56); and the additional genes of interest: MYC and BCL-2. The complete list of genes, target sequences, and detailed methods are available in supplement.
Immunohistochemistry and fluorescence in situ hybridization (FISH) Immunohistochemistry (IHC) was centralized in Milan for the DLCL04 trial and in Bologna for the real-life control group. Primary antibody source and dilutions are shown in Table S1 and a detailed description of IHC and FISH methods is provided in supplement.
The cut-off values of 50% and 40% positive neoplastic cells were applied for BCL2 and MYC respectively [14], and 50% for γH2AX and 8-OHdG.

Reagents, in vitro assays, and cell lines
Prexasertib was provided by the Eli Lilly company for in vitro studies, and was purchased from Selleckchem (Houston, Tx) for in vivo studies. AZD7762, MK-8776, Venetoclax and the MCL-1 inhibitor S63845 were purchased from Selleckchem (Houston, TX).
Detailed information on cell lines, 8-OHdG ELISA assay, Caspase 3/7 assay, cell cycle analyses, qPCR assays, proliferation assays (Cell Titer Glo, Promega), and western blot antibodies are provided in supplement. The Tet-OFF MYC P-4936 cell line [35,36], which carries a conditional, tetracycline-regulated MYC promoter, was provided by Dr. A. Younes lab (Memorial Sloan Kettering Cancer Center, New York, NY). Inducible overexpression of BCL2 in the SUDHL5 (BCL2 negative) cell line was performed by using the Cellecta InDOXible Tet-Activated cDNA Lentiviral Expression System (custom Cellecta). Detailed methods for in vitro studies are provided in supplement.

High-throughput screening experiments
High-throughput drug screening experiments were performed as previously described [37]. Plates were imaged after incubation with Alamar Blue on the LEADseeker TM Multimodality Imaging System (GE Healthcare, Piscataway, NJ). In order to evaluate synergy, we compared the observed activity to the expected activity of the combination at that dose level under Bliss independence model [38,39]. HTS experiments and analyses were performed at the Memorial Sloan Kettering Cancer Center, New York. Detailed information is available in supplement.

In vivo studies
Experiments involving animals were approved by the Italian Ministry of Health and have been performed in accordance with the applicable Italian laws (D.L.vo 26/14 and following amendments), the Institutional Animal Care and Use Committee, the institutional guidelines of the European Institute of Oncology and the ARRIVE guidelines [40].

Statistical analysis
Survival data and correlations were analyzed retrospectively. For survival analysis, we used the Kaplan-Meier method [42] to estimate overall survival (OS). The two-tailed Student t test and Wilcoxon Rank test were used to estimate statistical significance. Correlations and differences in patients' characteristics were analyzed with the chi-square and Fisher's exact test. The PRISM software was used for the statistical analyses (v7). Significance was set at P < 0.05. Combination index analysis was performed using the Chou-Talalay method [43].
T-GEP statistical analyses were calculated with the R software (v3.5.0) [44]. A detailed description is provided in supplement.

