Oncogenic RAS Simultaneously Protects Against Anti-EGFR Antibody-Dependent Cellular Cytotoxicity and EGFR Signaling Blockade

Home , Rituximab

ORIGINAL ARTICLE Oncogenic RAS simultaneously protects against anti-EGFR antibody-dependent cellular cytotoxicity and EGFR signaling blockade

S Kasper1, F Breitenbuecher1, H Reis2, S Brandau3, K Worm2,JKo¨ hler1, A Paul4, T Trarbach1, KW Schmid2 and M Schuler1

Monoclonal antibodies against the epidermal growth factor receptor (EGFR) are effective cancer therapeutics, but tumors harboring RAS mutations are resistant. To functionally dissect RAS-mediated resistance, we have studied clinically approved anti-EGFR antibodies, cetuximab and panitumumab, in cancer models. Both antibodies were equally cytotoxic in vitro. However, cetuximab, which also triggers antibody-dependent cellular cytotoxicity (ADCC), was more effective than panitumumab in vivo. Oncogenic RAS neutralized the activity of both antibodies in vivo. Mechanistically, RAS upregulated BCL-XL in cancer cell lines and in primary colorectal cancers. Suppression of BCL-XL by short hairpin RNA or treatment with a BH3 mimetic overcame RAS-mediated antibody resistance. In conclusion, RAS-mutant tumors escape anti-EGFR antibody-mediated receptor blockade as well as ADCC in vivo. Pharmacological targeting of RAS effectors can restore sensitivity to antibody therapy.

Oncogene (2013) 32, 2873–2881; doi:10.1038/onc.2012.302; published online 16 July 2012 Keywords: cetuximab; colorectal cancer; RAS; ADCC; anti-EGFR antibodies; BCL-XL

INTRODUCTION products are involved in activating the mitogen-activated protein Overexpression of the epidermal growth factor receptor (EGFR) is kinase pathway and additional pathways downstream of the EGFR. observed in several human cancers. It associates with adverse Hence, the current model of mutant RAS-mediated resistance to prognosis in patients with colorectal cancer (CRC) and head and antibody therapy focuses on the ability of oncogenic RAS to neck cancer (HNSCC), thus defining the EGFR as an attractive compensate for upstream signals lacking owing to EGFR blockade therapeutic target.1 Combining anti-EGFR antibodies cetuximab by the antibody. In analogy, receptor blockade is regarded as the or panitumumab with cytotoxic chemotherapy in patients with main effector mechanism of the two clinically approved anti-EGFR metastatic CRC increased remission rates, prolonged progression- antibodies, cetuximab and panitumumab. Cetuximab is a chimeric free survival and, in some clinical trials, increased overall survival. IgG1 antibody capable to mediate antibody-dependent cellular Importantly, more patients with isolated liver metastases were cytotoxicity (ADCC) in vitro and in vivo.12,13 In contrast, converted to a curatively resectable stage when chemotherapy panitumumab is unable to induce classical, natural killer (NK) 14 was complemented by an anti-EGFR antibody. In addition, cell-mediated ADCC owing to its IgG2 isotype. So far, no head-to- monotherapy with anti-EGFR antibodies has clinical activity in head comparison of these two antibodies in a clinical trial or in a metastasized CRC patients relapsing after prior chemo- preclinical in vivo model has been reported. Accordingly, it therapies.2–5 Adding the anti-EGFR antibody cetuximab to remains speculative whether the additional ADCC activity of standard chemotherapy increased response rates, progression- cetuximab results in enhanced therapeutic activity over receptor free survival and overall survival in patients with recurrent or blockade alone. Interestingly, Fc receptor-g polymorphisms, which metastatic HNSCC.6 Also, locoregional control of advanced HNSCC may determine the efficacy of ADCC, have been correlated with was improved when cetuximab was applied simultaneously with outcome following cetuximab-based treatment in a retrospective radiotherapy.7 Recently, several retrospective analyses of studies analysis of CRC patients. This correlation was maintained in RAS- conducted in CRC patients have suggested that the clinical benefit mutant and RAS-wild-type tumors.15 In vitro, RAS-mutant cancer from both clinically approved anti-EGFR antibodies, cetuximab cell lines were protected against direct antiproliferative actitivies and panitumumab, is restricted to patients suffering from tumors of cetuximab and panitumumab, but not against ADCC enabled devoid of somatic mutations of KRAS and additional RAS family by cetuximab.16 These lines of evidence argue that CRC oncogenes.4,8–11 Accordingly, more than 40% of all metastatic CRC susceptibility to cetuximab-mediated ADCC is not determined by patients are excluded from this therapeutic modality. the mutation status of RAS proto-oncogenes. However, no Anti-EGFR antibodies block the receptor to interfere with ligand apparent clinical benefit of cetuximab was derived in patients binding and possibly receptor dimerization, thus preventing signal suffering from RAS-mutant CRC despite the theoretical possibility transduction via the EGFR tyrosine kinase. The RAS family gene of preserved ADCC.

1Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany; 2Department of Pathology and Neuropathology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany; 3Department of Otorhinolaryngology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany and 4Department of General, Visceral and Transplant Surgery, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany. Correspondence: Professor Dr M Schuler, Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany. E-mail: [email protected] Received 12 July 2011; revised 1 May 2012; accepted 31 May 2012; published online 16 July 2012 RAS-mediated antibody resistance S Kasper et al 2874 A full molecular understanding of relevant effector mechanisms non-obese diabetic severe combined immunodeficiency (NOD/ of, as well as resistance mechanisms to, anti-EGFR antibody SCID) mice. Injection of either cell line reproducibly led to therapy will allow the development of strategies (i) for potentially outgrowth of flank tumors, which could be followed in vivo. Two synergistic combination treatments, and (ii) to overcome antibody models were established: (A) in a ‘therapeutic setting’, NOD/SCID resistance in RAS-mutant cancers. To this end, we have established mice bearing palpable flank tumors were treated twice weekly by preclinical models for the study of anti-EGFR therapy in vitro and intraperitoneal antibody injections (1 and 0.1 mg), and tumor in vivo. We find that ADCC indeed provides a therapeutic benefit growth was monitored. (B) In an ‘adjuvant setting’, intraperitoneal in addition to EGFR blockade in vivo. However, oncogenic RAS antibody injections (1 mg twice weekly) were initiated 1 day after completely abolishes both, receptor blockade and ADCC, by the subcutaneous implantation of A431 cells in NOD/SCID mice, cetuximab in vivo. Dissecting RAS-mediated signaling alterations in and tumor development and survival were monitored. Applying cancer models and primary CRC, we identify anti-apoptotic BCL-XL cetuximab, panitumumab and rituximab in therapeutic model A, as a clinically and functionally relevant target for therapeutic we observed a dose-dependent activity of both anti-EGFR modulation of anti-EGFR antibody resistance. antibodies (Figure 2 and data not shown). In the ‘adjuvant’ model B, cetuximab prevented tumor development more effectively than panitumumab, resulting in superior survival of cetuximab-treated RESULTS mice (Figures 3a–c). Diverging efficacies of cetuximab and panitumumab in cancer As both anti-EGFR antibodies were equally effective in vitro models in vitro and in vivo (Figure 1), where cytotoxic effects are solely mediated by EGFR Using a panel of EGFR-expressing cell lines, we confirmed that the blockade, we reasoned whether the superiority of cetuximab cytotoxic activity of the anti-EGFR antibodies, cetuximab and in vivo resulted from its ability to trigger ADCC. Canonical ADCC is panitumumab, in vitro strictly correlated with the extent of EGFR executed by Fc receptor-bearing NK cells, which kill antibody- expression and KRAS mutation status (Supplementary Table 1). For marked target cells by granule-mediated cytotoxicity.17 While further studies, we selected two cell lines, which exhibited a NOD/SCID mice are lacking B and T cells, NK cell activity is at least profound reduction in clonogenic survival upon antibody treat- partially preserved and thus can contribute to cetuximab activity ment in vitro (Figures 1a and b). In short-term assays, anti-EGFR in vivo.18 To formally prove this hypothesis, NOD/SCID mice were antibodies effectively induced apoptosis in Difi cells, whereas radiodepleted (1.5 Gy) of cellular ADCC effectors prior to anti-EGFR A431 cells primarily responded by reduced proliferation (Figures antibody treatment. Interestingly, the in vivo activity of cetuximab 1c and d). Both anti-EGFR antibodies, cetuximab and panitumu- was markedly reduced in irradiated NOD/SCID mice as compared mab, were equally effective in vitro showing significant cytotoxi- with untreated mice. In contrast, panitumumab was equally city as compared with the chimeric monoclonal anti-CD20 effective in irradiated and non-irradiated NOD/SCID mice antibody rituximab (IgG1), which served as negative control (not (Figure 3d; Supplementary Table 2). These findings strongly shown). suggest that ADCC mediated by radiosensitive cellular immune To study antibody therapy in a host context, tumors were effectors at least in part explains the superior therapeutic activity established by subcutaneous injection of A431 or Difi cells in of cetuximab as compared with panitumumab in vivo.

Cetuximab Panitumumab Medium Cet 1µg/ml Cet 10µg/ml

100

% of medium control 20

0 0 10 25 50 100 Cet 20µg/ml Cet 50µg/ml Cet 100µg/ml antibody concentration [ng/ml]

Cetuximab Panitumumab 100 100

80 80

60 60

40 40 sub G1 [%]

20 % of medium control 20

0 0 0 25 50 100 250 500 0 2 4 6 8 10 antibody concentration [ng/ml] cetuximab concentration [µg/ml] Figure 1. EGFR-positive cancer cells are sensitive to anti-EGFR antibody therapy in vitro. Difi (a, c) and A431 (b, d) cancer cell lines were grown in the presence of cetuximab or panitumumab at the indicated concentrations. (a) Percentage of clones (normalized to medium control) of Difi cells treated with cetuximab (black columns) or panitumumab (gray columns; mean±s.d. of three independent experiments). (b) Representative photomicrographs of clones of A431 cells grown in the presence of medium or cetuximab (Cet) at the indicated concentrations. (c) Fraction of apoptotic Difi cells with subgenomic DNA content (sub-G1) following treatment with cetuximab (black columns) or panitumumab (gray columns; mean±s.d. of three independent experiments). (d) Proliferation of A431 cells grown in the presence of increasing concentrations of cetuximab. Mean values (±s.d.) of three independent MTT assays.

Oncogene (2013) 2873 – 2881 & 2013 Macmillan Publishers Limited RAS-mediated antibody resistance S Kasper et al 2875

140 Rituximab Cetuximab degranulation in vitro (Supplementary Figure 3a). Thus, ADCC resistance due to HRASG12V-imposed changes in NK cell activation 120 seemed unlikely. As expected for an IgG2 antibody, panitumumab- marked EGFR-expressing HRASG12V or control cells had no effect

] 100

2 on NK cell activation in vitro (Supplementary Figure 3b). Genetic mouse models have deﬁned the mitochondrial path- 80 way of apoptotic caspase activation, which is regulated by the BCL-2 protein family, as a main death effector mechanism 60 following growth factor withdrawal in vivo.19–21 In keeping,

