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Monoagent DNA-alkylating chemotherapies like dacarbazine are among a paucity of medical treatments for advanced carcinoid tumors, but are limited by host toxicity and intrinsic chemoresistance through the base excision repair (BER) pathway via poly (ADP-ribose) polymerase (PARP). Hence, inhibitors of PARP may potentiate DNA-damaging agents by blocking BER and DNA restoration. We show that the PARP inhibitor ABT-888 (Veliparib) enhances the cytotoxic effects of dacarbazine in carcinoids. Two human carcinoid cell lines (BON and H727) treated with a combination of ABT-888 and dacarbazine resulted in synergistic growth inhibition signified by combination indices <1 on the Chou-Talalay scale. ABT-888 administered prior to varying dacarbazine doses promoted the suppression of neuroendocrine biomarkers of malignancy ASCL1 and CgA, shown by Western analysis. ATM phosphorylation and p21Waf1/Cip1 activation, indicative of DNA damage, were increased by ABT-888 when combined with dacarbazine treatment, suggesting BER pathway attenuation by ABT-888. PE Annexin V/7-AAD staining and sorting revealed a profound induction of apoptosis following combination treatment, which was further confirmed by increased PARP cleavage. These results demonstrate that ABT-888 synergizes dacarbazine treatment in carcinoids. Therefore, ABT-888 may help treat carcinoids unresponsive or refractory to mainstay therapies.
Carcinoids are heterogenous neuroendocrine tumors (NETs) that arise from the body’s enterochromaffin cells and vary in anatomical site. They are most commonly found in the gastrointestinal tract, with the small intestine being the site of highest occurrence, followed by those of the lung, comprising about 2% of all bronchopulmonary tract tumors1, 2. Carcinoids can occur either sporadically or as part of hereditary syndromes, but are collectively rare with an age-adjusted incidence of about 2–4 per every 100,000 people1, 3, 4. Interestingly, the incidence of small bowel and bronchopulmonary carcinoids appears to have increased over a recent five-decade period per pan-SEER reports, possibly from improvements in surveillance or secondary to an evolving disease. A significant portion of carcinoids present with distant metastases of which about half have primaries of unknown origin4. In fact, carcinoids are among the most prominent causes of isolated hepatic metastasis, second only to colorectal cancer.
Surgical resection can offer definitive cure in the absence of metastatic disease, though no current data exists supporting adjuvant systemic therapies. Cytoreductive resection may be considered when intent is palliative and complete resection is not possible. In such cases, chemotherapies such as somatostatin analogs have been demonstrated to abate hormonal symptoms and also improve time-to-progression5. Cytotoxic agents are typically reserved for tumors with high proliferative indices (Ki-67≥5%), historically involving combined 5-fluorouracil or doxorubicin with the alkylating agent streptozocin6, 7. An alternative alkylating agent known as dacarbazine along with its oral, less toxic formulation temozolomide have also shown moderate activity in advanced NETs in addition to melanoma and glioma8–12. Notably, prospective studies of dacarbazine-inclusive polychemotherapy have demonstrated limited efficacy among carcinoids relative to other NETs13–17. Dacarbazine exerts its effect by methylating the O6-guanine position of its target DNA thereby causing mismatch repair and eventual cell death. More commonly, however, dacarbazine methylates the N7-guanine and N3-adenine position, comprising 70% and 9% of adducts respectively, and which may be removed by base excision repair (BER) pathway18. Hence, through its ability to restore DNA to its normal state, robust BER activities have been associated with dacarbazine resistance18, 19.
The BER pathway is carried out by the enzyme poly (ADP-ribose) polymerase (PARP), a nick-sensing enzyme that recruits BER complex proteins to double stranded DNA break sites to initiate repair following base excision of, for instance, N7 and N3 adducts20. Inhibitors of PARP have been thus developed with the intent of circumventing dacarbazine resistance by blocking BER and promoting N7 and N3 methylation-induced cell death20, 21. This approach has been successfully demonstrated in malignancies of pulmonary, colonic, glial and hematopoetic origin both in vivo and in vitro22–27. A novel PARP inhibitor ABT-888, also known by its trade name Veliparib, has been shown to potentiate DNA-damaging chemotherapies in advanced solid tumors28–32. Importantly, studies have supported the use of ABT-888 in combination with alkylating agents like dacarbazine and its sister drug temozolomide across a spectrum of cancers including glioblastoma, leukemia, hepatocellular carcinoma, and metastatic melanoma with recent corroboratory clinical investigations33–43.
