Evaluation of azacitidine and entinostat as sensitization agents to cytotoxic chemotherapy in preclinical models of non-small cell lung cancer

Abstract

Recent clinical data in lung cancer suggests that epigenetically targeted therapy may selectively enhance chemotherapeutic sensitivity. There have been few if any studies rigorously evaluating this hypothesized priming effect. Here we describe a series of investigations testing whether epigenetic priming with azacitidine and entinostat increases sensitivity of NSCLC to cytotoxic agents. We noted no differences in chemosensitivity following treatment with epigenetic therapy in in vitro assays of viability and colony growth. Using cell line and patient derived xenograft (PDX) models, we also observed no change in responsiveness to cisplatin in vivo. In select models, we noted differential responses to irinotecan treatment in vivo. In vitro epigenetic therapy prior to tumor implantation abrogated response of H460 xenografts to irinotecan. Conversely, in vitro epigenetic therapy appeared to sensitize A549 xenografts (tumor growth inhibition 51%, vs. 22% in mock-pretreated control). In vivo epigenetic therapy enhanced the response of adenocarcinoma PDX to irinotecan. Taken together, these data do not support broadly applicable epigenetic priming in NSCLC. Priming effects may be context-specific, dependent on both tumor and host factors. Further preclinical study is necessary to determine whether, and in which contexts, priming with epigenetic therapy has potential to enhance chemotherapeutic efficacy in NSCLC patients.

Figure 1. Epigenetic changes associated with azacitidine and entinostat treatment

(A) Box plots of deltaBeta values depicting promoter region (+/− 1500 bp of transcription start site) demethylation (negative deltaBeta) relative to mock control (probes with Beta >0.5) at day 3 and day 10 following treatment with entinostat (E), Aza (A), or combo (C). (B) Percent HDAC2 enzyme activity after 24 h treatment with 50 nM entinostat, relative to mock control. Bars represent the mean of seven replicates +/− standard deviation. Statistical significance by two-tailed, unpaired t-test denoted as follows: **p < 0.01, ***p < 0.001. (C) Western blots depicting acetylated histone H4 (lysine 5, 8, 12, 16) and total histone H4 levels at the end of treatment (day 3) with mock (M), entinostat (E), Aza (A), or combo (C). (D) Quantified histone H4 acetylation, relative to mock control.

Figure 2. Epigenetic priming does not alter sensitivity of NSCLC cell lines to subsequent chemotherapy

Log dose response curves for NSCLC cell lines treated (in triplicate) with cisplatin, docetaxel, gemcitabine, or 17-AAG for 72 hours one week post epigenetic therapy. Individual curves represent the percentage of viable cells (+/− standard deviation) for each epigenetic pretreatment condition normalized to its own untreated control cells, such that the highest values for each pretreatment condition represent 100%, and 0 = 0%. Data shown from representative experiments.

Figure 3. Epigenetic priming does not potentiate the effects chemotherapy on colony growth

H358 and A549 cells were seeded on a solidified Matrigel layer six days after epigenetic therapy. Beginning the following day, cells were treated with chemotherapy for 72 hours. Drug was then removed and colonies were permitted to grow 2 – 4 additional days. (A) Representative H358 colonies following treatment with 10 nM gemcitabine or 10 nM 17-AAG. (B) H358 percent colony formation (+/− standard deviation) relative to untreated control (PBS or DMSO), calculated from one representative experiment with five replicates. (C) Representative A549 colonies following treatment with 600 nM cisplatin, 30 nM 17-AAG, or 1 nM docetaxel. (D) A549 percent colony formation (+/− standard deviation) relative to untreated control (PBS or DMSO), averaged from two independent experiments (total nine replicates). Statistical significance by ANOVA with Tukey's multiple comparison test denoted as follows: *p < 0.05, **p < 0.01.

Figure 4. Epigenetic therapy augments response of A549 xenografts, but abrogrates response of H460 xenografts, to irinotecan, and does not sensitize to cisplatin or docetaxel

Subcutaneous hind flank tumors were established in NOD/SCID mice from A549 or H460 cells treated in vitro with mock (M) or the combination of Aza and entinostat (C). (A) Mice bearing pretreated A549 tumors were treated with 2 mg/kg cisplatin (M-C and C-C) on day 2, 10 mg/kg irinotecan (M-I and C-I) on days 2 & 5, or saline vehicle (M-V and C-V), for three one-week cycles. Curves represent mean tumor volume +/− SEM. Statistical significance determined using a mixed effects model and REML. (B) Mice bearing pretreated H460 tumors were treated with 2.5 mg/kg docetaxel q4d × 2 escalated to 5 mg/kg docetaxel q4d × 2 (M-D and C-D), 10 mg/kg irinotecan q4d × 4 (M-I and C-I), or saline vehicle (M-V and C-V). Curves represent mean tumor volume +/− SEM.

Figure 5. Epigenetic therapy in vivo does not sensitize H358 xenografts to immediate subsequent chemotherapy

Nude mice bearing H358 xenografts were treated with 0.5 mg/kg Aza (sc, qd × 5) and 1 mg/kg entinostat (ip, day 5), or vehicle, for four one-week cycles. (A) Mean tumor volume (+/− SEM) over time (left) and individual tumor volumes at day 29 (right). Statistical significance determined by two-tailed unpaired t test. (B) Mean tumor volume (+/− SEM) from vehicle (left) and epigenetic (right) pretreated mice randomized on day 29 to receive 2 mg/kg cisplatin on day 2, 10 mg/kg irinotecan on days 2 & 5, or saline vehicle. Mice were treated for four one-week cycles.

Figure 6. Epigenetic therapy sensitizes a patient derived model of adenocarcinoma to repeat treatment with irinotecan, but does not sensitize to cisplatin

(A) LX7 bearing NOD/SCID mice were treated with 0.5 mg/kg Aza (sc) on days 1-3, 6-10, 13-17, and 1 mg/kg (ip) entinostat on days 3, 10, 17, or vehicle (n = 9 per arm). (B) NOD/SCID mice bearing LX7 tumors established from pooled vehicle (V) or epigenetic (E) pretreated tumors were treated with 10 mg/kg irinotecan (V-I and E-I) q4d × 3, 2 mg/kg cisplatin (V-C and E-C) q7d × 2, or saline vehicle (V-V and E-V). (C-D) Irinotecan tumors were allowed to grow following cessation of treatment. On day 32, mice were re-challenged with 10 mg/kg irinotecan on days 32, 36, and 40 (same dose and schedule as first cycle). * Two mice in the Vehicle – Irinotecan (V-I) arm were euthanized early (day 42 and 45 after final measurements) for n = 6 after day 45. Significance determined using a mixed effects model and REML. All growth curves depict mean tumor volume +/− SEM.