RESULTS
Replicative and oxidative stress biomarkers identify poor prognosis subsets of double expresser DLBCL In order to investigate the relationship between COO classification, MYC/BCL-2 status, and replicative/oxidative stress biomarkers, we first profiled two independent case series of chemoimmunotherapy-treated DLBCL with T-GEP, FISH, and immunohistochemistry: a discovery cohort from the DLC04 study [34], and a validation cohort of patients treated in real-life clinical practice. Patients characteristics are shown in Table 1 and were similar in the two cohorts (except for median age, significantly higher in the real-life cohort), with no significant differences in the overall outcome ( Figure S1). For T-GEP studies, we used a digital multiplex gene expression profiling platform (NanoString Technology) with a panel containing 22 genes (15 genes for COO subtyping according to LST algorithm [6] plus MYC and BCL2 and 5 housekeeping genes). T-GEP, FISH and immunohistochemistry (IHC) profiling for c-MYC, BCL-2, the phosphorylated form of H2AX at S139 (γH2AX, a biomarker of DNA damage and DDR activation) [45,46] and 8-hydroxy-2'-deoxyguanosine (8-OHdG, an oxidative DNA damage marker) [47] were available in 69 patients from the DLC04 study [34], and 66 patients treated in the real-life cohort ( Fig. 1 A, B).
The two cohorts displayed similar patterns of γH2AX and 8-OHdG expression (cut-off value 50% of positive cells, with 55% and 47% of cases showing dual nuclear positivity for γH2AX and 8-OHdG in the DLCL04 and real-life cohort respectively). The expression levels of γH2AX and 8-OHdG were significantly correlated, with the 8-OHdG positive subgroup showing a significantly higher fraction of γH2AX positive cases, as compared to the 8-OHdG negative subset. In line with this observation, the proportion of 8-OhDG-positive samples was significantly increased in the γH2AX-positive subset in both cohorts (Fig. 2 A,B).
In order to define the prognostic implications of γH2AX and 8-OHdG expression with respect to the COO and MYC/BCL-2 status, we analyzed the impact of these variables on OS rates in the two cohorts.
Although there was a significant correlation between MYC and BCL-2 mRNA (assessed with T-GEP) and protein levels (assessed by IHC) (Figure S2A), MYC/BCL-2 status evaluated by T-GEP outperformed IHC for prognostic stratification in both cohorts (Table S2 and Figure S2B) (patients were classified as high and low MYC or BCL-2 expressers based on the median normalized MYC and BCL-2 mRNA levels in the respective cohorts).
In both patients cohorts, dual nuclear positivity for γH2AX and 8-OHdG identified a MYC/BCL-2 mRNA Double Expresser (DE)-DLBCL subset characterized by dismal outcome (hereafter defined as DE-OX_high) (Fig. 2 C, D). Interestingly MYC/BCL-2 mRNA DE cases with low oxidative DNA damage (DE-OX_low) had a very favorable outcome, with OS rates comparable to non-DE cases (Fig. 2 C, D). Similar trends were observed in both cohorts (Fig. 2 C, D) and a cumulative analysis (n = 135 patients) confirmed a significantly worse OS rate for the DE-OX_high subgroup, compared to all other subgroups ( Figure S3A).
To further define the biologic characteristics of the DE-OX_high subgroup we investigated COO subtyping and the presence of MYC and BCL-2 rearrangements (HG-BCL w/DH) across different patients' subsets (DE-OX_high vs DE-OX_low). Interestingly we found that the DE-OX_high subgroups were enriched in HG-BCLs w/DH (Fig. 2E), showed a relative increase in ABC cases, and were characterized by relatively higher BCL-2 mRNA levels compared to the DE-OX_low subsets (Fig. 2F). Notably, all but one HG-BCL w/ DH clustered in the DE-OX_high subgroups ( Table 2). The outcome of DE-OX_HIGH subset was dismal irrespective of the DH status, with similarly poor OS rates observed in DH and non-DH cases ( Figure S3B). These data indicate that the expression of replicative and oxidative stress biomarkers such as γH2AX and 8-OHdG could define a subset of MYC/BCL-2 DE DLBCL with specific molecular features and characterized by a worse outcome.
Functional characterization of replicative and oxidative stress biomarkers following DDR inhibition in DLBCL To further investigate these findings we treated a panel of DLBCL cell lines with the CHK1/2 inhibitor Prexasertib [48], and assessed the effects of treatment on γH2AX and 8-OHdG levels. Prexasertib showed antiproliferative activity at sub-micromolar concentrations across multiple cell lines, irrespective of the cell doubling time, COO, MYC/BCL-2 rearrangements, TP53, and (Ataxia Telangiectasia Mutated) ATM status ( Fig. 3A and Figure S4A,B). As opposite, the in vitro efficacy of the DNA-damaging agent Doxorubicin was closely related to the TP53 wild-type status ( Figure S4C). As previously reported [48,49], treatment with Prexasertib resulted in reduced clearance of DNA damage foci and consequent upstream DDR activation as demonstrated by increased γH2AX and p-CHK1 S345 levels respectively (Fig. 3B). While in some cell lines Prexasertib and Doxorubicin had similar effects on cell viability and DNA damage accumulation, DDR inhibition by Prexasertib significantly increased γH2AX levels in cell lines where Doxorubicin failed to determine significant DNA damage ( Figure S4D). Furthermore, Prexasertib-induced DDR inhibition resulted in increased levels of DNA oxidation as assessed with a 8-OHdG ELISA assay (Fig. 3C,D). The extent of 8-OHdG induction was similar to that observed after treatment with known oxidative stress-inducing agents (Fig. 3C), such as Antimycin A [50], and was more prominent in Prexasertib-sensitive cell lines (Fig. 3D), showing a dose-dependent pattern ( Figure S4E). Prexasertib treatment induced significant apoptosis in DLBCL cell lines irrespective of TP53 status and was associated with significant accumulation of cells in S phase of the cell cycle, in line with previous observations [51] (Fig. 3E, F and Figures S4F and S5A-F). These data, together with our observations on γH2AX and 8-OHdG expression patterns in DLBCL tissues, suggest that oxidative stress could be a major source of inherent DNA damage contributing to constitutive DDR activation in DLBCL, and indicate that DDR inhibition induces oxidative DNA damage accumulation, cell cycle arrest and apoptosis in DLBCL cell lines.

