Histone Deacetylase Inhibitor Sensitizes Apoptosis-resistant Melanomas to Cytotoxic Human T Lymphocytes through Regulation of TRAIL/DR5 Pathway

Abstract

Modern immune therapies [PD-1/PD-L1 and CTLA-4 checkpoints blockade, and adoptive cell transfer (ACT)] have remarkably improved the response rates of metastatic melanoma. These modalities rely on the killing potential of cytotoxic T lymphocytes (CTL) as proximal mediator of anti-melanoma responses. Mechanisms of tumor resistance to and the predominant cytotoxic pathway(s) employed by melanoma-reactive CTL are important outcome determinants. We hypothesized that down-modulation of death receptors in addition to aberrant apoptotic signaling might confer resistance to death signals delivered by CTL. To test these two hypotheses, we used an in vitro model of MART CTL resistant melanoma sublines. TCR transgenic and patient-derived CTLs employed the TNF-related apoptosis-inducing ligand (TRAIL) cytotoxic pathway, through DR5. Further, rhTRAIL and Drozitumab (anti-DR5 agonistic mAb) were used to explicitly verify the contribution of the DR5/TRAIL pathway in killing melanomas. CTL-resistance was due to DR5 down-regulation and an inverted ratio of pro- to anti-apoptotic molecules, both of which were reversed by the histone deacetylase inhibitor (HDACi) SAHA. Apoptosis negative (c-IAP-2 and Bcl-_xL) and positive (DR5) regulators were potential incriminators partly regulating CTL sensitivity. These pre-clinical findings suggest that exposure to this chromatin remodeling drug of immune-resistant melanomas can skew towards an intracellular pro-apoptotic milieu, increase death receptor expression, and overcome acquired immune-resistance.

Introduction

Various modern immune therapies – CTLA-4 and PD-1/PDL-1 blockade, and adoptive cell transfer (ACT) – have increasing effectiveness in producing clinical responses in metastatic melanoma. In all of these modalities, the proximal mediators of anti-tumor response are tumor-reactive cytotoxic T cells (1).

Advanced melanomas are composed of heterogenous cell populations: the variable sensitivity of individual melanoma cells to apoptotic death signals delivered by these modalities may account for the variability of anti-tumor responses. Thus, delineation of the underlying mechanisms of CTL-resistance, and strategies to reverse resistance and enhance sensitivity, could potentially inform clinical trials and improve response rates.

TNF-related apoptosis-inducing ligand, TRAIL (Apo2L), frequently employed by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, is a potent and selective apoptosis-inducer in tumors while sparing normal cells. TRAIL and agonistic antibodies to its death receptors are attractive therapeutic approaches. Their potential use has been broadly evaluated in various cancers including melanoma (2–5), which are currently being evaluated in clinical trials (6–9). TRAIL engages death-inducing TRAIL-R1 (DR4) and TRAIL-R2 (DR5), and decoy TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) receptors. Upon ligation of TRAIL with trimeric death receptors, the adaptor protein Fas-associated death domain (FADD), and initiator caspases (2, 8, 10) are recruited to the cytoplasmic death domain (DD) of the receptor forming the multimeric death inducing signaling complex (DISC). This leads to caspase-8 activation, Bid cleavage, and subsequent apoptosis either through direct caspase-3 activation (extrinsic pathway) or mitochondrial destabilization and apoptosome formation (intrinsic pathway) (4, 5, 10). Despite its potent anti-tumor activity, some tumors are inherently resistant or acquire secondary resistance to TRAIL.

Histone deacetylase inhibitors (HDACi) activate transcription of genes responsible for creating an intracellular proapoptotic milieu (11), render tumors more recognizable targets by the immune system, and are considered immune sensitizers (12). We have recently reported significant improvement of anti-tumor activity of ACT by HDACi in an in vivo melanoma model (13), providing the rationale for further examining the sensitizing effects of HDACi on CTL-resistant melanomas. The FDA approved class II HDACi SAHA increases surface death receptors and regulates the expression of apoptosis-associated genes. As single agent or combined with other agents, SAHA has some anti-melanoma activity in vitro and in vivo (12, 13).

