# Identification of predictive factors of response to the BH3-mimetic molecule ABT-737: An ex vivo experiment in human serous ovarian carcinoma

Stéphanie Lheureux, Monique N'Diaye, Cécile Blanc-fournier, Audrey Emmanuelle Dugué, Bénédicte Clarisse, Soizic Dutoit, Florence Giffard, Edwige Abeilard, Mélanie Briand, Alexandre Labiche, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Stéphanie Lheureux, Monique N'Diaye, Cécile Blanc-fournier, Audrey Emmanuelle Dugué, Bénédicte Clarisse, et al.. Identification of predictive factors of response to the BH3-mimetic molecule ABT-737: An ex vivo experiment in human serous ovarian carcinoma. International Journal of Cancer, 2015, 136 (5), pp.E340-E350. 10.1002/ijc.29104. hal-05466304

HAL Id: hal-05466304

https://hal.science/hal-05466304v1

Submitted on 19 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Identification of predictive factors of response to the BH3-mimetic molecule ABT-737: An *ex vivo* experiment in human serous ovarian carcinoma

Stéphanie Lheureux<sup>1,2,3,4,5</sup>, Monique N'Diaye<sup>1,2,3</sup>, Cécile Blanc-Fournier<sup>1,2,3,6</sup>, Audrey Emmanuelle Dugué<sup>3,4</sup>, Bénédicte Clarisse<sup>3,4</sup>, Soizic Dutoit<sup>1,2,3</sup>, Florence Giffard<sup>1,2,3</sup>, Edwige Abeillard<sup>1,2,3</sup>, Mélanie Briand<sup>1,2,3</sup>, Alexandre Labiche<sup>1,2,3</sup>, Jean-Michel Grellard<sup>3,4</sup>, Hubert Crouet<sup>3,5</sup>, Sandrine Martin<sup>3,5</sup>, Florence Joly<sup>1,3,4,5,7</sup> and Laurent Poulain<sup>1,2,3</sup>

Ovarian cancers are addicted to Bcl-x<sub>L</sub> and Mcl-1, antiapoptotic members of the Bcl-2 family. Bcl-x<sub>L</sub> can be inhibited by the BH3-mimetic ABT-737. *In vitro*, ABT-737 can induce apoptosis of cancer cells, and its activity is potentiated by Mcl-1 inactivation. Herein, we assessed the sensitivity of human ovarian tumor nodes to ABT-737 when combined with carboplatin, which can indirectly inhibit Mcl-1. Fresh samples from 25 patients with high-grade serous ovarian cancer (HGSOC) who were chemo-naïve and had undergone surgery were prospectively exposed *ex vivo* to ABT-737 ± carboplatin. The treatment effect was studied on sliced tumor nodes by assessment of cleaved-caspase 3 immunostaining. We also studied the association between baseline Bcl-2 family protein expression (via immunohistochemistry) and the response of nodes to treatment. ABT-737 induced apoptosis as a single agent but its efficacy was not improved by the addition of carboplatin. Bim was frequently expressed (20/25) and its absence or low expression was associated with the absence of response to ABT-737, *p* value = 0.019 by Fisher's test and sensitivity = 93%, (95% confidence interval, 66–100). Moreover, we observed that in tumors in which Bim was expressed, a low expression of phospho-Erk1/2 or Mcl-1 improved the proportion of responses. This pilot study showed that ABT-737 has promise as monotherapy for HGSOC in a specific subgroup of tumors. Bim, Mcl-1, and phospho-Erk1/2 appeared to be relevant biomarkers that could be used for the selection of patients in the design of clinical trials using Navitoclax (an orally available compound related to ABT-737).

## Introduction

Ovarian cancer remains the leading cause of death from gynecological malignancies, with a 5-year survival rate less

**Key words:** ABT-737, apoptosis, Mcl-1, Bim, P-Erk, ovarian carcinoma

**Conflicts of interest:** No potential conflicts of interest were disclosed.

**Grant sponsor:** This work was supported by the "Ligue Contre le Cancer" (Calvados and Manche's committee), by Abbvie and by Lower-Normandy Lion's Club ("Operation Tulipes contre le Cancer")

**DOI:** 10.1002/ijc.29104

**History:** Received 6 Mar 2014; Accepted 16 Jul 2014; Online 28 Jul 2014

**Correspondence to:** Laurent Poulain, Centre de Lutte Contre le Cancer François Baclesse, 3 Avenue du Général Harris, BP5026, 14076 Caen Cedex 05, France, E-mail: l.poulain@baclesse.unicancer.fr

than 30%.<fnref n="1" /> Diagnosed at an advanced stage, standard therapy includes cytoreductive surgery and platinum based chemo-therapy. Yet, despite an initial good response to first line treatment, approximately 70% of patients will relapse. These patients will follow a chronic relapse/response pattern for a modest period of time before ultimately becoming chemoresistant. Although molecular chemoresistance mechanisms appear to be multifactorial, progression toward drug resistance can be achieved through the inactivation of apoptosis, generally considered as a hallmark of cancer.<fnref n="2" />