BCL-2 inhibition enhances the activity of DDR inhibitors in in vitro DLBCL models
Since constitutive expression of replicative and oxidative stress biomarkers defined a subset of DE-DLBCL characterized by worse outcome and increased BCL-2 gene expression levels, we next investigated the therapeutic implications of these findings in in vitro DLBCL models, by combining different DDR inhibitors with the selective BCL-2 inhibitor Venetoclax. In a preliminary experiment, using a combinatorial high-throughput drug screening (HTS) approach we first combined two DDR inhibitors (the CHK1/ CHK2 inhibitor AZD-7762 and the selective CHK1 inhibitor MK-8776) with the BCL-2 inhibitor Venetoclax in 10 DLBCL cell lines. As shown in Fig. 4A, BCL-2 inhibition significantly enhanced the antiproliferative activity of CHK inhibitors in BCL-2 positive cell lines (ABC-derived or GCB-derived harboring BCL-2 rearrangements, Figure S6 A-D). In general, better trends were observed with CHK1/2 inhibition (AZD-7762) than with selective CHK1 inhibition (MK-8776) (Fig. 4A). BCL-2 protein expression data and HTS results of individual cell lines are shown in supplement ( Figure S6 A-E). Individual matrices of 1 representative GCB cell line with BCL-2 translocation (SUDHL-4) and 1 ABC-derived cell line (HBL-1) treated with AZD7762 are represented in Fig. 4B (complete data are shown in HTS data supplement). Since we described a significant enrichment of HG-BCL w/DH cases in the DE-Ox high subset, we then extended our cell line panel including different DH DLBCL models, and validated HTS results by cell-titerglo assay using the CHK1/2 inhibitor Prexasertib. As observed with AZD-7762, BCL-2 inhibition significantly enhanced the efficacy of Prexasertib in multiple DLBCL cell lines (Fig. 4C,D and Figure S7). In line with our HTS data, the most favorable interactions were observed in BCL-2 positive DLBCL cell lines, including different BCL-2 rearranged and DH lymphoma models (Fig. 4 C and D). The observation that Prexasertib-induced apoptosis was less pronounced in BCL-2-rearranged and DH cell lines (Figure S5 C,D) further corroborates these findings. BCL-2 protein expression data in this validation panel are shown in supplement ( Figure S8A). Although the efficacy of Prexasertib in combination with Venetoclax in DE cell lines was not affected by baseline γH2AX  Figure S5. and 8-OHdG levels, cell lines with low levels of γH2AX and 8-OHdG showed a trend toward a decreased sensitivity to single-agent Venetoclax ( Figure S8B-G). Combinatory treatment with Prexasertib and Venetoclax synergistically induced apoptosis as demonstrated by time-dependent increase in caspase 3/7 cleavage in BCL-2 positive ABC and DH lymphoma cell lines (Fig. 4E). In line with the data shown in Fig. 2 and with the role of BCL-2 in regulating the oxidative stress response, these changes were associated with early induction of oxidative DNA damage (Fig. 4F). Taken together these data indicate that selective BCL-2 blockade enhances the in vitro activity of CHK inhibitors in multiple BCL-2 positive DLBCL models including HG-BCL w/DH, by inducing oxidative DNA damage and apoptosis. Of note, single-agent Prexasertib or oxidative stress-inducing agents such as doxorubicin or H2O2 did not determine significant BCL-2 induction in either BCL-2 negative or BCL-2 positive cell lines ( Figure S9A). Finally, the MCL-1 inhibitor S68345 enhanced Prexasertib efficacy in BCL-2 negative cell lines ( Figure S9B), suggesting that dual blockade of the DDR and alternative BCL-2 family members could be of value in DLBCLs with low BCL-2 expression.