tumor size [mm tumor size 40 pharmacological suppression of EGFR-mediated oncogenic signals by gefitinib triggered ‘mitochondrial’ apoptosis.22 20 Recently, we have shown that inhibition of this apoptotic pathway either by transgenic expression of BCL-XL or by 0 enhanced mitogen-activated protein kinase and protein kinase B 0 10 20 30 40 50 60 signaling protects cancer cells against tumor suppression by days post implantation activated T cells and NK cells in vivo.23–25 Against this background, Figure 2. Effective treatment of established transplant tumors by an we immunohistochemically profiled the expression of anti- anti-EGFR antibody in vivo. Palpable flank tumors were established apoptotic BCL-2, BCL-XL and MCL-1 in primary human CRC by subcutaneous injection of Difi cells in NOD/SCID mice. After 21 specimens. While at least one of these BCL-2 proteins was days (arrow), tumor-bearing mice were treated twice weekly by detectable in almost all cancer samples, there was a high intraperitoneal injections of cetuximab (1 mg, closed triangles) or prevalence of strong cytoplasmic expression of BCL-XL (Figures the control antibody rituximab (1 mg, closed circles). Tumor growth 6a and b). Interestingly, a significantly higher proportion of KRAS- was monitored by palpation and measured using a caliper. Mean mutant cancers stained positively for BCL-XL as compared with bidimensional tumor sizes þ s.d. of a representative experiment KRAS-wild-type tumors (median 97.5±6.25% vs 85.0±13.8%, (five mice per treatment group) are given. P ¼ 0.02). No significant differences in expression of BCL-2 (33.3% positive vs 30% positive, P ¼ 0.63) or MCL-1 (median 95.5±7.62% vs 95.0±8.67%, P ¼ 0.731) were observed between Oncogenic RAS confers resistance to ADCC in vivo and to EGFR KRAS-mutant and KRAS-wild-type cancers (Figures 6c and d and blockade in vitro and in vivo Supplementary Table 3). To assess the impact of oncogenic RAS on antibody effector Based on these findings in primary CRC, we studied the impact mechanisms in identical cellular backgrounds, we stably of oncogenic RAS on the expression of BCL-2 family proteins in G12V expressed clinically occurring RAS mutants, HRASG12V, KRASG12V EGFR-positive cancer cell lines. A431 cells expressing HRAS , G12V G12V and NRASG12V, in anti-EGFR antibody-sensitive A431 and Difi cells. KRAS or NRAS exhibited markedly higher BCL-XL RNA and Functional transgene expression was confirmed by immunoblot protein levels than control cells (Figures 6f and g and not shown). analyses of various phosphoepitopes indicating enhanced con- Blocking protein synthesis with cycloheximide revealed signifi- stitutive and ligand-induced mitogen-activated protein kinase cantly enhanced stability of BCL-XL, but not BCL-2 or MCL-1, in the G12V activation in cells expressing mutant RAS (Figure 4a and not presence of HRAS , arguing for transcriptional activation as shown). As expected each of the RAS mutants, equally protected well as posttranslational mechanisms of action (Figure 6e). Taken EGFR-positive cancer cells against cetuximab- and panitumumab- together, upregulation of anti-apoptotic BCL-XL is frequently induced cytotoxicity in vitro (Figures 4b–d and Supplementary observed in KRAS-mutant human CRC. Moreover, induction and Figure 1). Accordingly, we selected the HRASG12V model for further posttranslational stabilization of BCL-XL by oncogenic RAS might studies. To rule out oncogene-induced antigen suppression, we contribute to resistance against anti-EGFR antibody therapy. confirmed that EGFR expression was not lost in HRASG12V- To support this hypothesis at a functional level, we stably expressing cancer cells (Supplementary Figure 2). This argued expressed BCL-XL in Difi cells and A431 cells, both of which are for RAS-mediated resistance mechanisms at the level of intracel- devoid of endogenous mutations of HRAS, KRAS and NRAS lular signal transduction. (Figure 7a). BCL-XL effectively protected these RAS-wild-type HRASG12V-expressing tumors established in NOD/SCID mice cancer models against cetuximab-induced apoptosis in vitro were significantly less responsive to anti-EGFR antibody therapy (Figure 7b). In vivo, cetuximab and panitumumab were blunted in vivo (Figure 5a). Surprisingly, HRASG12V-expressing tumors in preventing the outgrowth of BCL-XL-expressing tumors in NOD/ exhibited identical growth rates under ‘adjuvant’ treatment with SCID mice (Figure 7c and Supplementary Table 4). For example, cetuximab and panitumumab in vivo (Figure 5b). Moreover, median size of A431-BCL-XL tumors following 15 days of 2 2 radiodepletion of cellular ADCC effectors did not alter tumor cetuximab treatment in vivo was 64.49 mm (±17.67 mm , 2 outgrowth from HRASG12V-positive cancer cells in NOD/SCID mice Figure 7c), as compared with 0 mm for A431 control tumors treated with cetuximab (Figure 5c), which was in stark contrast to (Figure 3a) (P ¼ 0.0018, unpaired t-test). Panitumumab-treated 2 the results obtained with RAS-wild-type cancer models (Figures 3a A431-BCL-XL tumors grew to a median size of 124.1 mm 2 and d). These results provide a strong argument for oncogenic RAS (±13.06 mm , Figure 7c) at 15 days, as compared with 2 2 simultaneously conferring resistance against cetuximab-induced 16.54 mm (±14.18 mm , Figure 3a) for A431 control tumors ADCC as well as cetuximab- and panitumumab-imposed EGFR (Po0.0001, unpaired t-test). Interestingly, no significant difference blockade in vivo. in growth rates of cetuximab- and panitumumab-treated A431- BCL-XL tumors was observed (Figure 7c), which was reminiscent of the results obtained by treating RAS-wild-type tumors growing in Oncogenic RAS protects against anti-EGFR antibody therapy by leukocyte-depleted mice (Figure 3d) or HRASG12V-expressing upregulation of BCL-XL tumors (Figure 5b). These findings argue that BCL-XL alike The above findings indicate that oncogenic RAS either triggers oncogenic RAS equally protects against EGFR blockade and ADCC two distinct resistance mechanisms against the cytotoxic effects of in vivo, and thus can substitute its upstream regulator in EGFR signaling blockade and ADCC, or mutant RAS confers cross- mediating resistance to anti-EGFR antibody therapy. resistance via a unifying molecular pathway. Cetuximab-marked To study the contribution of BCL-XL to RAS-mediated resistance HRASG12V-expressing cancer cells effectively stimulated NK cell at a functional level, we devised short hairpin RNA (shRNA)

& 2013 Macmillan Publishers Limited Oncogene (2013) 2873 – 2881 RAS-mediated antibody resistance S Kasper et al 2876 Rituximab Pantitumumab Cetuximab 300 100 Cetuximab 250

] Panitumumab 2 75 200 Rituximab

150 50 100 survival [%]

tumor size [mm 25 50

0 0 0 5 10 15 20 25 30 35 40 45 0 10 20 30 40 50 60 70 80 days post implantation Day