The Bcl-2 protein family plays a major role in regulating chemotherapy-induced apoptosis *via* the control of the mitochondrial apoptotic pathway. This family includes antiapoptotic proteins (Bcl-2, Bcl-x<sub>L</sub>, Bcl-w, Mcl-1, for example), effector proapoptotic proteins (such as Bax, Bak), and “BH3-only” members (including Bim, Puma, Noxa, Bad). Under cellular stress, BH3-only proteins initiate apoptosis by either blocking the activity of antiapoptotic members or directly activating effector proapoptotic members. This is mediated

<footnote n="1">¹ Normandy University, Caen, France</footnote>

<footnote n="2">² UNICAEN, Unité "Biologie et Thérapies Innovantes des Cancers Localement Agressifs" (EA 4656), Centre de Lutte Contre le Cancer François Baclesse, 14076 Caen Cedex 05, France</footnote>

<footnote n="3">³ Centre de Lutte Contre le Cancer François Baclesse, 3 Avenue du Général Harris, BP5026, 14076 Caen Cedex 05, France</footnote>

<footnote n="4">⁴ Service de recherche clinique, Centre de Lutte Contre le Cancer François Baclesse, 14076 Caen Cedex 05, France</footnote>

<footnote n="5">⁵ Comité d'Uro-gynécolgie Oncologique, Centre de Lutte Contre le Cancer François Baclesse, 14076 Caen Cedex 05, France</footnote>

<footnote n="6">⁶ Laboratoire d'Anatomie Pathologique, Centre de Lutte Contre le Cancer François Baclesse, 14076 Caen Cedex 05, France</footnote>

<footnote n="7">⁷ Inserm UMR S 1086—Cancers & Préventions, Centre de Lutte Contre le Cancer François Baclesse, 14076 Caen Cedex 05, France</footnote>
<table><caption>Table 1. Patients' characteristics</caption><thead><tr><th colspan="3">Characteristics of eligible patients</th></tr></thead><tbody><tr><td colspan="3"><strong>Age (years)</strong></td></tr><tr><td>Median</td><td colspan="2">67</td></tr><tr><td>Range</td><td colspan="2">[36–80]</td></tr><tr><td><strong>FIGO stage</strong></td><td><strong>n = 25</strong></td><td><strong>%</strong></td></tr><tr><td>IIb</td><td>23</td><td>92</td></tr><tr><td>IV</td><td>2</td><td>8</td></tr><tr><td><strong>Histology, n (%)</strong></td><td><strong>n = 25</strong></td><td><strong>%</strong></td></tr><tr><td>Serous</td><td>25</td><td>100</td></tr><tr><td>High grade</td><td>25</td><td>100</td></tr><tr><td><strong>Type of surgery</strong></td><td><strong>n = 25</strong></td><td><strong>%</strong></td></tr><tr><td>Complete</td><td>14</td><td>56</td></tr><tr><td>No complete</td><td>11</td><td>44</td></tr><tr><td><strong>First line of chemotherapy</strong></td><td><strong>n = 25</strong></td><td><strong>%</strong></td></tr><tr><td>Platinum/paclitaxel</td><td>19</td><td>76</td></tr><tr><td>Platinum/paclitaxel/bevacizumab</td><td>4</td><td>16</td></tr><tr><td>none</td><td>2</td><td>8</td></tr><tr><td><strong>Treatment free interval</strong></td><td><strong>n = 25</strong></td><td><strong>%</strong></td></tr><tr><td>&lt;6 months</td><td>9</td><td>36</td></tr><tr><td>&gt;6 months</td><td>16</td><td>64</td></tr></tbody></table>

sterile prewarmed complete culture medium (RPMI 1640 supplemented with 2 mM Glutamax™, 25 mM HEPES, 10% fetal calf serum, and 33 mM sodium bicarbonate (Fisher Scientific Bioblock, Illkirch, France) and 1% antibiotic) and treated with either vehicle (DMSO) or 10 µM ABT-737, with or without 20 µg/ml carboplatin in complete medium for 48 hr in a 5% CO₂ humidified atmosphere at 37°C. Slices were then fixed and frozen as described above for further analyses. Histology of all samples was confirmed by a certified pathologist.