Differential role of MYC and BCL-2 in modulating the efficacy of checkpoint kinase inhibition
To determine the effects of BCL-2 overexpression on the therapeutic efficacy of DDR inhibition, we generated a Tet-on inducible system to overexpress BCL-2 in the Prexasertib-sensitive BCL-2 negative cell line SUDHL-5. Cells were preincubated with doxycycline 1 μg/ml for 24 h to overexpress BCL-2, and then were treated with DMSO or Prexasertib for 24 h ( Figure S10A). BCL-2 overexpression, while not exerting significant effects on cell proliferation in untreated cells, significantly decreased the efficacy of Prexasertib in this system, indicating that BCL-2 promotes resistance to DDR inhibition ( Fig. 5A and S10A). These changes were associated with an attenuated induction of γH2AX and with a decreased caspase 3 cleavage in cells overexpressing BCL-2 (Fig. 5B,C and S10A,B). BCL-2 overexpression did not determine significant changes in cell cycle dynamics under Prexasertib treatment (Fig. 5D).
To assess the role of MYC in regulating the antiproliferative activity of Prexasertib, we used the P-4936 cell line, which carries a conditional, tetracycline-regulated (Tet-OFF) MYC promoter [35,36]. Cells were pre-treated with DMSO or with doxycycline for 6 h to abrogate MYC expression, and then incubated with Prexasertib for 24 h (Fig. 5E-H and S11A-C). MYC silencing with doxycycline significantly reduced cell proliferation and baseline γH2AX expression ( Figure S11A-C). Importantly MYC depletion reduced the antiproliferative effects of Prexasertib in these cells, which was associated with impaired γH2AX induction, decreased caspase 3 cleavage, and attenuated effects on cell cycle dynamics (Fig. 5E-H). These data suggest that MYC and BCL-2 may modulate the sensitivity to CHK1 inhibition in opposite ways: in fact, while high MYC expression could be associated with increased DDR activation and enhanced susceptibility to DDRi-induced DNA damage, overexpression of BCL-2 may significantly decrease the therapeutic activity of DDR inhibitors, providing mechanistic rationale of dual blockade of DDR and BCL-2 in DE and DH lymphomas.

BCL-2 inhibition enhances the activity of DDR inhibitors in vivo in DH PDX models
To assess whether our in vitro results could be confirmed in in vivo lymphoma models, we used a PDX mouse model harboring a double MYC and BCL-2 rearrangement and a 17p deletion (TP53 loss) [42]. Treatment with Prexasertib as single-agent significantly extended the survival of mice bearing DH lymphomas (Fig. 6A). Of note, a short course 3-week therapy schedule was sufficient to extend survival of several weeks in this mouse model. In order to assess the in vivo effects of CHK inhibition, lysates from bone marrow and spleens harvested after 6 h of vehicle or Prexasertib administration were subjected to western blotting. In line with our in vitro data, Prexasertib treatment resulted in increased γH2AX and p-CHK1 s345 levels, which are established biomarkers of DDR inhibition, indicative of DNA damage accumulation and ATRdependent CHK1 phosphorylation (Fig. 6B). In an independent experiment, we investigated the efficacy of Prexasertib in combination with the BCL-2 inhibitor Venetoclax. In vivo combination therapy with Prexasertib and Venetoclax exerted synergistic effects in our DH PDX model. Prexasertib as singleagent confirmed high antitumor activity resulting in extended survival. On the contrary, Venetoclax alone had no substantial antitumor activity. However, the combination of Prexasertib and Venetoclax resulted in enhanced tumor growth inhibition and prolonged survival, as compared to either drug administered as single agent (Fig. 6C-E). Interestingly these synergistic effects were observed after only one cycle (3 weeks) of combined treatment. We did not observe significant weight loss in mice treated with Prexasertib, Venetoclax, or the combination (Figure S12 A and B). Collectively these data suggest that combined DDR and BCL-2 inhibition could be an effective treatment strategy in DH lymphoma models, including those with defective p53 axis.