500 Rituximab Panitumumab Cetuximab 450 400 Median Survival ] Group Established Tumors 2 [days] 350 300 cetuximab 0/10 n.r. 250 panitumumab 9/10 47 200 150 tumor size [mm rituximab 8/10 25 100 50 0 0 5 10 15 20 25 30 35 days post implantation Figure 3. Cetuximab is superior over panitumumab in prevention of tumor outgrowth in vivo.(a) NOD/SCID mice received subcutaneous injections of A431 cells on day 0. Starting on day 1 (arrow), mice were treated twice weekly by intraperitoneal injections of panitumumab (open boxes), cetuximab (closed triangles), or the control antibody rituximab (closed circles, 1 mg each). Tumor development was monitored by palpation and tumors were measured using a caliper. Mean bidimensional tumor sizes ( þ s.d.) of five mice per treatment group are given. (b) Kaplan–Meier plots of tumor-free survival of NOD/SCID mice following subcutaneous injection of A431 cells. Mice were treated as in (a) with cetuximab (solid line), panitumumab (dashed line), or the control antibody rituximab (dotted line). Cetuximab-treated mice exhibited a significantly prolonged survival as compared with panitumumab-treated (P ¼ 0.0027) or rituximab-treated mice (P ¼ 0.0031, log rank test). (c) Numeric representation of tumor development and median survival (days) in relation to antibody treatment (10 mice per treatment group; n.r. denotes not reached). (d) Following total body irradiation (1.5 Gy) for in vivo leukocyte depletion, NOD/SCID mice were subcutaneously injected with A431 cells on day 0. Starting on day 1 (arrow), mice were treated with panitumumab (open boxes), cetuximab (closed triangles), or the control antibody rituximab (closed circles) as in (a). Tumor development was monitored by palpation and tumors were measured using a caliper. Mean bidimensional tumor sizes ( þ s.d.) of five mice per treatment group are given. Note that in contrast to experiment (a), cetuximab was not superior over panitumumab in the prevention of tumor growth in irradiated NOD/SCID mice.

technology in HRASG12V-expressing Difi cells (Figure 7d). Suppres- outcome following cetuximab-based therapy. This association was sion of endogenous BCL-XL restored sensitivity of HRASG12V- maintained when KRAS mutation status was taken into account.15 expressing cancer cells to cetuximab-induced apoptosis The availability of panitumumab, a fully human IgG2 antibody (Figure 7e). Moreover, subtoxic concentrations of ABT-737, a unable to trigger NK cell-mediated ADCC14 (Supplementary pharmacologic BH3 mimetic antagonizing the anti-apoptotic Figure 3b), allows to resolve this apparent discrepancy and to activities of BCL-2, BCL-XL and BCL-W, but not MCL-1,26–28 dissect the relative contribution of ADCC to the antitumor activity effectively sensitized HRASG12V-expressing cancer cells to of cetuximab. So far, no head-to-head comparison of cetuximab cetuximab-induced apoptosis (Figure 7f). This suggests that and panitumumab has been reported in the clinic. Here we have mutant RAS confers anti-EGFR antibody resistance by blocking devised EGFR-expressing cancer models to address the role of apoptotic caspase activation through a pathway regulated by ADCC in anti-EGFR antibody therapy in relation to resistance BCL-XL. mediated by oncogenic RAS. Transplanting anti-EGFR antibody-sensitive human cancer cells in NOD/SCID mice, we observed therapeutic superiority of DISCUSSION cetuximab over panitumumab in vivo, which was abolished by Somatic mutations of RAS proto-oncogenes are associated with depletion of cellular immune effectors. This is a strong indication clinical resistance to anti-EGFR antibody therapy in CRC.11 of ADCC contributing to cancer control by cetuximab at least in Accordingly, application of the clinically approved antibodies, the present model. Similar observations were reported from cetuximab and panitumumab, is restricted to patients suffering antibody treatment studies of leukemia xenografts in NOD/SCID from CRC, in which at least somatic KRAS ‘hot spot’ mutations have mice, which proved sensitive to depletion or stimulation of NK been ruled out. As most CRC express the EGFR antigen, patients cells in vivo.18 As NK cell activity of NOD/SCID mice is reportedly harboring RAS-mutant CRC in theory could still benefit from blunted,29 the relative contribution of ADCC to the cytotoxic effect antibody-mediated immunotherapy via complement-dependent of cetuximab might actually be underestimated by our cytotoxicity or ADCC. However, the lack of clinical activity of experimental systems. Nevertheless, expression of oncogenic cetuximab, which is able to activate NK cells mediating ADCC RAS interfered with both antibody effector mechanisms, EGFR (Supplementary Figure 3a) in KRAS-mutant CRC, argues against signaling blockade (panitumumab and cetuximab) and ADCC this hypothesis. Surprisingly, certain Fc receptor polymorphisms (cetuximab), in that growth of RAS-mutant tumors was enhanced were reported to independently associate with an improved and the difference in efficacy between these antibodies was

Oncogene (2013) 2873 – 2881 & 2013 Macmillan Publishers Limited RAS-mediated antibody resistance S Kasper et al 2877 HRAS G12V control control * HRAS G12V ++ - - ++ - -EGF KRAS G12V 100 +- +- +- +-Cetuximab NRAS G12V * 80 P-EGFR * * EGFR 60 * p-RAF-1 * * 40 RAF-1 * p-ERK 20 % of medium control * ERK 0 Actin 0 10 25 50 100 cetuximab concentration [ng/ml]

control HRAS G12V KRAS G12V control HRAS G12V 100 100 * 80 80 * * 60 * 60 * 40 40 * sub G1 [%] * * * 20 20 # % of medium control

0 0 0 10 25 50 100 500 0 50 100 500 panitumumab concentration [ng/ml] cetuximab concentration [ng/ml] Figure 4. Oncogenic RAS protects cancer cells against anti-EGFR antibody-mediated signaling blockade and cytotoxicity in vitro. A431 and Difi cells were retrovirally transduced to stably express the oncogenic RAS mutants HRASG12V, KRASG12V or NRASG12V.(a) Inhibition of constitutive and ligand-induced (EGF 10 ng/ml) phosphorylation of EGFR, and mitogen-activated protein kinase signal transducers RAF and ERK by cetuximab (1 mg/ml) in A431 cells expressing HRASG12V or controls. (b, c) Clonogenic survival of Difi cells expressing HRASG12V (gray columns), KRASG12V (checked bars), NRASG12V (striped bars) and controls (black bars) cultured in the presence of cetuximab (b) or panitumumab (c). Mean colony numbers ( þ s.d.) normalized to medium control from three independent experiments are given. (d) Induction of apoptosis by cetuximab in Difi cells expressing HRASG12V (gray columns) and controls (black bars). Mean percentages ( þ s.d.) of cells with subgenomic DNA content from three independent experiments are given. Asterisks (*) denote a statistically significant (Po0.05, t-test) difference in apoptosis observed in RAS-mutant cells as compared with control cells.