## Immunohistochemical analysis

Automated immunohistochemistry using a DakoCytomation Autostainer was performed on 4-µm-thick paraffin sections. The mouse monoclonal antibodies anti-Bcl2 (clone 124) and Mcl-1 (Y37) were obtained from Dako (Courtaboeuf, France) and Abcam (Paris, France), respectively. The rabbit polyclonal antibodies anti-Bax (#2774), cleaved caspase-3 (Asp175) and the rabbit monoclonal antibodies anti-Bak (D2D3), Bim (C34C5), Puma (D30C10), and Phospho-p44/42 MAPK (Thr202/Tyr204) (D13.14.4E) corresponding to Phospho-Erk1/2 and noted P-Erk, were obtained from Cell Signaling (Ozyme, Saint Quentin Yvelines, France). The rabbit polyclonal antibodies anti-Bcl-xL, Noxa and Phospho-Bim (Ser69/65), noted P-Bim, were from BD Biosciences (Le Pont de Claix, France), LabVision (Clinisciences, Nanterre, France), and Imgenex (Clinisciences), respectively.

Briefly, to unmask epitopes, deparaffinized slides were treated for 15 min by high-temperature heating antigen

retrieval technique in EDTA buffer 0.5 M pH8 for Bax, Bim (EL, L and S isoforms), Bcl2, cleaved caspase 3, Mcl-1, Puma, and Noxa antibodies, and in Citrate buffer 0.07M pH6 for Bak, Bcl-xL, P-Bim, and P-Erk antibodies. Sections were incubated 1 hr at room temperature with the following antibodies: anti-Bak 1:800, Bax 1:400, Bcl-xL 1:500, Bim 1:200, Bcl-2 1:50, cleaved caspase-3 1:200, Mcl-1 1:000, Noxa 1:100, P-Bim 1:200, P-Erk 1:400, or Puma 1:50.

After washes, slides were incubated with Perox Detect System (Novocastra, Leica Microsystems, Nanterre, France), according to manufacturer instructions. Staining was performed with DAB chromogen and sections were counterstained with Hematoxylin QS (Vector Laboratories, Clinisciences, Nanterre, France). Stained slides were then digitized using an Aperio ScanScope slide scanner.

Based on our preliminary optimization, marker quantification, or classification was assessed as follows by an independent certified pathologist:

**Cleaved caspase 3.** The percentage of tumor cells positive for cleaved caspase 3 were evaluated.

**Bcl-2, Bcl-xL, Mcl-1, Bax, Bak, Bim, Noxa, and Puma.** The intensity of staining was considered as their expressions were relatively homogeneous in the whole sections and thus the expression of each protein was scored as high or low.

**P-ERK and P-Bim.** Contrary to the other proteins, expression of the phosphorylated forms of ERK and Bim were strongly heterogeneous in the tumor nodes. We then assessed these parameters using the percentage of marked cells. When a percentage of stained cells ≥ 20% was observed, staining was considered as high.

## Statistical Analysis

Descriptive analyses of biological and clinical data were performed on “intent to treat.” The levels (%) of caspase 3 expression between two kinds of exposure were compared using the Wilcoxon signed-rank test. The correlation between the levels (%) of caspase 3 under ABT-737 and basal apoptotic cell death was assessed and tested by the Spearman's method. Concerning the predictive factors analysis, each gene expression was classified in several classes (from 0 to 4) and then defined as low or high. This was done in order to include at least five patients per group with the most discriminating cutoff value, regarding the response to ABT-737. The threshold of the expression of cleaved caspase 3 was setup to 25%, and thus considering each patient as a nonresponder with an expression below 25%. A univariate analysis was performed using Mann-Whitney tests to compare the distribution of cleaved caspase 3 in low and high expression class of each gene. Gene combinations were sought by classification methods (JRip: RIPPER-repeated incremental pruning to produce error reduction—propositional rule learner and J4.8: unpruned C4.5 decision tree) in the whole cohort and then in the 25 data established when removing one patient at a
time. Statistical analyses were performed using STATA© v.10 and R© v.3.0 software. The type I error rate was 0.05 for each analysis.

<figure><img src="image_3.png" /><figcaption>Figure 1. Response to treatments. Evaluation of cleaved caspase 3 immunostaining proportion before treatment (CONT) or after a 48 hr culture (UNT) or treatment with DMSO, 10 µM ABT-737, 20 µg/ml Carboplatin (CA), or both (ABT-737 + CA), *P* corresponding to the *p*-value of the associated Wilcoxon signed-rank test.</figcaption></figure>

## Results

### Patient characteristics

Tumor samples from 25 chemotherapy naïve ovarian cancer patients enrolled from November 2010 to May 2012 were obtained and deemed evaluable for the *ex vivo* study. Patient demographics are listed in Table 1. The mean age of the population was 67 years. All patients presented with advanced ovarian cancer with high grade serous carcinoma. Following initial surgery, most patients received a platinum/taxane combination as first-line chemotherapy treatment.