Several melanoma lines, which present the melanoma antigenic epitope MART-1_27–35 in the context of HLA A*0201 and are sensitive to MART-specific CTL (F5 CTL) killing, were serially exposed to F5 CTL yielding completely resistant (R) sublines. These R sublines expressed intact MART-1/A*0201 complex, had reduced DR5 expression and an inversion of apoptotic genes programs favoring resistant phenotype. Pretreatment of R sublines with SAHA increased DR5 expression, restored the gene expression profile to favor an intracellular proapoptotic milieu, and restored CTL sensitivity of R sublines through TRAIL/DR5. This study provides rational molecular basis for combining small molecule sensitizing agents in modern melanoma immune therapy protocols.

Materials and Methods

Cell lines and sublines

Human melanoma lines were established from surgical specimens as described. The generation of F5 CTL R lines has been reported previously (14, 15). Briefly, P cells were grown in the presence of step-wise increasing numbers of F5 CTLs for a total of 8 weeks (2–3 weeks for each E:T). Thirty percent to 50% of melanoma cells survived the first cycle of selection (20:1, 2 weeks), percentage of which drastically reduced during subsequent selection cycles until no further killing was observed. Remaining viable melanoma cells were then subjected to two consecutive rounds of limiting dilution analysis. Single cells were propagated and maintained in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS). After immunoselection, sublines were maintained in medium containing excess (10:1) F5 CTLs, but were grown in F5 CTL-free medium at least 1 week prior to analysis. Cultures were incubated in controlled atmosphere incubator at 37°C with saturated humidity at 0.25 × 10⁶ cells/mL and were used at 50% to 70% confluency for each experiment. Cultures were routinely (once/month) checked for mycoplasma contamination (Lonza).

Reagents

Antibodies specific to MART-1 and anti-cytochrome C and smac/DIABLO were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and DAKO (Carpinteria, CA), respectively. Mouse anti-actin mAb was obtained from Chemicon. rhTRAIL was purchased from Peprotech (NJ). DR5, Bcl-xL and c-IAP-2 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DR5 expression plasmid and Drozitumab were kindly provided by Dr. Avi Ashkenazi (Genentech Inc., San Fransisco, CA) under Material Transfer Agreement (MTA). Blocking Abs and fluorochrome conjugated Abs for FACS analyses were purchased from eBiosciences (San Diego, CA). Suberoylanilide hydroxanic acid (SAHA) procured commercially, was diluted in dimethyl sulfoxide (DMSO). DMSO concentration did not exceed 0.1% in any experiment.

Quantitative real-time PCR (qPCR)

Samples were analyzed with iQ SYBR Green Supermix using iCycler Sequence Detection System (BioRad) using RT² profiler apoptosis PCR arrays. Total RNA was extracted from 10⁷ cells for each condition with RNeasy mini kit (Qiagen) and quantified by 3.1.2 NanoDrop ND-1000 spectrophotometer. Three micrograms of total RNA was reverse transcribed to first-stranded cDNA for 1 hour at 42°C with 200 units SuperScript II RT and 20µM random hexamer primers. Amplification of 2.5µL of cDNAs was performed using gene-specific primers. Expression of each molecule was calculated with the assumption that control samples were considered as 1.

Transduction of CD8 CTLs with F5 MART-1 TCRα/β retrovirus

Nonadherent population of healthy donor human peripheral blood mononuclear cells (PBMC) were cultured in AIM-V media supplemented with 5% human AB serum, αCD3 antibody (50 ng/mL), and IL-2 (300 IU/ mL) for 48 hours. CD3⁺CD8⁺ CTLs were isolated by EasyStep Negative Selection enrichment kits (Stem Cell Technologies, Vancouver, Canada) according to manufacturer’s instructions. CTLs were transduced with MSCV MART-1 TCR as described (14, 15). CD8⁺ CTLs with more than 95% MART-1 TCRα/β expression were used in all experiments.