300 control HRAS G12V 450 Cetuximab Panitumumab 300 Cetuximab Panitumumab 400 250 250 350 ] ] ] 2 2 200 2 300 200 250 150 150 200 100 150 100 tumor size [mm tumor size tumor size [mm tumor size tumor size [mm tumor size 100 50 50 50 0 0 0 0 10 20 30 40 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 days post implantation days post implantation days post implantation Figure 5. Oncogenic RAS protects tumors against anti-EGFR antibody-mediated direct cytotoxicity as well as ADCC in vivo.(a) Tumor growth following injection of Difi cells expressing HRASG12V (open boxes) or control cells (closed triangles) in NOD/SCID mice. After 21 days (arrow), mice were treated biweekly with intraperitoneal injections of cetuximab (0.1 mg). Mean bidimensional tumor sizes ( þ s.d.) of five mice per group are given. (b) NOD/SCID mice received subcutaneous injections of A431 cells expressing HRASG12V on day 0. Starting on day 1 (arrow), mice were treated with biweekly intraperitoneal injections of cetuximab (closed triangles) or panitumumab (open boxes, 1 mg each) and tumor growth was monitored. Mean bidimensional tumor sizes ( þ s.d.) of five mice per group are given. (c) Following total body irradiation (1.5 Gy) for in vivo leukocyte depletion, NOD/SCID mice were subcutaneously injected with A431 cells expressing HRASG12V on day 0. Starting on day 1 (arrow) mice were treated with biweekly intraperitoneal injections of cetuximab (closed triangles) or panitumumab (open boxes, 1 mg each) and tumor growth was monitored. Mean bidimensional tumor sizes ( þ s.d.) of five mice per group are given.

neutralized. Hence, it can be concluded that oncogenic RAS their ability to execute the ‘mitochondrial’ pathway of apoptotic mediates protection against direct (inhibition of signal caspase activation.23–25,30 Deregulated growth and survival factor transduction) and indirect (immunological) cytotoxic activities of signaling is a common phenomenon in human cancer.31 anti-EGFR antibodies in vivo. Frequently, this results in increased expression and function of These ﬁndings are in line with recent observations from our anti-apoptotic BCL-2 family proteins, as observed for BCL-XL in our group and others indicating that the responsiveness of cancer present model of RAS-mediated antibody resistance. As direct cells to cytotoxic cellular immune effectors is also determined by pharmacological targeting of oncogenic RAS so far has not been

& 2013 Macmillan Publishers Limited Oncogene (2013) 2873 – 2881 RAS-mediated antibody resistance S Kasper et al 2878

12810/05 BCL-XL 6282/02 10427/02 39486/04 6282/03 22272/06 21442/02 22439/04 20556/04 1577/04 22197/04 BCL-2- BCL-2+ 9902/03 8677/01 39019/04 26955/06 13123/03 16893/04 11631/99 57494/04 8524/03 20136/05 17532/04 BCL-XL + BCL-XL ++ BCL-XL +++ 125/04 3365/0B 21442/02 62106/03 10543/04 22197/04 18311/04 21578/04 10199/05 25801/03 4235/00 MCL-1 + MCL-1 ++ MCL-1 +++ 0 2 4 6 8 10 12 Immunoreactive Score

57494/04 MCL-1 KRAS mutant KRAS wild type 9902/03 BCL-XL IRS 12 22197/04 7% BCL-XL IRS 9 6282/02 18% 22272/06 27% BCL-XL IRS 8 9% 40% 10427/02 64% BCL-XL IRS 6 20136/05 9% 1577/04 13% 13% BCL-XL IRS 4 39019/04 8677/01 12810/05 N=11 N=15 17532/04 KRAS mutant KRAS wild type 16893/04 MCL-1 IRS 12 10199/05 10% 14% 14% MCL-1 IRS 9 20556/04 10% MCL-1 IRS 8 21578/04 40% 14% 11631/99 MCL-1 IRS 6 10543/04 40% 57% 62106/03 MCL-1 IRS 4 21442/02 3365/0B N=10 N=7 18311/04 22197/04 4235/00

0 2 4 6 8 10 12 G12V G12V G12V Immunoreactive Score S

NRAS KRAS HRA control

BCL-XL

6h 4h 2h 0h time with Actin CHX

BCL-2 3 BCL-XL transcript level * 2.5 MCL-1 2 *

1.5 BCL-XL 1

Actin relative expression 0.5

+ - + - + - + - HRAS G12V 0 control HRAS G12V HRAS G12V -Puro -Hygro Figure 6. Upregulation of anti-apoptotic BCL-XL by oncogenic RAS. (a) Representative photomicrographs of immunohistochemically detectable BCL-2, BCL-XL and MCL-1 expression in tissue microarrays from 47 human CRC specimens. (b) Immunoreactive score (IRS) of BCL- XL expression in CRC specimens from 33 patients in relation to KRAS and BRAF mutation status. Each bar represents the IRS of an individual sample. Black bars, KRASwild type/BRAFwild type tumors; blue bars, BRAFmutant tumors; red bars, KRASmutant tumors; gray bars, mutation analysis not informative. (c) IRS of MCL-1 expression in CRC specimens from 24 patients in relation to KRAS and BRAF mutation status. Each bar represents the IRS of an individual sample; color coding of bars as in (b). (d) Distrubution of immunhistochemical staining intensity (IRS) for BCL-XL (upper diagrams) and MCL-1 (lower diagrams) in KRASwild type and KRASmutant tumors. (e) Expression of anti-apoptotic BCL-2, MCL-1 and BCL-XL in A431 cells stably expressing HRASG12V ( þ ) and control cells ( À ). To prevent new protein synthesis, cells were treated with cycloheximide (CHX, 10 mg/ml) for the indicated periods. (f) Expression of BCL-XL in A431 cells stably expressing the oncogenic RAS mutants HRASG12V, KRASG12V or NRASG12V and A431 control cells. (g) Relative BCL-XL transcript level in Diﬁ cells expressing HRASG12V from two different retroviral vectors (pBabe.puro and pBabe.hygro) as compared with control cells. Expression levels of BCL-XL were normalized to the housekeeping gene beta-ACTIN. Mean change ( þ s.d.) of BCL-XL transcript levels from three independent experiments are given. An asterisk (*) denotes a statistically signiﬁcant (Po0.05, t-test) difference.