### The ABT-737/carboplatin combination is more effective than carboplatin alone, but ABT-737 is also effective as a single agent

We evaluated the response to treatments using cleaved caspase 3 detection by immunohistochemistry. Whereas most of the tumors had undergone little apoptosis following carboplatin exposure (median caspase 3 = 10%), the carboplatin/ABT-737 combination induced significantly more cell death (median caspase 3 = 40%), thus achieving the primary objective of our study (Fig. 1). The two control conditions, *ex vivo* culture and exposure to DMSO, induced no or little apoptosis (median caspase 3 < 10%).

<figure><img src="image_4.png" /><figcaption>Figure 2. Illustration of cleaved caspase 3 immunostaining after treatment (right panel) and corresponding proportion evaluation (left panel), in low or high response cases.</figcaption></figure>
In 14/25 tumors, we observed that 20 to 80% of the cancer cells underwent apoptosis following exposure to ABT-737 when used as a single agent (median caspase 3 = 30%). Treatment with ABT-737 alone was significantly more potent than carboplatin alone. The efficacy between treatments with ABT-737 used alone or in combination with carboplatin was not statistically different. Therefore, we observed two main cases: (i) in 14/25 tumors, ABT-737 induced cell death when administered as a single agent as well as in combination with carboplatin (Fig. 2, top panel); and (ii) in 11/25 patient tumors, no significant response was observed to ABT-737 treatment, nor was a response observed to the ABT-737/carboplatin combination (Fig. 2, bottom panel). Interestingly, stromal cells remained intact in response to both ABT-737 and the combination.

## Identification of predictive factors to ABT-737 response

To further investigate the relationship between the intrinsic features of tumors and response to ABT-737, we used immunochemistry to assess basal apoptosis rate (cleaved caspase 3) and Bcl-2 family member protein expression. The node fragments used to perform these analyses belonged to the same tumor nodes as those sliced to evaluate the response to treatment.

*Apoptotic cell death.* Using the proportion of cleaved caspase 3 positive cancer cells, the basal apoptosis rate and response to ABT-737 did not correlate with one another (Spearman's rho = -0.037, *p* = 0.86). Of note, none or little caspase 3 cleavage was observed in tumors at basal level (Fig. 1).

*Bcl-2 family proteins.* We studied the correlation between the expression of eight Bcl-2 protein family members and response to ABT-737. We studied both antiapoptotic (Bcl-2, Bcl-x<sub>L</sub>, Mcl-1) and proapoptotic (multidomains members Bax and Bak as well as BH3-only members Noxa, Puma, and Bim) proteins whose expression was assessed by immunohistochemistry. Univariate analysis is presented in the Figure 3a. The expression of Bim was the most significant variable among all studied proteins. Indeed, Bim was frequently expressed (20/25) and its absence or low expression was associated with a lower response to ABT-737 [*p* value = 0.0081 by Mann-Whitney test and sensitivity to detect responders = 93, 95% CI (66–100), specificity = 36, 95%CI (10–70), negative predictive value (NPV) = 80, 95%CI (28–100) and positive predictive value (PPV) = 65, 95%CI (40–85)]. Among the five patients with a low expression of Bim, only one had a response to ABT-737, and from the 20 patients with a high expression of Bim, 13 were responders. These findings suggest that high Bim expression may be correlated to ABT-737 efficacy.

As Bim can be phosphorylated (P-Bim) and inactivated by phospho-Erk1/2 (P-Erk), we also studied P-Bim and P-Erk expression to evaluate whether these parameters were correlated with the absence of response to ABT-737 among the

high Bim expressing tumors. Their expression was assessed by immunochemistry using the percentage of marked cells. P-ERK level was correlated with P-Bim level (rho = 0.55, *p*-value = 0.0048), but these two parameters were not significantly associated to the response to treatment on their own (Fig. 3b).

## Appreciation of basal Bim expression heterogeneity in tumor nodes

As ovarian cancer is a heterogeneous disease, we assessed the variations of Bim expression between different tumor nodes from a same patient. Our findings show that in the majority of cases (20/25 patient tumors), Bim expression level was homogeneous in the four different peritoneal carcinomatosis tumor nodes tested (Fig. 4a). Figure 4b illustrates the different cases (homogeneous low or high expression and partial heterogeneity between tumor nodes).