Cell-mediated Cytotoxicity assay

Melanoma cultures were trypsinized for 5 minutes, washed once in cold PBS and labeled with 100 µCi of Na₂⁵¹CrO₄ for 1 hour (37°C/5%CO2). After 3× washes, 10⁴ cells were added to V-bottom 96-well plates and used immediately as described (14, 15). Percentage of specific ⁵¹Cr-release was measured as: % cytotoxicity = (experimental release − spontaneous release)/(total release − spontaneous release) × 100.

Immunoblot analysis

A total of 10⁷ cells were grown in complete medium (±inhibitors), lysed at 4°C in RIPA buffer [50 mmol/L Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 µmol/L NaCl] supplemented with protease inhibitor cocktail (Complete Mini; Roche) and subjected to standard immunoblot analysis as described (14–16). The relative intensity of bands, hence, relative alterations in expression was assessed by densitometric analysis of digitized images obtained from multiple independent blots. For confirmation, membranes were stripped and reprobed with anti β-actin mAb.

Evaluation of Active Caspase-3 Levels by Flow Cytometric Analysis

Melanoma cells were treated under the conditions explained for PI staining. At the end of the incubation period, the cells were washed once with ice-cold 1× PBS/0.1% BSA and were resuspended in 100 µl ice-cold 1× PBS/0.1% BSA. Fifty microliters of cell suspension (containing 2×10⁶ cells) were aliquoted to each sample and fixed with the perm/fix solution (PharMingen) for 20 min. Thereafter, the cells were washed twice with 1× perm/wash (PharMingen) solution and stained with the FITC-labeled anti-active caspase-3 antibody (1:1000 predetermined dilution) for 30 min (light protected). Thereafter, the samples were washed once with 1× perm/wash solution followed by flow cytometric analysis (Coulter Electronics, Miami, FL). As negative control, the cells were stained with isotype control (pure IgG1) under the same conditions described above (16).

Gene knock-down by small interfering RNA (siRNA)

Cells were seeded in the wells of 6-well plates in 2 mL antibiotic-free growth medium and incubated until cell confluency reached 50%. A total of 8–10 µL (40 nM) of various siRNA constructs (cIAP-2, Bcl-_xL, DR5) or a relevant amount of a control siRNA solution was mixed with 4 µL of Lipofectamin 2000 in OptiMeM solution (Invitrogen). After 6 hours supernatant was aspirated, 2 mL of fresh medium was added and transfection was performed for 80 hours according to the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, CA (14, 15)).

Statistical analysis

Assays were set up in duplicates or triplicates and results were expressed as mean ± standard error of the mean (SEM). Statistical analysis and P values were calculated by two-tailed paired t test with confidence interval (CI) of 95% for determination of significance of differences between treatment groups (P < 0.05: significant). ANOVA was used to test significance among the groups using InStat 2.01 software.

Results

MART TCR-transduced T cells (F5 CTL) induce apoptosis in melanoma targets through TRAIL/DR5 pathway

Human peripheral blood T cells, transduced to high efficiency (>95%) with a retroviral vector encoding MART TCR α/β chains (F5 CTL), efficiently recognize and kill three representative MART-1⁺/A*0201⁺ melanoma cell lines with cytotoxic indices of 43.4±3.2% (M202), 68.7±1.8% (M249), and 61.3±1.9% (M329) (Figure 1A). These melanoma lines were predominantly killed in a caspase-dependent manner and through DR5 receptor as blocking antibodies to DR5, but neither to DR4 nor FasR, inhibited killing (Figure 1B). Treatment of these targets with rhTRAIL or Fas agonistic antibody (CH-11) resulted in apoptosis induction by the former, but not the latter, as measured by active caspase-3 levels. In addition, antagonistic TRAIL Ab significantly reduced the cytotoxic potential of F5 CTL in ⁵¹Cr-release assays (Figure 1C). Blocking FasL on F5 CTLs did not affect their cytotoxic potential (data not shown). These results indicate that F5 CTL primarily use TRAIL/DR5 pathway in killing TRAIL-sensitive melanomas.

Figure 1. Death receptor-mediated killing of melanomas.