Oncogene (2013) 2873 – 2881 & 2013 Macmillan Publishers Limited RAS-mediated antibody resistance S Kasper et al 2879

100 control BCL-XL

60 * * A431 BCL-XLA431 control Difi BCL-XL Difi control * 40

BCL-XL sub G1 [%] * 20 Actin

0 0 25 50 100 500 cetuximab concentration [ng/ml]

400 cetuximab 350 panitumumab 502 501 500 499 ctrl parental shRNA clone ] 300 2 250 BCL-XL 200 150 Actin

tumor size [mm 100 50 0 0355 10 15 20 25 30 days post implantation

control HRAS G12V 60 ctrl. shRNA BCL-XL 100 50 * * 80 40 60 30 40 to ctrl. [%]

20 sub G1 [%]

10 20

relative induction of apoptosis 0 0 Cetuximab 25ng/ml 50ng/ml Cetuximab - + - + - + - + ABT-737 --++ --++ Figure 7. BCL-XL protects against cetuximab-mediated cytotoxicity in vitro and in vivo.(a) A431 and Difi cells were retrovirally transduced to stably express a human BCL-XL cDNA. (b) Induction of apoptosis by cetuximab treatment in Difi cells expressing BCL-XL (gray columns) or control cells (black columns). Mean percentages ( þ s.d.) of cells with subgenomic DNA content from three independent experiments. An asterisk (*) denotes a statistically significant (Po0.05, t-test) protection against apoptosis by BCL-XL. (c) NOD/SCID mice received subcutaneous injections of A431 cells expressing BCL-XL on day 0. Starting on day 1 (arrow), mice were treated with biweekly intraperitoneal injections of cetuximab (closed triangles) or panitumumab (open boxes, 1 mg each) and tumor growth was monitored. Mean bidimensional tumor sizes ( þ s.d.) of five mice per group are given. (d) BCL-XL protein levels in Difi-HRASG12V cells stably expressing four different lentiviral shRNA expression vectors targeting BCL-XL (499–502), a control shRNA vector (ctrl) and parental Difi-HRASG12V cells. (e) Difi-HRASG12V cells stably expressing shRNA vector 499 (black columns) or a control vector (gray columns) were treated with cetuximab (25 and 50 ng/ml). Mean relative percentages ( þ s.d.) of apoptotic cells with subgenomic DNA content from three independent experiments are given. An asterisk (*) denotes a statistically significant (Po0.05, t-test) difference. (f) Difi-HRASG12V cells (black columns) or control cells (gray columns) were treated with cetuximab (50 ng/ml), the BH3-mimetic ABT-737 (10 nM), or combinations of both. Mean percentages ( þ s.d.) of apoptotic cells with subgenomic DNA content from three independent experiments are given.

successfully achieved, modulation of downstream effectors is an previously.32 Lentiviral vectors encoding short hairpin RNA (shRNA) from alternative therapeutic strategy. Using subtoxic concentrations of the MISSION TRC-Mm 1.0 library were purchased from Sigma-Aldrich a BH3 mimetic, which antagonizes the activity of BCL-XL, we were (Munich, Germany); clones TRCN 0000033499-502 were used for BCL-XL indeed able to sensitize HRASG12V-expressing cancer cells to suppression, and the MISSION pLKO.1-puro non-target shRNA plasmid cetuximab-induced cytotoxicity. This provides proof-of-concept (SHC016-1EA) served as negative control. Clinical grade cetuximab (Erbitux, Merck Serono, Darmstadt, Germany), panitumumab (Vectibix, Amgen, for a rational combination therapy approach to expand the Thousand Oaks, CA, USA) and rituximab (Mabthera, Roche, Grenzach- population of cancer patients potentially beneﬁtting from anti- Wyhlen, Germany) were purchased from the pharmacy of the University EGFR antibody therapy. Hospital Essen; ABT-737 was provided by Abbott Laboratories (Abbott Park, IL, USA). For RNA expression analysis, total RNA was isolated (High Pure RNA Isolation Kit, Roche Diagnostics, Mannheim, Germany) and reversely transcribed into cDNA (Transcription High Fidelity cDNA Synthesis Kit, MATERIALS AND METHODS Roche Diagnostics) following the manufacturer’s instructions. Quantitative Cell lines and reagents RT–PCR analysis was performed on a LC480 instrument (Roche Diagnostics) The human EGFR-positive cancer cell lines A431 and Diﬁ were obtained using SYBR Green 1 Master chemistry (Roche Diagnostics) and primers for from DSMZ (Braunschweig, Germany) and Dr Robert Coffey (Nashville, TN, human BCL-XL:50-GGCTGGGATACTTTTGTGGA-30,50-TGTCTGGTCATTTCC- USA), respectively. All cells were cultured in DMEM medium supplemented GACTG-30 and human ACTIN: 50-TCAGCTGTGGGGTCCTGT-30,50-GAAGGGA- 33 with 10% fetal bovine serum (PAA, Coelbe, Germany), L-glutamine, CAGGCAGTGAG-30 as previously described. The following primary penicillin and streptomycin (Invitrogen, Frankfurt, Germany). Stable antibodies were used for immunoblotting and immunolabeling following expression of the HRASG12V, KRASG12V, NRASG12V and BCL-XL complementary standard protocols: RAS, BCL-2 (C2), RAF-1, phospho-RAF-1S338 (all from DNAs (cDNAs) was achieved by retroviral transduction as described Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-ERK1/2, ERK1/2,