## Improvement of Bim predictive value by combination with other genes expression

As 7/20 high Bim expressing tumors did not respond to ABT-737, we studied the combination of Bim expression with other Bcl-2 family members. Data from these investigations show that several variables significantly improved the prediction of response to ABT-737 among Bim expressing tumors. Indeed, adding the expression of Mcl-1 or P-Erk (Fig. 5a) to the expression of Bim improved response prediction (Fig. 5b). In tumors in which Bim was expressed, we observed that a low expression of Mcl-1 or phospho-ERK1/2 was significantly associated with a better response to ABT-737 (Fig. 5b).

Protein expression combinations of Bim, Mcl-1, and P-Erk were found to improve response prediction with both classification methods (Fig. 5c). Using this approach, only four responders were not well classified (*p* = 0.00055). Thus, sensitivity reached 71, 95%CI (42–92); specificity was 100, 95%CI (72–100); NPV was 73, 95%CI (45–92); and PPV was 100%, 95%CI (69–100).

## Discussion

As previously demonstrated, ovarian carcinoma shows strong overexpression of antiapoptotic proteins Bcl-x<sub>L</sub> and Mcl-1.<sup>4,5,7,16</sup> Together, these two proteins cooperate to prevent apoptosis in ovarian cancer cells.<sup>8</sup> In this context, the BH3-mimetic ABT-737 molecule appears to be a promising therapy as it inhibits Bcl-x<sub>L</sub>, a key antiapoptotic protein in ovarian cancer; however, its activity appears to be impacted by Mcl-1 inactivation.<sup>13</sup>

Here, we studied the relevance of ABT-737 alone or in combination with carboplatin in a panel of human ovarian tumor samples. The tumor panel was composed of HGSOC which is the most frequent histological subtype.<sup>23</sup> To address the issue of heterogeneity of disease, we evaluated serial slices obtained from the same tumor node which were used to assess response to different treatments. The strategy to use
<figure><img src="image_5.png" /><figcaption>Figure 3. Predictive factors. Cleaved caspase 3% obtained with ABT-737 according to (a) the expression of different Bcl-2 family genes and (b) the expression of the phosphorylation of Bim and Erk proteins. Boxplots represent median, quartiles, and extreme values. The dotted line shows the cutoff below which the patient was considered as a nonresponder (25+). *N* represents the number of patients in the box-plot and *P* the *p*-value of the Fisher's exact test.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4. Appreciation of Bim expression level in various tumor nodes of a same patient. (a) Waterfall plot of the number of nodules with a high or low Bim expression level among the four nodules of each patient (One barplot representing one patient, the nodule used in all analyses is colored in grey); (b) illustration of cases with homogeneous low Bim staining (left panel), homogeneous high Bim staining (right panel) or heterogeneous staining (middle panels).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Improvement of Bim predictive value by Mcl-1 and P-Erk expression level. (a) Illustration of low or high Mcl-1 and P-Erk expression level. (b) Cleaved caspase 3% obtained with ABT-737 according to Bim/Mcl-1 and Bim/P-Erk combined expressions. Boxplots represent median, quartiles, and extreme values. The dotted line shows the cutoff below which the patient was considered as a nonresponder (25+). *N* represents the number of patients in the boxplot and *P* the *p*-value of the Fisher's exact test. (c) Representation of the decisional tree that was found to be the most related to the response to ABT-737. Barplots represent the number of patients.</figcaption></figure>
ex vivo tumor samples is of importance as cancer cell lines differ substantially from patient tumors at the genetic, transcriptomic, and proteomic levels, all of which could largely influence the response to treatments²⁴ This is one of the reasons why numerous molecules or strategies that demonstrated activity *in vitro* and *in vivo* on cancer cell lines did not exert antitumor activity in the patient,²⁵ and justifies the use of models closer to patient tumors. The three-dimensional (3D) architectural integrity and microenvironment are preserved and drug penetration is facilitated. Furthermore, cellular viability is preserved as less than 10% apoptosis was observed in control samples. Similar 3D tumor models have been previously used in order to study the potential of innovative therapeutics ex vivo in prostate and breast tumors.¹⁵,²⁶

We observed that single-agent ABT-737 induced massive apoptosis in the majority of cases tested and its combination with carboplatin did not significantly improve its efficacy. This result supports the concept that ABT-737 alone should be effective in patients and as such, better-tolerated than its combination with carboplatin. Using concentrations from our own and previously published *in vitro* studies,¹⁶,²⁷⁻²⁹ we have shown similar preliminary data in human ovarian cancer cells xenografted into nude mice and subsequently exposed *ex vivo* to ABT-737.¹⁶ Similarly, in an *ex vivo* study performed on five human prostate tumor samples, Bray et al. observed a partial response to ABT-737 that was strongly enhanced when it was combined with cisplatin.¹⁵ It should be noted that these concentrations are, in some cases, completely unable to induce apoptosis—not only in stromal cells but also in tumors cells—whereas in other cases, more than 80% of the cancer cells appeared as cleaved-caspase 3 positive. Moreover, this ABT-737 concentration (10 µM) has also been used in a similar *ex vivo* evaluation by Bray et al. in prostate cancer. However, solid tumors and more particularly ovarian cancers are less sensitive to ABT-737 than hematological malignancies. This observation could be responsible for therapeutic failure with Navitoclax in the future. This however remains to be investigated through a dedicated clinical trial.

