Optimal targeting of BCL-family proteins in head and neck squamous cell carcinoma requires inhibition of both BCL-xL and MCL-1

Abstract

Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares - Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9μM (range 6.6μM - 13.9μM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7μM (range, 8.8μM to 12.7μM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.

Keywords: A-1210477; BCL-xL; MCL-1; head and neck squamous carcinoma; navitoclax.

Figure 1. Patient cohort and gene expression data

(A) Diagram describing the selection process for patients deemed failures and responders used for gene expression analysis. (B) Diagram of results from sparse Partial Least Squares-Discriminant Analysis (sPLS-DA). Red – failure cases, green – responder cases. (C) Heatmap of gene expression profiles from genes selected on the first component of the sPLS-DA model shows HNSCC treatment responders largely cluster separately from failures.

Figure 2. Baseline profiling of radiation and cisplatin response in HNSCC cell lines

Results from clonogenic survival assays showing radiation sensitive (A) and radiation resistant (B) head and neck squamous cell carcinoma cell lines – average surviving fraction with standard error of the mean (S.E.M., error bars) is calculated from triplicate experiments; (C) Graphical representation of surviving fraction after 2 Grey dose of radiation (SF 2Gy), with cell lines arranged from most sensitive to most resistant; (D–K) Representative results from MTT assays of cisplatin in head and neck squamous cell cancer cell lines organized from most sensitive (D–G) to most resistant (H–K) to cisplatin. (L) Graphical representation of the average IC50, with S.E.M. (error bars) (MTT assay done in triplicate), cell lines arranged according to radiation sensitivity. There was no significant correlation between radiation sensitivity and IC50 dose for cisplatin.

Figure 3. Baseline BCL-xL expression and inhibition of BCL-xL with ABT-263 (navitoclax) treatment of HNSCC cells, alone and in combination with radiation or cisplatin

(A) Western blot demonstrating consistent high expression of BCL-xL and rare expression of BCL-2 in HNSCC cells. (B) Average IC50 values, with S.E.M. (error bars), after treatment with ABT-263 (navitoclax) assessed from cell viability assays using MTT. (C) Percent Survival calculated from clonogenic survival assays, with S.E.M. (error bars), after treatment of HNSCC cells with 2μM or 4μM ABT-263 (navitoclax) alone or in combination with 2 Gy radiation. (D) Average IC50 values, with S.E.M. (error bars), for cisplatin given in combination with 2μM and 4 μM ABT-263 (navitoclax).

Figure 4. Evaluation of MCL-1 expression in HNSCC

(A) Western blot demonstrating baseline MCL-1 expression in HNSCC cell lines, compared to BCL-xL and BCL-2 expression as demonstrated previously. Cell lines most resistant to radiation (HN5, PCI15B, Detroit562) demonstrate high MCL-1 expression. (B) Box and whisker plot representing gene expression transcript levels for BCL-2, BCL-xL, and MCL-1 as measured on gene expression microarrays in the HNSCC patient cohort, stratified by “responders” and “failures”. Lines represent median average transcript levels; Boxes represent 25^th and 75^th percentile, whiskers represent 2^nd and 98^th percentile. Dots represent outlying data points. (C) Western blot demonstrating MCL-1 protein expression at baseline, and 24 hours after treatment with 4μM ABT-263 (navitoclax). (D) Quantification of MCL-1 expression shows consistent increases of average MCL-1 expression, with standard deviation (error bars), after treatment with ABT-263 (navitoclax).

Figure 5. Inhibition of MCL-1 in HNSCC cells with A-1210477, alone and in combination with ABT-263 (navitoclax)

(A) Average IC50 values, with S.E.M. (error bars), for A-1210477 in HNSCC cell lines. (B) Average IC50 values, with S.E.M. (error bars), for ABT-263 (navitoclax) in combination with 2.5 μM and 5μM A-1210477. Note that baseline IC50 values for A-1210477 (A) vary slightly from control values (B) as these were carried out as independent experiments with triplicate data for each.

Figure 6. Bliss Independence analysis to evaluate synergistic activity between ABT-263 (navitoclax) and A-1210477

Cell viability curves and Bliss Independence analysis scores after treatment with ABT-263 (navitoclax) and A-1210477 across varying doses in PCI15B (A), Detroit 562 (B), MDA686LN (C), and HN30 (D).