4A). All treatment arms significantly inhibited xenograft growth, particularly lapatinib/trastuzumab, LJM716/trastuzumab and the 3-drug combination. Mice treated with any of these three combinations exhibited a close to total response with tumors measuring <25 mm3 after 3 weeks of treatment (Fig. 3A). Treatment was halted at this time and tumor regrowth was monitored. To assess the effect of treatment on survival, mice were followed until they reached a tumor burden of 2,000 mm3, time when they had to be humanely euthanized according to institutional guidelines. At 34 weeks of SPP1 follow-up with no treatment, less than 40% of mice in the lapatinib/trastuzumab and the LJM716/trastuzumab arms were alive whereas 93% of mice in the lapatinib/trastuzumab/LJM716 group were so (gene-amplified and mutant cells. Treatment with LJM716 alone inhibited ACP-196 (Acalabrutinib) proliferation, defined as >25% growth inhibition (GI) relative to control, in 6/18 (33%) cell lines as measured by the cell content of ATP (CellTiterGlo assay). Treatment with BYL719 induced >25% GI in 9/18 (50%) cell lines, particularly those with hotspot mutations (i.e. H1047R, E545K) in PIK3CA (Fig. 4A, cell lines marked reddish). In 12/18 (67%) cell lines, treatment with the combination of LJM716 with BYL719 resulted in >25% growth inhibition (Fig. 4A). Combination activity exceeded that enacted by either agent in isolation in 11/18 (61%) cell lines. Analysis using the Chalice software package confirmed that combination treatment resulted in synergistic action of the two compounds (Suppl. Fig 1). We confirmed these results in a second assay where cells are plated in monolayer followed by crystal violet staining. We observed a statistical decrease in growth of 4 of 5 HER2+ breast malignancy cell lines treated with LJM716 and BYL719 compared to either single agent (Fig. 4B, Suppl. Fig 2). Comparable results were observed in ACP-196 (Acalabrutinib) single cells plated in 3-D Matrigel and assessed for colony formation for 14C21 days, where 5/5 cell lines treated with LJM716 and BYL719 exhibited a statistically larger reduction in growth compared to either single agent (Fig. 4C, Suppl. Fig 3). Finally, we examined the effect of the combination and single drugs on cell signaling at 1C24 h. Treatment with BYL719 as a single agent increased P-HER3 in all four cell lines examined (Fig. 4D), consistent with the reported observation that inhibition of PI3K/AKT results in compensatory upregulation of active HER3 (24, 25). BYL719 reduced both S473 and T308 P-AKT, although in some cases this inhibition was partial. In BT474 and MDA361 cells, more potent inhibition of S473 P-AKT S473 was achieved with the combination of LJM716/BYL719 (at 24 h) than with either single agent. A similar result was observed with HCC1954 cells treated for 1 h with the combination (Fig. 4D). Treatment with the combination did not impact P-ERK in three of the four cell lines. Open in a separate windows Physique 4 LJM716 and p110 inhibitor synergistically inhibit tumor cell growth and PI3KA. Heatmap representing percent growth inhibition for the outlined cell lines 5 days after treatment with 33 nM (5 g/ml) of LJM716, 330 nM BYL719 or the combination, relative to untreated cells as assessed by the CellTiterGlo Assay. Values for LJM716 were the average of two impartial dose-titration curves. Synergistic inhibition (synergy score 2.0) was observed for the following cell lines: EFM192A, AU565, SKBR-3, BT474, MDA361 and MDA453 (all with HER2 gene amplification). Percent inhibition relative to IgG-treated (control) cells is usually visualized in the form of a warmth map colored from blue (0% inhibition) to reddish (100% inhibition). Cell lines harboring hotspot mutations are highlighted in reddish. B. Cells were plated (1C5104 cells/well) in 6-well plates and treated in triplicate with DMSO, 10 g/ml LJM716 1 M BYL719. Media and drugs were replenished every 3C4 days. Cells were stained with crystal violet when the DMSO-treated (control) monolayers became confluent, ranging from 14C21 days. Quantification of integrated intensity (% control) is usually shown (*, test). C. Cells were seeded in Matrigel and allowed to grow in ACP-196 (Acalabrutinib) the absence or presence of 10 g/ml LJM716 and/or 1 M BYL719 as indicated. Cell media and drugs were replenished every 3 days. Images shown were.
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