To identify markers of ABT-737 efficacy in HGSOC, we investigated the impact of basal Bcl-2 family members' expressions on response to ABT-737 in our model using immunohistochemistry. Bcl-2 overexpression has been associated with ABT-737 response; yet, whilst the involvement of Mcl-1 in resistance to ABT-737 has been extensively described,¹³,¹⁶ we did not observe any link between antiapoptotic members and response to ABT-737. These findings may be explained by the fact that HGSOC naturally expresses high levels of these proteins, in particular, Bcl-X~L~. As such, it may be difficult to differential between tumors that respond to ABT-737 versus tumors that do not respond. Of relevance to these findings, it has been shown in other studies that response may not correlate with Mcl-1 expression.²⁵,³⁰

Concerning the multidomain proapoptotic proteins, neither Bak nor Bax expression were correlated to ABT-737

response in our study. Still, in small cell lung cancer (SCLC), acquired resistance has been associated with a decrease in Bax expression.²⁵ This may indicate that Bcl-2 family members' protein expression is tissue specific. In contrast, Bim protein expression was strongly associated with response to ABT-737. Bim has been described as a tumor suppressor gene³¹ acting as both a direct activator of multidomain members Bax/Bak and as a BH3-only protein sensitizer allowing the desequationation of these proteins from their antiapoptotic relatives.³¹,³² The importance of Bim protein expression as a predictive factor of response to various treatments has been suggested in other tumor types. For example, a high Bim mRNA level has been correlated with a response to kinase inhibitors in EGFR mutant lung cancers³³ and paclitaxel.³⁴ In response to BH3-mimetic molecules, the response to ABT-263 (orally available compound related to ABT-737) in nine patients with chronic lymphocytic leukemia was associated with high basal Bim/Mcl-1 ratio.³⁵

Identifying predictive factors that allow for appropriate patient selection remains a critical preliminary step in the success of BH3-mimetic-based strategies. The phase II clinical trial performed with ABT-263 used as a single agent in patients with relapsed SCLC that did not stratify patients, led to negative results. However, the authors were able to retrospectively show that Bcl-2 gene amplification was correlated with increased apoptosis suggesting that biomarker could constitute an attractive strategy in the selective treatment of patients.³⁶ Subsequent investigations into the impact of Bcl-2 family members on the response to ABT-263 are required to improve patient selection and identify pertinent subpopulations for treatment using BH3 mimetic molecules.

In ovarian carcinomas, the expression of Bim seems to constitute a robust biomarker. Indeed, it should be noted that Bim expression appears highly homogenous in all tumor nodes studied in the same patient (20/25 patients) despite the fact that ovarian carcinomas are considered as particularly heterogeneous. This finding is important as it rules out the notion that a false evaluation of Bim expression could occur due to the choice of the tumor node. We were able to determine that in the five remaining cases, Bim expression level was somehow heterogeneous between the four tumor nodes.

The supplementary nodes have not been exposed to treatment. Consequently, we were not able to assess the response to ABT-737 in these tumor nodes. However, these results show that it will be important to assess Bim expression in multiple tumor biopsies in future clinical trials, as the heterogeneity of this expression could itself be associated to a low response to the drug or to an earlier recurrence. This point would be essential to investigate in the context of a clinical trial that thus should study Bim expression in several biopsies and try to define the link between such and heterogeneity between tumor nodes and the response to treatment.

Among “high Bim” expressing tumors, 7/20 did not respond to ABT-737 and these tumors could be identified by the combination of Bim, Mcl-1, and P-Erk expression levels.
Indeed, all nonresponding tumors of this subgroup (high Bim) were identified in the group in which Mcl-1 and P-Erk were concomitantly highly expressed. In contrast, tumors highly expressing only one of these proteins responded to treatment. Using this combination protein expression approach, only 3/20 "high Bim" tumor samples remained classified as nonresponding when in actuality the ex vivo experiment showed a response to treatment.