A. ⁵¹Cr-labeled parental and resistant (R) cells were incubated with F5 CTLs at various E:T ratios in 6-hour standard ⁵¹Cr-release assay. B. Caspase and death Receptor blockade. Parental cells were either left untreated or pretreated with zVAD-fmk (1µmol/L – 18 hours) or antagonistic anti–FasR, –DR4, and –DR5 (1–2µmol/L – 6 hours), coincubated with F5 CTLs and subjected to ⁵¹Cr-release assay. C. TRAIL killing of melanomas. Parental cells were incubated with (i) rhTRAIL or (ii) agonistic FasL Ab (CH-11) (5–25ng/ml–18hr) and percentage apoptosis was assessed by active caspase-3 levels using FACS analysis. The FasL sensitive DT140 murine lymphoma line was used as control. (iii) F5 CTL either left untreated or pretreated with TRAIL blocking Ab (1µmol/L–6hr) prior to coincubation with melanomas in ⁵¹Cr-release assay. Results are presented as mean ± SEM of duplicate samples (n = 3). * P values <0.05 significant.

CTL resistant melanomas have reduced DR5 expression

These three F5 CTL-sensitive melanoma cell lines – M202, M249, M329 – were subjected to serial cycles of in vitro exposure to these cytotoxic transgenic T cells to generate sublines resistant to killing (Figure 1A). Some of the characteristics of these cells have been described previously (14, 15) and the details for their generation are described in the Materials and Methods section. The respective resistant sublines (designated M202R1, M249R1, R2 and M329R1) retained surface expression of A*0201 and MART-1 expression, and were comparable to the parental sensitive cells in presentation of MART-1/A*0201 complex as measured by IFN-γ and IL-2 cytokine release recognition assay (Figure 2A). The three resistant sublines could not be killed with rhTRAIL (Figure 2B) and all had >50% reduction in levels of surface DR5 compared with their parental counterparts. Expression levels of surface DR4 and FasR very low and remained unchanged (Figure 2C). These findings raised the question whether CTL resistance in these cells might be due, in part, to DR5 down regulation.

Figure 2. Recognition of R cells by F5 CTLs.

A. Melanomas (10⁶) and F5 CTL were cocultured overnight at 1:1 and 0.5:1 E:T ratios. Amount of cytokine released was measured by ELISA. The MART-1⁻/A*0201⁻ melanoma line M238 and the myelogenous line K562 were used as control. B. rhTRAIL resistance. CTL R melanomas were treated with rhTRAIL (0–25 ng/ml –18hr) and apoptosis was evaluated by active caspase-3 levels. C. Death receptor surface expression. Melanomas were stained with anti–DR4, –DR5, –FasR fluorochrome-conjugated mAbs (1µmol/L) and subjected to FACS analysis. As negative control cells were stained with isotype control. Results are presented as mean ± SEM of duplicate (quadruplicate for ELISA) samples (n = 3). * P values <0.05 significant.

Adequate levels of DR5 expression are necessary for CTL killing but do not fully account for resistance

We transiently transfected the resistant cell line M202R1 with two concentrations of a DR5 expression plasmid (17), which while restoring DR5 surface expression to the levels seen in the parental M202 (and even above) (Figure 3A), did not render these cells sensitive to rhTRAIL killing (Figure 3B). Recognizing that these melanoma targets were killed through TRAIL/DR5, we knocked down DR5 levels in sensitive M202 melanoma cells using siRNA. As DR5 expression was reduced, somewhere between 70%–40% of normal levels, we observed a precipitous drop in TRAIL-induced apoptosis (Figure 3C). These results, taken together, suggest that resistant cells could not be killed regardless of the levels of DR5 expression, whereas otherwise sensitive cells required adequate levels of DR5 for killing. Thus, DR5 expression in M202R –about 40% of that of the parental M202– clearly contributes to CTL-resistance, but is not the full explanation.

Figure 3. Role of DR5 in killing.