DISCUSSION
In an effort to understand the functional basis of the intrinsic chemoresistance associated with increased MYC and BCL-2 levels in a significant fraction of DLBCL, we hypothesized that: 1) Overexpression of DDR and oxidative DNA damage markers (γH2AX and 8-OHdG) could identify poor prognosis DLBCL subsets. 2) Pharmacologic inhibition of the DDR and antioxidant response through combined CHK1/2 and BCL-2 blockade could unleash endogenous replicative and oxidative stress resulting in synergistic therapeutic activity in MYC/BCL-2 positive DLBCL.
To address these hypotheses, we first profiled two independent DLBCL cohorts with T-GEP, FISH, and IHC, in order to define COO subtyping, MYC, and BCL-2 status and expression levels of  Table 2). These data indicate that a subgroup of ABC DLBCL and HG-BCL w/DH are characterized by high levels of inherent oxidative DNA damage, these features being associated with increased BCL-2 expression levels. Given the poor prognosis of these DLBCL subsets, the well-established oncogenic cooperation between MYC and BCL-2, and the known role of BCL-2 in oxidative stress response, these data are in line with a model whereby BCL-2 overexpression and constitutive DDR activation could provide a tolerance mechanism to MYC-induced replicative and oxidative stress. Since antracyclines exert their cytotoxic activity at least in part by increasing ROS levels [52,53] and determining oxidative DNA damage, lymphoma subsets displaying inherent tolerance to oxidative DNA damage through constitutive DDR activation and BCL-2 overexpression could be intrinsically resistant to current antracycline-based chemotherapeutic regimens. The results of our in vitro experiments support this hypothesis since DDR inhibition by Prexasertib determined oxidative DNA damage accumulation (Fig. 3), which was further enhanced by the addition of Venetoclax (Fig. 4). Notably, treatment with ROS-inducing agents (Antimycin A) and Prexasertib determined accumulation of oxidative DNA damage to similar extents, suggesting that constitutive DDR activation could have a major role in preventing intolerable levels of DNA damage and genomic instability induced by endogenous oxidative stress (Fig. 3). BCL-2 blockade with Venetoclax enhanced the antilymphoma activity of checkpoint kinase inhibitors in multiple BCL-2 positive cell lines (including ABC and DH DLBCL models) resulting in increased apoptosis (Fig. 4). While enforced BCL-2 expression significantly decreased the efficacy of single-agent DDR inhibition by attenuating DNA damage accumulation and apoptosis induction (Fig. 5), on the contrary, MYC overexpression was associated with increased sensitivity to DDR inhibitors, and enhanced apoptotic response in line with previous reports [21][22][23]. Interestingly, while BCL-2 overexpression did not exert significant effects on cell proliferation, ectopic MYC expression was associated with increased cell proliferation and enhanced γH2AX expression, indicative of increased replicative stress and DDR activation ( Figure S11). These observations underline the intrinsic correlation between MYC and the DDR, and the importance of dual targeting of the DDR and BCL-2 in MYC/BCL-2 positive lymphoma in order to maximize the therapeutic efficacy. These data were confirmed in vivo, in a double hit PDX model with TP53 loss (Fig. 6). Interestingly single-agent Venetoclax had negligible antilymphoma activity in vivo in line with data from early phase clinical trials in DLBCL [54]. The recent demonstration of synergy between Venetoclax and Tygecycline in DH lymphoma models is in line with our findings, indicating that therapeutic strategies based on synthetic lethal targeting of oxidative stress could be of value in DH-DLBCL [55].
In summary, these data indicate that increased tolerance to replicative and oxidative stress through DDR activation and BCL-2 overexpression could be a unifying feature of poor prognosis MYC positive DLBCL subsets such as ABC and HG-BCL w/DH, which could be the basis for a tailored therapeutic approach. In this light, novel therapies based on dual targeting of DDR and antioxidant response could determine significant improvements in DLBCL therapy. This strategy, based on unleashing endogenous MYCrelated replicative and oxidative stress rather than inducing exogenous DNA damage, represents a significant innovation, which could provide less toxic alternatives to conventional chemotherapy. Fig. 5 Role of BCL-2 and MYC in modulating sensitivity to DDR inhibition. A Cell Titer Glo assay showing the effects of 3 doses of Prexasertib (3, 6, and 12 nM) for 24 h in the absence (EMPTY VECTOR) or in the presence of BCL-2 (BCL-2 EXP). SUDHL-5 cells (transfected with EMPTY VECTOR or a BCL-2 TET-ON inducible system (BCL-2 EXP) were preincubated with doxycycline 1 μg/ml for 24 h and then treated with Prexasertib at the indicated doses (see also Figure S10A). Error bars represent standard error of the mean (S.E.M) of triplicate experiments. Differences between groups were calculated with the Student T test. *p < 0.05, **p < 0.01. B On the left, representative western blot assay showing the effects of Prexasertib (6 nM) (PREX) on γH2AX induction and caspase 3 cleavage (CL. CASP 3) in the presence or absence of BCL-2, after 6 and 24 h of incubation. On the right, quantitative densitometry analyses (ImageJ software, western blots are shown in Figure S10B) showing normalized γH2AX levels vs vinculin after 6 h of incubation with Prexasertib in the presence or absence of BCL-2: γH2AX induction by Prexasertib was significantly attenuated in the presence of BCL-2. Error bars represent standard error of the mean (S.E.M) of triplicate experiments. Differences between groups were calculated with the Student T test. *p < 0.05, **p < 0.01.