Both Mcl-1 and P-Erk are negative regulators of Bim activity. Indeed, Mcl-1 has been shown to sequester Bim thereby impeding its proapoptotic activity as a direct activator of Bax/ Bak.³⁷ Bim could also be considered to be an Mcl-1 inhibitor as it is able to release Bak from Mcl-1 in an integrated model.³⁸ Furthermore, P-Erk1/2 is able to phosphorylate Bim on Ser-69, thus impeding its proapoptotic activity and eventually leading to its proteosomal degradation.³⁹ It could be hypothesized that in tumors with high Mcl-1 and P-Erk expression levels, a strong cooperation between these two proteins leads to a global inactivation of Bim, despite its high expression level. However, when one of these proteins is expressed at low levels, the other protein is unable to counteract the high Bim expression level, thus allowing ABT-737 activity.

Collectively, these findings suggest that the overall level of the free dephosphorylated form of Bim should be considered as the most important event leading to apoptotic cell death in response to its release from Bcl-xL by ABT-737 as

previously suggested.⁴⁰ All events leading to Bim inactivation or degradation could have a negative impact on response to ABT-737. Furthermore, our study also showed that considering several Bcl-2 protein family members' and the cellular events impinging upon their expression could be more informative than investigating individual proteins at the time of drug exposure.

In summary, using freshly collected patient tumor samples we have shown that ABT-737 successfully induces apoptotic cell death when administered as a single agent in HGSOC. Using a simple and easy-to-interpret method, we report the potential use of Bim as a robust marker for predicting response to ABT-737 in human ovarian cancer. Moreover, its predictive value is strongly improved by the concomitant consideration of P-Erk and Mcl-1 expression levels. This pilot study showed that ABT-737 has promise as a monotherapy for HGSOC in a specific subgroup of tumors and that patient selection using this strategy may be attractive in the design of future clinical trials using Navitoclax for the treatment of ovarian cancers.

## Acknowledgements

We acknowledge Abbvie Laboratory for providing us with ABT-737, Emma Arriola (Abbvie US) and Elisabeth Rouffiac (Abbvie France) for their constant support for the realization of this study. We thank Dr Nicolas Elie from quantitative histo-imaging and microscopy core facilities of the University of Caen Basse-Normandie (Structure Fédérative 4206 ICORE) for his technical support and Katherine Karakasis for manuscript review.

## References

1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. *CA Cancer J Clin* 2010;60:277-300.

2. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. *Cell* 2011;144:646-74.

3. Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. *Oncogene* 2007; 26:1324-37.

4. Marone M, Sambia G, Mozzetti S, et al. Bcl-2, bax, bcl-XL, and bcl-XS expression in normal and neoplastic ovarian tissues. *Clin Cancer Res* 1998;4:517-24.

5. Williams J, Lucas PC, Griffith KA, et al. Expression of Bcl-xL in ovarian carcinoma is associated with chemoresistance and recurrent disease. *Gynecol Oncol* 2005;96:287-95.

6. Villedieu M, Louis MH, Dutoit S, et al. Absence of Bcl-xL down-regulation in response to cisplatin is associated with chemoresistance in ovarian carcinoma cells. *Gynecol Oncol* 2007;105:31-44.

7. Shigemasa K, Katoh O, Shiroyama Y, et al. Increased MCL-1 expression is associated with poor prognosis in ovarian carcinomas. *Ipn J Cancer Res* 2002;93:542-50.

8. Brotin E, Meryet-Figuiere M, Simonin K, et al. Bcl-XL and MCL-1 constitute pertinent targets in ovarian carcinoma and their concomitant inhibition is sufficient to induce apoptosis. *Int J Cancer* 2010;126:885-95.

9. Simoes-Wust AP, Schurpf T, Hall J, et al. Bcl-2/bcl-xL bispecific antisense treatment sensitizes breast carcinoma cells to doxorubicin, paclitaxel and cyclophosphamide. *Breast Cancer Res Treat* 2002;76:157-66.

10. Ni CT, Letai A. Mimicking the BH3 domain to kill cancer cells. *Oncogene* 2008;27(Suppl 1):S149-57.

11. Oltersdorf T, Elmore SW, Shoemaker AR, et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. *Nature* 2005;435: 677-81.

12. Del GMV, Brown JR, Certo M, et al. Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737. *J Clin Invest* 2007;117:112-21.

13. Tahir SK, Yang X, Anderson MG, et al. Influence of Bcl-2 family members on the cellular response of small-cell lung cancer cell lines to ABT-737. *Cancer Res* 2007;671176-83.

14. Tagscherer KE, Fassl A, Campos B, et al. Apoptosis-based treatment of glioblastomas with ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins. *Oncogene* 2008;27:6646-56.

15. Bray K, Chen HY, Karp CM, et al. Bcl-2 modulation to activate apoptosis in prostate cancer. *Mol Cancer Res* 2009;7:1487-96.

16. Simonin K, N'diaye M, Lheureux S, et al. Platinum compounds sensitize ovarian carcinoma cells to ABT-737 by modulation of the Mcl-1/Noxa axis. *Apoptosis* 2013;18:492-508.