Given the clinical refractoriness of carcinoids to dacarbazine-based therapies and mounting evidence regarding resistance mechanisms implicating DNA-damage responses, we sought to investigate the in vitro interaction between ABT-888 and dacarbazine in carcinoid cell lines.
Human gastrointestinal carcinoid cells (BON), were gifted by Drs. Courtney M. Townsend, Jr. of the University of Texas Medical Branch (Galveston, TX, USA) and B. Mark Evers of the University of Kentucky (Lexington, KY, USA). Human bronchopulmonary carcinoid (H727) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). BON and H727 cells were grown in DMEM/F-12 (Life Technologies, Grand Island, NY, USA) and RPMI/F-12 (Life Technologies, Grand Island, NY, USA), respectively, at a 5% CO2 and 37°C atmosphere. Media was supplemented with 100 IU/mL penicillin, 100µg/mL streptomycin (Life Technologies, Grand Island, NY, USA) and 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). ABT-888 (Selleck Chemicals, Houston, TX, USA) and dacarbazine (Sigma-Aldrich) were stored in aliquots of 10mM in DMSO at -80°C, and freshly thawed before use. Cells were plated at sub-confluency the day prior to treatment, and then incubated in fresh medium containing ABT-888 (0–10µM) for 24 hours, after which dacarbazine was added (0–1000µM) for 2 additional days. DMSO concentrations were normalized across all treatment groups.
Total BON cell lysates following dacarbazine ± ABT-888 treatment were prepared and analyzed by Western blotting as previously described44. Each antibody was diluted as follows: 1:2000 for mammalian achaete-scute complex-like1 (ASCL1) (BD Pharmingen, San Diego, CA, USA), 1:3000 for chromogranin A (Zymed Laboratories, San Francisco, CA, USA), 1:10,000 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA), and 1:1000 for p21Waf1/Cip1, cleaved poly (ADP-ribose) polymerase (PARP), phosphorylated ATM, total ATM, and Survivin (Cell Signaling Technology, Beverly, MA, USA). Antibody signals were detected using Supersignal West Femto, Dura, or Pico (Pierce, Rockford, IL, USA) chemiluminescence systems and manufacturers’ instructions were adhered to.
BON and H727 cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) rapid colorimetric assay. Both cell lines were seeded in 96-well plates, and treatment groups were plated in sextuplicate. To assess viability following treatment, media was replaced with 25µL of 0.5µg/mL MTT in serum-free media for 3.5 hours at 37°C, followed by addition of 75µL DMSO before measuring optical densities. The remainder of our protocol was followed as previously described45.
BON cells treated with dacarbazine (0–600µM) ± ABT-888 (0-10µM) were collected and incubated with PE Annexin V and 7-Aminoactinomycin D (7-AAD) fluorescein solutions (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s protocol. The FACSCalibur™ (BD Biosciences, San Jose, CA, USA) fluorescent-activated cell-sorting (FACS) instrument was used for quantitative fluorescent sorting, and FlowJo v10.0.8 (TreeStar Inc., Ashland, OR, USA) was used for subsequent analysis.
Student’s t-test was used to compare means between groups and all data are represented as mean ± SEM. CompuSyn® (Paramus, NJ, USA) was used to calculate combination indices (CI) following dacarbazine ± ABT-888 treatment as per the Chou-Talalay method for drug interactions. CI of greater than, equal to, or less than 1 signify antagonistic, additive, or synergistic interactions respectively between two treatments46.
We set out to determine if BON and H727 cells could be sensitized to dacarbazine with the addition of ABT-888. We observed that both BON and H727 prolifieration were dose-dependently suppressed following dacarbazine doses of up to 1000µM, with IC50 values of 218.2µM and 268.6µM, respectively. To determine the correct dose range for concomitant ABT-888 treatment, its monotherapeutic effects were first established using viability assays. ABT-888’s IC50 in BON and H727 exceeded 50µM, with near 92% of cells still viable at 20µM. We conservatively chose ABT-888 doses of 5µM and 10µM for combination treatment with dacarbazine, as these exerted minimal cytotoxic effect alone, and were within the range necessary, as previously reported, to inhibit its molecular targets and achieve combinatorial benefits37, 39, 43. Co-treatment of BON cells with 5µM ABT-888 potentiated dacarbazine-induced cytotoxicity as indicated by CI<1 following 500µM dacarbazine and above. When ABT-888 co-treatment was increased to 10µM, cells appeared further sensitized to dacarbazine, with CIs falling <1 following dacarbazine doses of 400µM and above (Table 1). H727 cells responded similarily, with improved sensitization to dacarbazine at 10µM ABT-888 relative to 5µM. Doses of dacarbazine above 750µM and 400µM interacted synergistically with 5µM and 10µM ABT-888, respectively, signified by CI<1 (Table 2). These curves are represented in Figure 1A-B.