A. M202R1 cells were transfected with DR5 expression plasmid (1, 10ng) using standard techniques. Surface DR5 expression was measured by FACS analysis. Cells were then treated with rhTRAIL (5–25ng/ml-18hr) and were subjected to apoptosis assay measured by active caspase-3 levels. M202 cells were used as control. Results are presented as mean ± SEM of duplicate samples (n = 2). C. DR5 knockdown. M202 parental cells were transfected with DR5 or scrambled siRNA, subjected to immunoblot for confirmation of specific gene-silencing, apoptosis (active caspase-3) assay, and DR5 expression by FACS analysis (presented as %MFI). Cells at 50–60% confluency were used for transfection. Results are representative of two independent experiments.

Pretreatment of resistant melanomas with the HDACi SAHA restores their sensitivity to killing

Pretreatment of the resistant melanoma sublines with SAHA (1µmol/L – 48hr) restored their sensitivity to killing by TCR transgenic MART CTL. SAHA also sensitized the resistant cells to killing by two patient-derived MART-specific CD8⁺ CTL, by rhTRAIL and DR5 agonistic mAb Drozitumab (p < 0.05) (8) (Figure 4A–D, Suppl. Figure 1A–E). Anti-DR5 antagonistic mAb (2µmol/L–6hr) significantly reduced CTL sensitivity of SAHA-pretreated R sublines (Figure 4E, Suppl. Figure 1F).

Figure 4. SAHA-mediated sensitization of CTL R sublines to killing.

Cells (10⁶) were left either untreated or pretreated with SAHA (1µmol/L – 48hr) and used in killing assay using A. TCR transgenic, B. two patient-derived MART-1 specific CTLs as effectors (⁵¹Cr-release assay), C. rhTRAIL (0–25ng/ml) or B. cross-linked Drozitumab (0–50ng/ml) for 18 hours; apoptosis was evaluated by active caspase-3 levels. E. DR5 blockade. SAHA-pretreated CTL R cells were washed, incubated with antagonistic DR5 mAb (2µmol/L – 6hr) and used in cytotoxicity assays. Results are presented as mean ± SEM of duplicate samples (n = 3). * P values <0.05 significant.

Kinetics of SAHA-mediated immunosensitization

SAHA-mediated sensitized M202R1 cells became progressively resistant over 72hrs period after drug withdrawal (Figure 5A). DR5 surface expression paralleled CTL sensitivity (Figure 5B), reinforcing a strong correlation between SAHA-mediated DR5 induction and CTL sensitivity. Treatment of both the parental and SAHA-treated CTL-resistant cells with MHC-I blocking mAb (1µmol/L-20min) significantly reduced the recognition (Figure 5C) and killing (Figure 5D) of melanomas by F5 CTL. These results indicate that F5 CTLs recognize melanomas in an MHC-I restricted fashion. Obviously, in the absence of recognition, MART-1/A*0201-specific F5 CTLs fail to induce cytotoxicity in melanoma tumor targets.

Figure 5. Kinetics of SAHA effects on melanomas.

M202R1 cells were grown in media supplemented with SAHA (1µmol/L – 48 hours). The cells were then grown in SAHA-depleted media for various intervals (24–72hr), subjected to A. ⁵¹Cr-release assay suing F5 CTL as effectors. B. stained with anti–DR5 mAb and subjected to FACS analysis. Results are presented as mean ± SEM of duplicate samples (n = 3). Melanomas were either left untreated or treated with SAHA (1µM -48hr). The cells were then treated with anti-MHC I blocking Ab (1µM-20 min) and used in C. recognition assay as measured by IFN-γ release of overnight cocultures of melanomas and F5 CTLs (1:1 E:T ratio), or D. incubated with various E:T ratios of F5 CTLs in a 6h ⁵¹Cr-release assay. Results are presented as mean ± SEM of duplicate samples (n = 2). * P values <0.05 significant.