17. Konopleva M, Contractor R, Tsao T, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. *Cancer Cell* 2006;10:375-88.

18. Kang MH, Wan Z, Kang YH, et al. Mechanism of synergy of N-(4-hydroxyphenyl)retinamide and ABT-737 in acute lymphoblastic leukemia cell lines: Mcl-1 inactivation. *J Natl Cancer Inst* 2008;100:580-95.

19. Simonin K, Brotin E, Dufort S, et al. Mcl-1 is an important determinant of the apoptotic response to the BH3-mimetic molecule HA14-1 in cisplatin-resistant ovarian carcinoma cells. *Mol Cancer Ther* 2009;8:3162-70.

20. Willis SN, Chen L, Dewson G, et al. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. *Genes Dev* 2005;19:1294-305.

21. Thomas LW, Lam C, Edwards SW. Mcl-1; the molecular regulation of protein function. *FEBS Lett* 2010;584:2981-9.

22. van Delft MF, Wei AH, Mason KD, et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. *Cancer Cell* 2006; 10:389-99.

23. Coleman RL, Monk BJ, Sood AK, et al. Latest research and treatment of advanced-stage epithelial ovarian cancer. *Nat Rev Clin Oncol* 2013;10: 211-24.

24. Daniel VC, Marchionni L, Hierman JS, et al. A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro. *Cancer Res* 2009;69: 3364-73.

25. Hann CL, Daniel VC, Sugar EA, et al. Therapeutic efficacy of ABT-737, a selective inhibitor of BCL-2, in small cell lung cancer. *Cancer Res* 2008;68:2321-8.

26. Seveno C, Loussouarn D, Brechet S, et al. gamma-Secretase inhibition promotes cell death, Noxa upregulation, and sensitization to BH3 mimetic ABT-737 in human breast cancer cells. *Breast Cancer Res* 2012;14:R96.

27. Zhu S, Cohen MB, Bjorge JD, et al. PI3K inhibition potentiates Bcl-2-dependent apoptosis in
renal carcinoma cells. *J Cell Mol Med* 2013;17:377-85.

28. Zhu H, Yang W, He LJ, et al. Upregulating Noxa by ER stress, celastrol exerts synergistic anti-cancer activity in combination with ABT-737 in human hepatocellular carcinoma cells. *PLoS One* 2012;7:e52333.

29. Cristofanon S, Fulda S. ABT-737 promotes tBid mitochondrial accumulation to enhance TRAIL-induced apoptosis in glioblastoma cells. *Cell Death Dis* 2012;3:e432.

30. Hauck P, Chao BH, Litz J, et al. Alterations in the Noxa/Mcl-1 axis determine sensitivity of small cell lung cancer to the BH3 mimetic ABT-737. *Mol Cancer Ther* 2009;8:883-92.

31. Faber AC, Ebi H, Costa C, et al. Apoptosis in targeted therapy responses: the role of BIM. *Adv Pharmacol* 2012;65:519-42.

32. Davids MS, Letai A. Targeting the B-cell lymphoma/leukemia 2 family in cancer. *J Clin Oncol* 2012;30:3127-35.

33. Faber AC, Corcoran RB, Ebi H, et al. BIM expression in treatment-naïve cancers predicts responsiveness to kinase inhibitors. *Cancer Discov* 2011;1:352-65.

34. Savry A, Carre M, Berges R, et al. Bcl-2-enhanced efficacy of microtubule-targeting chemotherapy through Bim overexpression: implications for cancer treatment. *Neoplasia* 2013;15:49-60.

35. Roberts AW, Seymour JF, Brown JR, et al. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. *J Clin Oncol* 2012;30:488-96.

36. Rudin CM, Hann CL, Garon EB, et al. Phase II study of single-agent navitoclax (ABT-263) and biomarker correlates in patients with relapsed small cell lung cancer. *Clin Cancer Res* 2012;18:3163-9.

37. Juin P, Geneste O, Gautier F, et al. Decoding and unlocking the BCL-2 dependency of cancer cells. *Nat Rev Cancer* 2013;13:455-65.

38. Moldoveanu T, Follis AV, Kriwacki RW, et al. Many players in BCL-2 family affairs. *Trends Biochem Sci* 2014;39:101-11.

39. Luciano F, Jacquel A, Colosetti P, et al. Phosphorylation of Bim-EL by Erk1/2 on serine 69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function. *Oncogene* 2003;22:6785-93.

40. Faber AC, Li D, Song Y, et al. Differential induction of apoptosis in HER2 and EGFR addicted cancers following PI3K inhibition. *Proc Natl Acad Sci USA* 2009;17:106:19503-8.