To determine the influence of combinatorial ABT-888 and dacarbazine treatment on carcinoid bioactivity we next investigated its effect on expression of neuroendocrine biomarkers of malignancy chromogranin A (CgA), and the basic helix-loop-helix transcription factor achaete-scute complex-like1 (ASCL1), biologically relevant markers of neuroendocrine tumors. CgA is a glycopeptide inherent to neuroendocrine tissue and often used clinically as a biomarker for disease prognosis47. ASCL1 is an evolutionarily conserved transcription factor central to neuroendocrine development and highly expressed in carcinoids48. As shown in Figure 1C, ASCL1 and CgA expression responded to dacarbazine monotherapy at doses up to 600µM in BON cells, with CgA only moderately suppressed and ASCL1 reduction more pronounced. This dose range for dacarbazine was chosen since it encompassed both the IC50 in BON cells, as well as the window within which combination indices reflected synergy (Table 1–2). Following ABT-888 co-treatment, CgA expression was reduced markedly further, and ASCL1 expression was near entirely diminished (Figure 1C). Collectively, these data indicate that ABT-888 potentiates the anticancer effects of dacarbazine on the neuroendocrine phenotype of carcinoids.
Through its mechanism of action, ABT-888 is purported to enhance apoptotic induction by impairing the mechanisms that mend dacarbazine-induced DNA damage37. To assess the extent of apoptosis in response to ABT-888 and dacarbazine, PE Annexin V/7-AAD was used to probe BON cells following 72 hours of treatment (as described in the Methods section). The percentage of cells in apoptosis (upper right quadrant), preapoptosis (lower right quadrant), necrosis (upper left quadrant) and those still viable (lower left quadrant) were quantified using flow cytometry. Experimental replicates were then averaged and are conveyed in the adjacent bar graph (Figure 2A). ABT-888 alone showed no appreciable increase in apoptosis. However, cells treated with ABT-888 and dacarbazine exhibited marked apoptotic induction relative to dacarbazine alone, almost approaching significance (p=0.07). To corroborate this, the degree of PARP cleavage was assessed following combinatorial treatment. Figure 2B shows considerable induction of cleaved PARP expression in BON cells treated with increasing dacarbazine doses in the setting of ABT-888 co-treatment, while dacarbazine alone only produced modest PARP cleavage. Once again, ABT-888 alone had no effect. These data further suggest that ABT-888 sensitizes cells to dacarbazine cytotxocity and apoptosis.
To further elucidate the mechanism by which ABT-888 and dacarbazine were inducing cell death, we next examined its effect on components of the ataxia telangiectasia mitogen factor (ATM)-mediated DNA-damage response, including ATM and p21Waf1/Cip1. Upon DNA damage, ATM is autophosphorylated and activates several downstream checkpoint proteins, ultimately leading to cell cycle arrest49, 50. Through its delayed response, ATM phosphorylation also leads to activation of p21Waf1/Cip1 and other p53 target genes, ultimately causing cell cycle arrest or apoptosis51. We observed that combination treatment increased levels of phospho-ATM and p21Waf1/Cip1, suggesting DNA damage from ABT-888 and dacarbazine treatment. Additionally, combination treatment potentiated suppression of the Inhibitor of Apoptosis protein Survivin, a marker of cancer cell malignancy that binds to and inhibits caspase cleavage, blocks G2/M phase progression and thereby promotes cell replication52. Together these data indicate that DNA damage occurs secondary to ABT-888 and dacarbazine combination treatment, thus mediating apoptotic induction (Figure 3).