Mechanism of SAHA action in restoring CTL sensitivity

SAHA treatment of resistant melanoma cell lines largely restores levels of DR5 expression (Figure 6A), and with kinetics that closely parallels those in killing assays. DR5 overexpression data (Figure 3) argue against this being the only operative mechanism. However, when DR5-overexpressed cells were treated with SAHA, CTL sensitivity was restored (Figure 6B, C). These data suggest that the sensitizing effect of SAHA is mediated via at least two mechanisms: induction of surface DR5 and inhibition of intracellular resistant factors. From our previous work (14, 15), we identified inverted apoptotic signaling pathways as an important mechanism to CTL-induced apoptosis. Focused apoptosis qPCR arrays showed expression patterns of several categories of apoptotic genes were altered in the CTL R cells compared to parental cells, consistent with an anti-apoptotic resistant phenotype (Table 1). Treatment of these CTL-resistant sublines with SAHA increased the expression levels of positive regulators of apoptosis, caspases, TNF/TNFR family and death domain proteins and decreased the expression levels of several negative regulators of apoptosis (Table 1, Supplemental Table 1). These events ultimately force the generation of a proapoptotic intracellular environment.

Figure 6. DR5 induction alone is insufficient for killing.

Resistant melanomas were stained with anti–DR4, –DR5, –FasR fluorochrome-conjugated mAbs (1µmol/L) and subjected to FACS analysis. As negative control, cells were stained with isotype Ig control. DR4 and Fas R expression levels were low and SAHA did not increase their expression. B. DR5 overexpressing M202R1 cells (adopted from figure 3) were grown in complete media (±SAHA) and subjected to C. cytotoxicity assay. M249(CTL R1) cells were left either untreated or treated with cIAP-2 or Bcl-_xL siRNA or DR5 overexpression constructs. D. Efficacy of gene knock-down (KD) of two independent experiments is shown by western blot. E. Cells were then incubated with rhTRAIL (15ng/ml-18hr) and cytotoxicity was assessed by measuring levels of active caspase-3. Results are presented as mean ± SEM of duplicate samples (n = 3). Untreated M249(CTL R1) and M249 cells were used as control. * P values <0.05 significant.

Table 1.

Differential expression of apoptotic genes in M249 CTLR cells and their regulation by SAHA as measured by real time quantitative PCR-based apoptosis array

	Gene name	CTL R vs. Parental mRNA fold change	CTL R + SAHA vs. CTL R mRNA fold change
Negative apoptosis regulator	ACC-1/ACC2 (A1)	4.43	3.05
	Bcl2L1 (Bcl-xL)	3.31	−5
	Bcl-B/BOO (Bcl2L10)	2.68	6.6
	Bcl-W (Bcl-2 like)	4.04	−6.22
	BAR/RNF47	−2.5	4.5
	AIP/AIP2 (BIRC3)	13.03	−18.11
	BNIP-2/NIP2	−2.8	2.8
	AKT	−2	5.25
Positive apoptosis regulator	Apaf-1	−2.1	4
	Bcl2L8 (BAD)	−5.1	8.15
	BAK	−3	5.3
	Bcl2L4 (Bax)	−5.3	5
	DFF-45/DFF1	−2.54	4.41
	TNFSR6/Apo-1 (Fas)	−4.42	3.5
	CD18/D12S370 (LTβR)	−3.6	5
	TNFSF10 (TRAIL)	−23.05	7.3
	TRAF4/LART-1	−3	4.5
	DP5/Harakiri (BH3 only)	−6.52	6.72
	LTα/TNFβ	6.1	−10.1
	DAPK/DKF2P	−3.62	4.5
Caspase activator	CINCIN1/CARD6	−2.51	3.9
	ICE/IL1BC (Casp1)	−4.54	13.1
	CASP-2/ICH1L	−2	7.3
	ICEREL11 (Casp4)	3.01	2.51
	ALPS2B/CAP4 (Casp8)	−2.3	4.81
death domain	GIG3/MORT-1 (FADD)	−2.8	6.9
	TNFRSF10B (TRAIL DR5)	−4.5	5.14
	TRADD	−2.15	3.5
	TRAF2/TRAD	−3.02	3.81
DNA damage	TRP53 (p53)	−6.39	8.9
	p73	−3.04	3.1

To further identify apoptotic gene(s) products potentially responsible for enhanced sensitivity observed by SAHA, we performed siRNA gene knock-down (KD) experiments on two anti-apoptotic molecules (Figure 6D). In DR5 overexpressing CTL R cells, c-IAP-2 and Bcl-_xL knock-down alone, or Bcl-2 family inhibitor (2MAM-A3) slightly enhanced TRAIL sensitivity. TRAIL sensitivity of c-IAP-2 and Bcl-_xL double KD resistant cells was superior to individual KD; an effect that was modestly potentiated by 2MAM-A3 (Figure 6E, Suppl. Figure 2). These results further confirm that simultaneous down-regulation of negative (e.g., Bcl-_xL and c-IAP-2) and upregulation of positive (DR5) apoptotic regulators are required to overcome TRAIL-resistance.