& 2013 Macmillan Publishers Limited Oncogene (2013) 2873 – 2881 RAS-mediated antibody resistance S Kasper et al 2880 BCL-XL (54H6), EGFR, phopho-EGFRY1068 (all from Cell Signaling Technology, ACKNOWLEDGEMENTS Danvers, MA, USA), actin (C4, ICN, Irvine, CA, USA), MCL-1 (Epitomics, We thank Sandra Hoffarth, Sarah-Luise Stergar, Kirsten Bruderek, Jeannette Burlingame, CA, USA), EGFR-Phycoerythrin (R&D-Systems, Minneapolis, Markowetz, Anna Even, Miriam Backs, Ali Sak, Sabine Harde and the staff of the MN, USA). Central Animal Facility, and the Pathology and Molecular Pathology Laboratories, University Hospital Essen, for their support. Robert Coffey, Roman Thomas, Hyatt Balke-Want, Scott W Lowe, Gary P Nolan and Abbott are acknowledged for providing Animal models reagents. This work was funded by grants from the Wilhelm Sander-Stiftung All animal studies were conducted in compliance with institutional (2005.136.3, MS), the Deutsche Forschungsgemeinschaft (SCHU 1541/5-1, MS), guidelines and German Animal Protection Law, and were approved by Mercator Research Center Ruhr-MERCUR (An-2011-0031, SK) a CESAR Research the responsible regulatory authority (Landesamt fu¨r Natur, Umwelt und Fellowship (SK), the Wiedenfeld-Stiftung (SK) and the IFORES program of the Medical Verbraucherschutz Nordrhein-Westfalen, Az. G969/08). NOD/SCID mice Faculty of the University Duisburg-Essen (MS). (Charles River Laboratories, France) received single subcutaneous flank injections of 2 Â 106 A431 or 1 Â 107 Difi cells suspended in 200 ml saline. Animals were monitored for tumor development twice weekly, and tumor growth was bidimensionally quantified using a caliper. Antibodies were REFERENCES dissolved in 200 ml saline and were administered as biweekly intraper- 1 Mayer A, Takimoto M, Fritz E, Schellander G, Kofler K, Ludwig H. The prognostic itoneal injections. For in vivo leukocyte depletion, mice received a single significance of proliferating cell nuclear antigen, epidermal growth factor receptor, fraction of total body irradiation (1.5 Gy) from a cobald source. Kaplan– and mdr gene expression in colorectal cancer. Cancer 1993; 71: 2454–2460. Meier plots for tumor-free survival were analyzed using the log rank test. 2 Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A et al. For statistical analysis of tumor size the unpaired t-test was used. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004; 351: 337–345. 3 Jonker DJ, O’Callaghan CJ, Karapetis CS, Zalcberg JR, Tu D, Au HJ et al. Cetuximab Cellular assays for the treatment of colorectal cancer. N Engl J Med 2007; 357: 2040–2048. 3 For clonogenic survival analysis, 5 Â 10 A431 or Difi cells were seeded in 4 Amado RG, Wolf M, Peeters M, Van CE, Siena S, Freeman DJ et al. Wild-type KRAS six-well plates in the presence of cetuximab, panitumumab or rituximab. is required for panitumumab efficacy in patients with metastatic colorectal Following incubation for 7 to 14 days, colonies were fixed with ethanol cancer. J Clin Oncol 2008; 26: 1626–1634. (70% v/v) for 30 min, stained with Coomassie brilliant blue and 5 Van CE, Peeters M, Siena S, Humblet Y, Hendlisz A, Neyns B et al. Open-label phase automatically counted using an Infinity-100 System and the Vision Capt III trial of panitumumab plus best supportive care compared with best supportive software (Vilber Lourmat, Eberhardzell, Germany). Proliferation was care alone in patients with chemotherapy-refractory metastatic colorectal cancer. quantified by means of the MTT assay according to the manufacturer’s J Clin Oncol 2007; 25: 1658–1664. instruction (Roche, Mannheim, Germany). Apoptosis was quantified by 6 Vermorken JB, Mesia R, Rivera F, Remenar E, Kawecki A, Rottey S et al. Platinum- flow cytometric determination of cells with subgenomic DNA content based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med 2008; following hypotonic lysis and staining with propidium iodide as previously 359: 1116–1127. 32 described. All results were obtained from at least three independent 7 Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB et al. Radiotherapy experiments. For statistical analysis, the unpaired t-test was used. plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006; 354: 567–578. 8 Karapetis CS, Khambata-Ford S, Jonker DJ, O’Callaghan CJ, Tu D, Tebbutt NC et al. NK cell isolation and degranulation assay K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl For NK cell isolation, lymphocytes from healthy donors were enriched by JMed2008; 359: 1757–1765. Ficoll centrifugation and purified using an NK cell isolation system 9 Van CE, Kohne CH, Hitre E, Zaluski J, Chang CCR, Makhson A et al. Cetuximab including magnetic bead-coupled specific antibodies from Miltenyi Biotec and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl (Bergisch Gladbach, Germany) according to the manufacturer’s instruc- JMed2009; 360: 1408–1417. tions. Upregulation of surface CD107a was measured to quantify NK cell 10 Bokemeyer C, Bondarenko I, Makhson A, Hartmann JT, Aparicio J, de BF et al. degranulation. Unstimulated or stimulated (200 U rhIL-2 for 24 h) NK cells Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first- were co-incubated with cetuximab- or panitumumab (1, 3 and 10 mg/ml)- line treatment of metastatic colorectal cancer. J Clin Oncol 2009; 27: 663–671. coated A431 cells at an effector:target cell ratio of 1:1 for 1 h in the 11 De RW, Claes B, Bernasconi D, De SJ, Biesmans B, Fountzilas G et al. Effects of presence of FITC-coupled anti-CD107. Following addition of monensin, KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus cells were further incubated for 5 h and then stained with fluorochrome- chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retro- conjugated antibodies against CD56, CD16 or isotype control antibody spective consortium analysis. Lancet Oncol 2010; 11: 753–762. (BD Biosciences, Heidelberg, Germany). Cells were washed, fixed and 12 Kurai J, Chikumi H, Hashimoto K, Yamaguchi K, Yamasaki A, Sako T et al. Antibody- analyzed by flow cytometry following standard protocols. dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines. Clin Cancer Res 2007; 13: 1552–1561. 13 Hara M, Nakanishi H, Tsujimura K, Matsui M, Yatabe Y, Manabe T et al. Interleukin- Analysis of primary tumor samples 2 potentiation of cetuximab antitumor activity for epidermal growth factor Clinical data and surplus tumor specimens were retrieved from 47 patients receptor-overexpressing gastric cancer xenografts through antibody-dependent with metastatic CRC. Tissue microarrays were prepared from formalin-fixed, cellular cytotoxicity. Cancer Sci 2008; 99: 1471–1478. paraffin-embedded tumor samples. Sections were subjected to hematox- 14 Schneider-Merck T, Lammerts van Bueren JJ, Berger S, Rossen K, van Berkel PH, ilin & eosin staining and immunohistochemical analyses following Derer S et al. Human IgG2 antibodies against epidermal growth factor receptor diagnostic protocols of the Department of Pathology and Neuropathology, effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to University Hospital Essen. Tumor-specific expression of each protein was IgG1, only by cells of myeloid lineage. J Immunol 2010; 184: 512–520. quantified applying an immunoreactive score.34 For statistical analysis, the 15 Bibeau F, Lopez-Crapez E, Di FF, Thezenas S, Ychou M, Blanchard F et al. Impact of Mann–Whitney test (for BCL-XL and MCL-1) and the Fisher–Yates test (for Fc{gamma}RIIa-Fc{gamma}RIIIa polymorphisms and KRAS mutations on the clin- BCL-2) was used. Tumor DNA was isolated from formalin-fixed, paraffin- ical outcome of patients with metastatic colorectal cancer treated with cetuximab embedded tumor sections following microdissection, and KRAS and BRAF plus irinotecan. J Clin Oncol 2009; 27: 1122–1129. mutation status was determined by PCR amplification of the relevant 16 Barriere J, Fischel J, Formento P, Renee N, Francoual MFJ, Chefour M et al. exons followed by direct Sanger sequencing. All studies involving patient Cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against data or biosamples were approved by the Ethics Committee of the Medical tumor cell lines characterized for EGFR expression and K-ras mutation. J Clin Oncol Faculty of the University Duisburg-Essen. 2010; 27: e14583. 17 Kagi D, Ledermann B, Burki K, Seiler P, Odermatt B, Olsen KJ et al. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 1994; 369: 31–37. CONFLICT OF INTEREST 18 Piloto O, Nguyen B, Huso D, Kim KT, Li Y, Witte L et al. IMC-EB10, an anti-FLT3 Martin Schuler has served as consultant to Amgen; Tanja Trarbach has received monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe research funding, consulting and speaker honoraria from Merck Serono and Amgen. combined immunodeficient engraftment of some acute lymphoblastic leukemia The other authors declare no conflict of interest. cell lines and primary leukemic samples. Cancer Res 2006; 66: 4843–4851.