Despite recent advances in targeted therapies, there is limited consensus on standardized treatment approaches for metastatic carcinoid tumors53. In the setting of distant spread, 5-year survival is only about 38.5% compared to 78.2% for local disease, based on late SEER data1. Patients with disseminated bronchopulmonary carcinoids fare very poorly as well with survival approaching only 15% after 5 years, as do those with atypical pulmonary carcinoids with only 25% mortality54. Therapeutic management of carcinoids is also made challenging given their long natural histories raising concerns for treatment toxicity. Carcinoids are known to be slow-growing, insidious cancers that follow a subtle yet malignant clinical course. Neuroendocrine in origin, they comprise cells packed with neurosectretory granules containing biogenic and vasoactive peptides including seratonin, histamine, and gastrin, that upon release lead to the debilitating carcinoid syndrome4. Other markers of neuroendocrine malignancy include chromogranin A (CgA), and the neuroendocrine-specific transcription factor ASCL147, 55. Cytotoxic therapies have been thought to play a role in treatment of patients with locally advanced or metastatic tumors wherein surgical resection is not curative7.
In most carcinoid patients, agents like dacarbazine and its sister drug temozolomide have shown little if any benefit compared to patients with other NETs. One particular study showed that only 1 of 14 carcinoid patients achieved objective response to temozolomide and thalidomide therapy14. A subsequent study similarily revealed no improvement among carcinoid patients receiving temozolomide and bevacizumab, while 33% of pancreatic NET patients achieved tumor response15. Temozolomide-based combination therapy was later associated with an only 2% partial or complete response among carcinoid patients compared to 34% of those with pancreatic NETs13. While other studies have associated monotherapy and combinatorial temozolomide regimens with up to 70% efficacy for pancreatic NETs, this approach has generally failed to show efficacy in carcinoids16, 17. Studies have attributed the treatment-refractoriness of carcinoids to its discrepant expression of mismatch repair mechanisms that may confer resistance. Archival specimens of carcinoids resistant to temozolomide were less likely to be deficient in the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) than NETs responsive to treatment13. Since temozolomide’s and dacarbazine’s mechanism relies upon DNA methylation at the O6-guanine position causing DNA mismatch and subsequent apoptosis, sensitivity to its effects would improve in the setting of impaired MGMT activity as demonstrated in patients with advanced glioblastomas and melanomas56–58. The BER pathway has also been acknowledged as a major contributor to temozolomide resistance in other cancer models, but moreover, its disruption in conjuction with temozolomide has been shown to sensitize cells to treatment18, 19, 22–27. Pharmacological inhibiton of BER by the PARP inhibitor ABT-888 has been demonstrated to enhance the antitumor effects of dacarbazine and temozolomide, and have advanced to phase I and II clinical trials for pediatric and adult gliomas, hepatocellular carcinoma, and metastatic melanoma with mixed success33–42. To improve carcinoid susceptibility to alkylating agent therapy for such candidate patients, we investigated the ability of the PARP inhibitor ABT-888 to enhance the activity of dacarbazine in carcinoids.
Our data show that in gastrointestinal and bronchopulmonary carcinoid cell lines, ABT-888 effectively sensitized cells to dacarbazine cytotoxicity, generating combination indices <1 (Tables 1–2) signifying synergistic interaction, and reduced the expression of neuroendocrine biomarkers of malignancy in BON cells (Figure 1). In addition to potentiating apoptosis (Figure 2), ABT-888 co-treatment profoundly increased levels of phosphorylated ATM and p21Waf1/Cip1, indicating induction of the ATM-mediated DNA-damage response pathway50 (Figure 3). Activation of ATM following DNA damage in response to PARP blockade is central to recruitment of DNA repair proteins, as has been illustrated following pharmacological inhibition of PARP59. Other reports have also reported on PARP’s participation in the DNA-damage response triggering ATM phosphorylation and recruitment60, 61. Relevant the interpretation of our findings, Tanaka et al. demonstrated that phospho-ATM is an accurate indicator of DNA damage following chemotherapy-induced apoptosis49. Further aligned with our study, Liu et al. reported that the extent of cytotoxicity following ABT-888 and temozolomide treatment in several cancer lines was proportional to the degree of DNA damage as represented by levels of ATM’s immediate target γH2AX43. Additionally, as a downstream target of p53 activation following ATM phosphorylation, p21Waf1/Cip1 is a key regulator of G2/S checkpoint passage by maintaining G2-phase arrest in the setting of cellular stress51. Of note, its expression has been shown to be a direct function of DNA damage given its inextricable link to the ATM-mediated DNA repair process62. Previous reports have confirmed that pharmacologic inhibitors of PARP including ABT-888 cause a G2/S phase arrest state during replicative stress, confirmed by an upregulation of p21Waf1/Cip1 63–65. Combined with the simultaneous suppression of the Inhibitor of Apoptosis gene Survivin alongside PARP cleavage, these results strongly suggest that ABT-888 optimizes dacarbazine-induced cytotoxicity at synergistic doses by inducing DNA damage and subsequent alteration in cell cycle kinetics, suggested by ATM pathway activity, to promote cellular demise.