We also observed cytosolic accumulation of cytochrome c and Smac/DIABLO (Suppl. Figure 3A lanes 4 and 8), which paralleled their mitochondrial depletion (lanes 12 and 16), as well as mitochondrial depolarization (Suppl. Figure 3B) only by combination of SAHA plus rhTRAIL or Drozitumab, indicating that combination treatment reduces mitochondrial membrane potential (ΔΦm), induces mitochondrial collapse and cytosolic release of apoptotic molecules.

Discussion

Resistance to apoptotic death signals delivered by cytotoxic T cells is likely to become an important limiting factor with increasingly effective immune therapies. We report herein that TCR trangenic CTLs primarily use the TRAIL apoptotic pathway, via DR5 receptor, in killing sensitive melanomas. CTL-resistant melanomas had both DR5 down-regulation and aberrant expression of apoptotic molecules. Clinically achievable concentration of the FDA approved chromatin remodeling drug SAHA increased surface DR5 expression, favorably inverted the expression of apoptotic genes, and restored the sensitivity of R melanomas to MART CTL. TCR transgenic MART CTLs were used as a reliable and reproducible source of highly potent melanoma-reactive CTLs. Acquisition of CTL-resistance and its reversal by SAHA was also confirmed by using more clinically relevant naturally occurring T cells expressing endogenous MART-1 TCR derived from metastatic melanoma patients. Further, rhTRAIL and Drozitumab were used to explicitly verify the contribution of the DR5/TRAIL pathway in killing melanomas. Apoptosis negative (c-IAP-2 and Bcl-_xL) and positive (DR5) regulators were potential candidates partly regulating CTL sensitivity. Combination of SAHA and rhTRAIL or Drozitumab destabilized mitochondria and facilitated cytosolic release of apoptotic molecules.

Immune effector cells eradicate tumor cells mainly by apoptosis. Tumors, in turn, have adopted various apoptosis-resistant mechanisms. Alterations in gene expression and cell signaling dynamics account for resistance to specific CTL-killing, where selective targeting of aberrant pathways or apoptosis-related proteins can overcome resistance to immune effector mechanisms (18–23). Expression of theses resistant factors is regulated by the NF-ΦB, MAPK, PI3/AKT, JAK/STAT and other signal transduction pathways that are constitutively activated/deregulated in melanomas (24, 25). Our results indicate that continuous exposure to melanoma-reactive CTLs results in the generation of R cells with diminished DR5 expression and altered apoptotic gene profile (consequence of NF-ΦB and ERK1/2 activation (14, 15)). Loss or down-modulation of death receptors and alterations in the ratio of pro- to anti-apoptotic gene products are the most frequently observed TRAIL-resistance mechanisms (10, 26, 27). In fact, superior contribution of DR5 than DR4 to TRAIL killing is reported (28). Thus, an agent that simultaneously upregulates DR5 and modulates apoptotic gene programs, to favor a sensitive phenotype, can potentially restore CTL sensitivity.

Chromatin remodeling drugs including SAHA increase acetylation of both histones and cytoplasmic proteins, thus, exerting multiple effects on gene transcription and protein function (29) specifically: transcribing genes responsible for creating an intracellular pro-apoptotic milieu (11), increasing expression of: surface death receptors (30–32), MHC molecules (33), tumor Ags recognized by CTL (34), and NK ligands recognized by NKG2A (35, 36), while having minimal toxicity to normal cells (34). Epigenetic drugs render tumors more recognizable targets by the immune system (37), thus, could serve as immune sensitizers (12). Significant enhancement of anti-tumor activity of ACT by HDACi in an in vivo melanoma model was recently reported by our group (13), therefore, in this study we examined whether SAHA can regulate the TRAIL pathway and sensitizes CTL-resistant melanomas.

