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. 2014 Dec 26;9(12):e115228.
doi: 10.1371/journal.pone.0115228. eCollection 2014.

HSP90 inhibition enhances antimitotic drug-induced mitotic arrest and cell death in preclinical models of non-small cell lung cancer

Affiliations

HSP90 inhibition enhances antimitotic drug-induced mitotic arrest and cell death in preclinical models of non-small cell lung cancer

Brenda C O'Connell et al. PLoS One. .

Abstract

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.

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Conflict of interest statement

Competing Interests: The authors would further like to clarify that at the time the work was done the authors were employees and shareholders of Infinity Pharmaceuticals, Inc. This does not alter their adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Combination of IPI-504 and docetaxel (DTX) improves efficacy in NSCLC xenograft models.
NCR nu/nu homozygous male mice were subcutaneously implanted with (A) H1993, (B) A549, (C) H292 and (D) H522 cells and treated with DMSO vehicle (black circles), IPI-504 (green squares), DTX (blue diamonds), or the combination of IPI-504 and DTX (red triangles). IPI-504 was administered by IP injection at a dose of 50 mg/kg, twice a week for a total of 6 doses. DTX was administered at 5 mg/kg (H522) or 15 mg/kg (H1993, A549, H292) by IP injection, weekly for a total of 3 doses. Numbers on graphs represent average percent tumor growth inhibition relative to vehicle-treated arm. Where indicated, * denotes statistical significance (p<0.05) between combination treatment and DTX treatment arms measured at day 30 (H1993, H549, H522) using Student's T-test.
Figure 2
Figure 2. In vitro synergy observed in NSCLC cell lines.
(A) Cell death was measured 48 h post treatment with non-fixed drug ratio combinations of IPI-504 and docetaxel (DTX) by 7AAD in H292 cells. Combination indices (CI) values were calculated using CalcuSyn software (Biosoft) with values <0.5 indicative of strong synergy. Error bars represent standard deviation (n = 4). Values for all combination treatments were statistically significant compared to single agent treatments as determined by Student's T-test (p<.01). (B) Cell death was measured 30 h post drug treatment in H1993 cells using the Caspase-Glo3/7 luminescence assay. Representative data are shown (n = 2). (C) Cells were treated with fixed drug ratios of IPI-504 and DTX for 72 h; cell proliferation was measured with Alamar Blue. Shown are normalized isobolograms. D =  dose, ED50 =  dose required to attain 50% growth inhibition. Points on the graph refer to the ratio of D/ED50 for DTX on the x-axis vs D/ED50 for IPI-504 on the y-axis. Data points which fall on the diagonal represent additivity; above the diagonal, antagonism; below the diagonal, synergy. (D) Treatment with PLK1 inhibitor (BI2436) sensitizes H292 cells to IPI-504. H292 cells were treated for 72 h with a dose titration of IPI-504 alone (blue diamonds) or in combination with 5 nM BI2536 (red squares) followed by cell death assay (7AAD). Error bars represent standard deviation (n = 2).
Figure 3
Figure 3. Mitotic delay upon treatment of cells with IPI-504 and docetaxel (DTX) combination.
EC50 doses for IPI-504 were determined by 72 h Cell Titer Glo (S1A Fig.). H292 (A, B left panel) and A549 (B, right panel) cells were treated with DMSO (blue diamonds), IPI-504 (red squares), DTX (green triangles) or IPI-504/DTX combination (purple circles) and harvested at the indicated time points for presence of the mitotic marker, pH3. Representative data are shown (n = 2).
Figure 4
Figure 4. Mitotic checkpoint bypass with Aurora kinase inhibition partially rescues combination effects of IPI-504 and docetaxel (DTX).
H292 cells were treated for 30 h (A, C) or 48 h (B) with indicated dose combinations of IPI-504 (175 nM), DTX (20 nM) and the Aurora kinase inhibitor ZM447439 (9 µM). Cells were harvested for (A) flow cytometry (pH 3) and phase contrast imaging, (B) cell death (7AAD), and (C) immunoblot analysis. An arrow denotes a slow migrating, phosphorylated form of Securin. Error bars for mitotic index and cell death represent standard deviation of replicates from two independent experiments.
Figure 5
Figure 5. Identification of components of the anaphase promoting complex as specifically depleted from H292 cells treated with IPI-504 and docetaxel (DTX) combination.
(A) Stable Isotope Labeling of Amino Acids in Culture (SILAC) schematic. Metabolically labeled H292 cells were treated for 24 h with the combination of 300 nM IPI-504 and 10 nM DTX or vehicle (DMSO) followed by identification of the HSP90 interactome by mass spectrometry. (B) Raw data from the SILAC study. Data points in the upper right and lower left quadrants represent proteins that are up-regulated and down-regulated, respectively in the HSP90 interactome upon treatment with the combination as compared to vehicle. The two arrows in the upper right quadrant represent independent peptide fragments with unique sequences targeting HSP70. (C) Immunoblot analysis verifies depletion of ANAPC3 and ANAPC4 upon 24 h treatment of H292 cells with 300 nM IPI-504 and 10 nM DTX combination. GR  =  Glucocorticoid Receptor. (D) Loss of APC/C components by RNAi sensitizes H292 cells to DTX mediated cell death (72 h, 7AAD) (left panel) and increases the mitotic index (30 h, pH 3) (right panel). H292 cells were transfected with nontargeting scrambled siRNA (blue diamonds) or siRNA's targeting ANAPC3 (red squares), ANAPC4 (green triangles), or ANAPC3/ANAPC4 combination (purple circles). (E) Immunoblot analysis showing knockdown efficiency. Representative data are shown (n = 2).
Figure 6
Figure 6. Model for IPI-504 and docetaxel (DTX) MOA.
In the absence of drug treatment, the mitotic checkpoint criteria are met and APC/C dependent degradation of mitotic inhibitors Securin, AURKB, and Cyclin B allow cells to progress through mitosis (left panel). IPI-504 and DTX combination treatment results in accumulation of mitotic inhibitors and impaired mitotic progression (right panel). Elevated levels of AURKB and Securin block the metaphase to anaphase transition whereas elevated levels of Cyclin B block mitotic exit. The potential contributions of APC/C as an HSP90 client protein, or other HSP90 client proteins to the mitotic arrest observed upon combination treatment are indicated with a question mark.

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