Common treatment strategies for managing locally advanced or metastatic carcinoids, although not standardized, have employed the use of alkylating agents such as streptozocin-based regimens that are FDA approved for these indications7. However, given carcinoids prolonged performance status and indolent growth pattern, the routine use of such treatment approaches often raises toxicity concerns despite their purported therapeutic potential53. Even the alternative alkylating agent temozolomide, which has been explored clinically for treating NETs including in phase II trials, has resulted in limited therapeutic benefit in carcinoids specifically, as described in the above discussion13–17. Clinical challenges surrounding temozolomide and similar cytotoxic drugs are reflected by a study by Ramirez et al. who although endorse survival benefits from temozolomide and capecitabine therapy in metastatic NET patients, add that adverse reactions forced dose reductions in 24% of patients66. Therefore, translating these treatments into effective and rational therapeutic regimens is challenging, particularly due to the long natural histories of carcinoids making formal treatment comparisons difficult to devise. Alternatively, the use of compounds designed to sensitize patients to lower doses of cytotoxic therapies may be of value in this setting. Inhibitors of DNA repair like ABT-888 designed to enhance the therapeutic indices of alkylating agents may offer an option to circumvent long-term toxicity and improve treatment tolerability. Because drug sensitivity has been linked to inherent impairments in the DNA damage response, it is likely, as our data suggests, that manipulating synthetic lethality through BER-pathway inhibition may improve treatment of carcinoids by targeting malignant cells with dysregulated repair mechanisms that confer resistance while spairing healthy cells18, 19. Moreover, we demonstrate that lone ABT-888 treatment has limited toxicity, further supporting its candidacy as a treatment adjunct with favorable toxicity. Initial human trials showed that only a single dose was necessary to achieve adequate plasma concentration necessary for effective PARP inhibition67. Since 2007, the National Cancer Institute has evaluated ABT-888 in 88 clinical trials for the treatment of several cancer types including melanoma, gliomas, hepatocellular carcinoma, pulmonary and colorectal cancers. Select trials have combined ABT-888 and temozolomide therapy, and despite offering no clinical suggestion of synergy, have demonstrated excellent tolerability among both pediatric and adult populations34, 35, 40, 42. Several studies report modest antitumor activity with some approaching significance, though their general failure to demonstrate favorable drug interaction may be attributed factors like acquired resistance through BER pathway overexpression41, 42. Previous in vitro reports have revealed that enhanced DNA repair mechanisms and homologous recombination capacity in response to ABT-888 and temozolomide therapy may underlie a learned resistance to this regimen68. Hence, the effects of combination therapy may be more profound when these intrinsic resistance mechanisms are inherently or therapeutically disabled. Additionally, given the low number of recruited patients in some of these trials, their results may be considered exploratory rather than confirmatory.
In summary, ABT-888 potentiates dacarbazine-induced cytotoxicity in carcinoid cell lines, while altering the neuroendocrine phenotype. Hence, this therapeutic strategy may be a viable option for circumventing treatment refractoriness while controlling syndrome symptomatology. Given the current clinical characterization and use of ABT-888 and dacarbazine’s more tolerable form temozolomide, these findings warrant further investigations into the clinical use of combinatorial treatment for management of locally advanced and metastatic carcinoids.
FUNDING SOURCES: 1) NIH National Research Service Award T32 GM07215 (Y.S.), 2) American Cancer Society MEN2 Thyroid Cancer Professorship 120319-RPM-11-080-01-TBG (H.C.), 3) American Cancer Society Research Scholar Award RSGM TBE-121413 (H.C.), and 4) Caring for Carcinoid Foundation Grant from the American Association for Cancer Research (H.C.).
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST: No potential conflicts of interest exist.
DEDICATED AUTHOR CONTRIBUTIONS: Yash Somnay executed all experiments with assistance from Harpreet Gill and Jon Blake Matsumura. Drs. Sam Lubner and Herbert Chen guided the project’s trajectory and interpretation of findings.
CONFLICTS OF INTEREST
The authors declare no financial disclosures or potential conflicts of interest.