While DR5 played an essential role in killing sensitive melanomas, surprisingly, its overexpression alone was insufficient for restoring sensitivity. Sensitization was evident only when DR5 overexpressed R cells were exposed to SAHA. Downregulation of anti-apoptosis genes (e.g., IAP and Bcl-2 family, AKT), concurrent with upregulation of DR5 as well as other positive regulators of apoptosis [e.g., caspases and caspase activators (e.g., CARD6, Casp-1, 2, 4, 8), death domain proteins (FADD, DR5, TRADD, TRAF2), TRAIL, TNF superfamily members (TNF, LTA, TNFRSF)], DNA damage molecules (TRP53, p73), and apoptosis inducers (e.g., Bax, Bad, Bak) suggests that SAHA sensitizes the R melanomas via combinatorial cooperation among several groups of apoptotic genes. However, cell fate is ultimately determined by an imbalance in the ratio of pro- to anti-apoptotic proteins. In fact, knock-down studies revealed that reduction of various anti-apoptotic proteins alone or combined with Bcl-2 family inhibitor fail to significantly enhance TRAIL sensitivity. Exposure of these cells (double KD ± 2MAM-A3) to SAHA overcame the resistance indicating the necessity of additional gene regulatory effects of SAHA. While DR5 overexpression alone was insufficient, knock-down of both anti-apoptotic genes in DR5 overexpressing R cells (±2MAM-A3) enhanced TRAIL sensitivity. Failure of these manipulations to fully restore TRAIL sensitivity of R cells at the levels of parental cells indicates that concurrent enhanced expression of DR5 and reduced expression of Bcl-_xL and c-IAP-2 are required, but insufficient, to fully overcome resistance. Yet other unidentified resistant factor(s), whose regulation is modified by SAHA, are involved. Obviously, SAHA favors the generation of a pro-apoptotic milieu, which predestines melanomas to die upon receiving CTL-delivered death signals. Consistent with our observations, death receptor induction, caspase activation, and reduction of anti-apoptotic Bcl-2 members by SAHA sensitizes various human tumors to TRAIL (38–43).

TCR transgenic and patient-derived MART CTL recognition of parental (14, 15) and SAHA-treated R cells was accomplished in an MHC-I restricted manner. Tumor recognition via TCR is a prerequisite for the subsequent delivery of death signals by various means (TRAIL, FasL, perforin/granzyme). In the absence of peptide/MHC recognition and conjugate formation, TRAIL expressing MART-1/A*0201 specific CTLs (patient-derived as well as TCR engineered) fail to engage DR5 and transmit the death signal. Hence, a reduction in killing is observed by MHC-I blockade. The partial involvement of the perforin/granzyme pathway is plausible; blocking the TRAIL pathway by various approaches did not entirely abrogate killing. Consistent with reports by other investigators (44–48), our data, however, indicate TRAIL being the predominant cytotoxic pathway preferentially employed by MART CTLs.

In conclusion, TCR transgenic and patient-derived CTL mainly use the TRAIL pathway in killing melanomas, exclusively via DR5 receptor. CTL-resistance was due to DR5 down-regulation and aberrant expression of apoptotic molecules. Brief exposure to the HDACi SAHA increased surface DR5 expression, regulated the expression of apoptotic molecules, and restored the sensitivity of R melanomas to patient-derived MART-specific and TCR transgenic CTL, rhTRAIL, and Drozitumab. Anti-apoptotic cIAP-2 and Bcl-_xL, and proapoptotic DR5 are potential candidates partly regulating CTL sensitivity. Further, combination of SAHA and rhTRAIL or Drozitumab destabilized mitochondria and facilitated apoptosis to proceed. These results are encouraging as one can safely design and implement a clinical trial using a combination of ACT, rhTRAIL (dulanermin), or Drozitumab [both being clinically evaluated (4, 7–9, 49, 50)], in conjunction with SAHA, to reduce the apoptosis threshold of melanomas and to achieve better clinical outcomes.