Oncogene (2013) 2873 – 2881 & 2013 Macmillan Publishers Limited RAS-mediated antibody resistance S Kasper et al 2881 19 Strasser A, Harris AW, Vaux DL, Webb E, Bath ML, Adams JM et al. Abnormalities of induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 2006; 10: the immune system induced by dysregulated bcl-2 expression in transgenic mice. 389–399. Curr Top Microbiol Immunol 1990; 166: 175–181. 28 Wesarg E, Hoffarth S, Wiewrodt R, Kroll M, Biesterfeld S, Huber C et al. Targeting 20 Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Bax-deficient mice BCL-2 family proteins to overcome drug resistance in non-small cell lung cancer. with lymphoid hyperplasia and male germ cell death. Science 1995; 270:96–99. Int J Cancer 2007; 121: 2387–2394. 21 Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem R et al. Apaf1 is required 29 Shultz LD, Schweitzer PA, Christianson SW, Gott B, Schweitzer IB, Tennent B et al. for mitochondrial pathways of apoptosis and brain development. Cell 1998; 94: Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid 739–750. mice. J Immunol 1995; 154: 180–191. 22 Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S et al. EGFR mutations in 30 Ravi R, Fuchs EJ, Jain A, Pham V, Yoshimura K, Prouser T et al. Resistance of lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; cancers to immunologic cytotoxicity and adoptive immunotherapy via X-linked 304: 1497–1500. inhibitor of apoptosis protein expression and coexisting defects in mitochondrial 23 Huber C, Bobek N, Kuball J, Thaler S, Hoffarth S, Huber C et al. Inhibitors of death signaling. Cancer Res 2006; 66: 1730–1739. apoptosis confer resistance to tumour suppression by adoptively transplanted 31 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; cytotoxic T-lymphocytes in vitro and in vivo. Cell Death Differ 2005; 12: 317–325. 144: 646–674. 24 Marques CA, Hahnel PS, Wolfel C, Thaler S, Huber C, Theobald M et al. An immune 32 Schuler M, Maurer U, Goldstein JC, Breitenbucher F, Hoffarth S, Waterhouse NJ escape screen reveals Cdc42 as regulator of cancer susceptibility to lymphocyte- et al. p53 triggers apoptosis in oncogene-expressing fibroblasts by the mediated tumor suppression. Blood 2008; 111: 1413–1419. induction of Noxa and mitochondrial Bax translocation. Cell Death Differ 2003; 10: 25 Hahnel PS, Thaler S, Antunes E, Huber C, Theobald M, Schuler M et al. signaling 451–460. sensitizes cancer to cellular immunotherapy. Cancer Res 2008; 68: 3899–3906. 33 Kasper S, Breitenbuecher F, Heidel F, Hoffarth S, Markova B, Schuler M et al. 26 Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al. Targeting MCL-1 sensitizes FLT3-ITD-positive leukemias to cytotoxic therapies. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature Blood Cancer J 2012; 2: e60. 2005; 435: 677–681. 34 Remmele W, Stegner HE. [Recommendation for uniform definition of an immu- 27 van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabotar PE noreactive score (IRS) for immunohistochemical estrogen receptor detection (ER- et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently ICA) in breast cancer tissue]. Pathologe 1987; 8: 138–140.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

& 2013 Macmillan Publishers Limited Oncogene (2013) 2873 – 2881