Figure 1.
Crizotinib-resistant patient sample does not indicate ROS1 gene amplification or histologic change.
(A) Pre-treatment and post-resistance patient samples analyzed by break-apart FISH assay for ROS1. Red probes are to the 5′ region of ROS1 and green probes to the 3′ region. Values represent the mean number of signals per cell. The single 3′ signal (values underlined) is indicative of the ROS1 fusion gene copy number. (B) RT-PCR, using primers to SDC4 and ROS1 that span the fusion point, performed on pre-treatment and post-resistance tumor samples. SD2;R32 is the ‘long’ variant (fusion of SDC4 exon 2 to ROS1 exon 32) and SD2;R34 is the ‘short’ variant (fusion of SDC4 exon 2 to ROS1 exon 34). (C) Hematoxylin and eosin staining of pre-treatment and post-resistance patient samples.
Table 1.
Figure 2.
HCC78-TR cells are resistant to ROS1 inhibition.
Cells were treated with TAE684 (A), crizotinib (B), or pemetrexed (C) as single-agents for 3 days and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3–7). Calculated IC50 values for TAE684: parental HCC78 = 0.14 µM, HCC78-TR = 1.09 µM, H322 = 1.42 µM, and HCC4006 = 1.15 µM. Calculated IC50 values for crizotinib: parental HCC78 = 0.79 µM, HCC78-TR = 1.95 µM, H322 = 4.13 µM, and HCC4006 = 3.03 µM. Calculated IC50 values for pemetrexed: parental HCC78 = 11 nM and HCC78-TR = 14 nM. HCC78-TR cells were significantly less sensitive than parental HCC78 cells to TAE684 (p<0.000005) and crizotinib (p<0.05) but not pemetrexed (p>0.05) as determined by student’s paired t-test.
Figure 3.
HCC78-TR cells do not exhibit ROS1 gene amplification or morphological changes compared to parental HCC78 cells.
(A) Parental HCC78 and HCC78-TR cells analyzed by break-apart FISH assay for ROS1. Red probes are to the 5′ region of ROS1 and green probes to the 3′ region. Values represent the number of signals per cell. The single 3′ signal (values underlined) is indicative of the ROS1 fusion gene copy number. In the HCC78-TR line, two populations existed that differed based on the number of 5′ signals detected. (B) Representative bright field images of parental HCC78 and HCC78-TR cells.
Figure 4.
Growth and survival signaling pathway activation is refractory to ROS1 inhibition in HCC78-TR cells.
Cells were treated with TAE684 (A) or crizotinib (B) for 4 hours. Lysates of the cells were then analyzed by Western blot using the indicated antibodies.
Figure 5.
Growth and survival signaling pathway activation has become reliant on EGFR activity in the HCC78-TR cells.
(A) Phospho-RTK (top) and phospho-kinase (bottom) array analyses performed on untreated parental HCC78 and HCC78-TR cells. Proteins of interest are labeled. Unlabeled spots at the corners of both sets of arrays are the positive control. (B) Parental HCC78 and HCC78-TR cells were treated with TAE684, gefitinib, or a combination of both for 4 hours. Lysates of the cells were then analyzed by Western blot using the indicated antibodies. (C) Parental HCC78 (P) and HCC78-TR (T) cells were left untreated, treated with 1 uM gefitinib for 4 hours, or treated with 100 ng/mL EGF for 10 minutes. Lysates of the cells were then analyzed by Western blot using the indicated antibodies.
Figure 6.
HCC78-TR cells have become sensitive to EGFR inhibition.
(A) Parental HCC78 and HCC78-TR cells were treated with gefitinib as a single-agent for 3 days and then analyzed by MTS assay. (B) Parental HCC78 and HCC78-TR cells were co-treated with 500 nM TAE684 and gefitinib, and HCC827, H322, and H358 cells were treated with single-agent gefitinib for 3 days and then analyzed by MTS assay. Calculated IC50 values: parental HCC78 (below 50% with single-agent TAE684), HCC78-TR = 0.86 µM, HCC827 = 0.04 µM, H322 = >5 µM, and H358 = 1.0 uM. All values represent the mean ± SEM (n = 4).
Figure 7.
EGF stimulation desensitizes parental HCC78 cells and CUTO-2 cells to ROS1 inhibition.
(A) Parental HCC78 and HCC78-TR cells were treated with TAE684 for 3 days with or without the addition of 100 ng/mL EGF and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3). Calculated IC50 values for TAE684: parental+vehicle = 0.18 µM, parental+EGF = 0.57 µM, HCC78-TR+vehicle = 1.39 µM, and HCC78-TR+EGF = 1.45 µM. EGF significantly desensitized parental HCC78 but not HCC78-TR cells to TAE684 as determined by student’s paired t-test (p<0.05). (B) Parental HCC78 cells were treated with TAE684 for 4 hours, EGF for 10 minutes, or a combination of both. Lysates of the cells were then analyzed by Western blot using the indicated antibodies. (C) CUTO-2 cells were treated with TAE684 for 4 days with or without the addition of 100 ng/mL EGF and then analyzed by MTS assay. Values represent the mean ± SEM (n = 3). Calculated IC50 values for TAE684: +vehicle = 0.2 µM and +EGF = 0.81 µM. EGF significantly desensitized CUTO-2 cells to TAE684 as determined by student’s paired t-test (p<0.01). (D) CUTO-2 cells were treated with TAE684 for 4 hours, EGF for 10 minutes, or a combination of both. Lysates of the cells were then analyzed by Western blot using the indicated antibodies. Phosphorylated ROS1 bands were below the limit of detection and were therefore not included.