Comparative drug screening in NUT midline carcinoma

Abstract

Background: The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed.

Methods: On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts.

Results: In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)-NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease.

Conclusions: These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease.

Figure 1

Comparative efficacy of anticancer agents in NMC cell lines. (A) Unsupervised hierarchical clustering of anticancer agents from the Prestwick Chemical Library (plus vincristine, FP, and JQ1) based on cytotoxicity (% cell kill) in NMC lines at 10 μM. Multiple drug classes are represented, as indicated by the coloured key; (B) The most effective drug classes were identified by averaging the 10 μM cytotoxic effect of agents within each class across all three NMC cell lines (heatmap ranked by mean % cell kill); (C) In vitro cytotoxicity of the bromodomain inhibitor JQ1 in NMC cell lines (mean±s.e.m.), with five independent experiments per cell line; (D) Relative efficacy of the indicated drugs in NMC cell lines, with data representing the averaged values (±s.e.m.) for the three NMC cell lines (PER-403, PER-624, and PER-704) with the exception of JQ1 which is shown without error bars for clarity (refer to C for individual cell line response to this agent).

Figure 2

Comparative efficacy of anticancer therapies in NMC vs non-NMC cell lines. (A) Mean IC50 (±s.e.m.) of the indicated agents in three NMC (PER-403, PER-624, and PER-704) and two non-NMC cell lines (PER-535 and SAOS2), ***P<0.001, unpaired t-test, corrected for multiple testing; (B) In vitro cytotoxicity of FP in NMC (grey symbols) and non-NMC cell lines (black symbols), with 3–4 independent experiments per cell line; (C) Heatmap of averaged IC50 values across the five cell lines for the indicated drugs (n=3–5 independent experiments per drug and per cell line); (D) In vitro cytotoxicity of NMC (grey symbols) and non-NMC cell lines (black symbols) to simvastatin (n=4–5 independent experiments per cell line); (E) Survival of NMC (light bars) and non-NMC cell lines (dark bars) 96 h after γ-irradiation at the indicated doses (n=5 independent experiments per cell line).

Figure 3

In vivo drug treatment of NMC xenografts. (A) Comparison of engraftment kinetics for PER-403, PER-624, and PER-704 (mean±s.e.m.); (B) Effect of FP (5 mg kg⁻¹ per day × 4 weeks) on PER-624 in vivo tumour growth (difference to day 21, P<0.001, two-way ANOVA); (C) Effect of FP (5 mg kg⁻¹ per day × 4 weeks) on overall survival in PER-624 xenografted mice (P<0.005, log-rank test); (D) Effect of FP (5 mg kg⁻¹ per day × 4 weeks) on PER-403 in vivo tumour growth (mean±s.e.m.); (E) Effect of vincristine (VCR, 0.5 mg kg⁻¹ per week) on tumour growth in PER-624 xenografts (P<0.001, two-way ANOVA); (F) Effect of vincristine (VCR, 0.5 mg kg⁻¹ per week) on tumour growth in PER-403 xenografts (P<0.001, two-way ANOVA).

Figure 4

Comparison of control and FP-treated tumours from (A) PER-624 or (B) PER-403 xenografts. Images show haematoxylin & eosin (H&E) staining, IHC for NUT (α-NUT), and cytokeratin (AE1/3), with brown immunopositive stain and blue haemotoxylin nuclear counterstain. In each case, tumours were poorly differentiated, with cells demonstrating large nuclei and prominent nucleoli, scant cytoplasm with little cytokeratin and extensive speckled nuclear staining for NUT. There was no discernable difference between tumours from control and FP-treated xenografts in either line.

Figure 5

Histological comparison of NMC xenograft tumours (PER-403 and PER-624) with the primary patient tumours from which each cell line was derived; left panel, haematoxylin & eosin (H&E); right panel, anti-NUT IHC (α-NUT, brown; haematoxylin counterstain, blue), magnification × 40, bars 50 μm. All tumours were poorly differentiated, with cells demonstrating large nuclei with prominent nucleoli, scant cytoplasm and extensive speckled nuclear staining for NUT. A small but similar proportion (∼10%) of cells in each tissue was negative for NUT. Alveolar structures are visible in the primary tumour (top panel) used to derive PER-403.