A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation

<div><p>Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a <em>MET</em> amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.</p> </div

METi inhibits c-Met phosphorylation in both GTL16 and resistant GTL16R1 and GTL16R3 clones.

<p>Heatmap of Reverse Phase Protein Array (RPPA) showing GTL16 and resistant clones GTL16R1 and GTL16R3 relative level of total and phosphorylated proteins. Red indicates higher intensity vs green which indicates lower intensity. Cells were treated with DMSO vehicle or METi (2.5 µM) for 1 hr.</p

The c-Met inhibitor synergizes with either RAFi or MEKi.

<p>(A) Cell Viability of resistant GTL16R1 and GTL16R3 clones treated with RAFi or MEKi as single agents. Resistance of GTL16R1 and GTL16R3 clones to RAFi single agent treatment suggests that c-Met is involved in signaling independent of Raf. MEKi treatment inhibits cell viability as a single agent with IC50 values of 1.5 nM and 4.5 nM for GTL16R1 and GTL16R3, respectively. (B) METi and RAFi synergistically inhibit tumor growth of GTL16R1 and GTL16R3 resistant lines. Wildtype GTL16 (WT), resistant GTL16R1 and GTL16R3 clones were cultured with a combination of METi and RAFi at the indicated nM concentrations. Matrix grid represents various concentration combinations. Single agent activity can be seen for METi and RAFi on the respective edges of the plot. Tumor cell growth inhibition (TGI) or ΔBLISS independence is indicated on the Z-axis and shaded according to the value in the legend. TGI ranges from low (no effect on growth) to high (complete suppression of growth). ΔBLISS ranges from low (complete independence/additivity) to high (synergy). (C) METi and MEKi can synergize in GTL16R1 and GTL16R3. Wildtype GTL16 (WT), resistant GTL16R1 and GTL16R3 clones were cultured with a combination of METi and MEKi at the indicated nM concentrations. Matrix grid represents various concentration combinations. Single agent activity can be seen for METi and MEKi on the respective edges of the plot. Tumor cell growth inhibition (TGI) or ΔBLISS independence is indicated on the Z-axis and shaded according to the value in the legend. TGI ranges from low (no effect on growth) to high (complete suppression of growth). ΔBLISS ranges from low (complete independence/additivity) to high (synergy).</p

Chromosomal rearrangement at 7q32 and 7q34 results in a highly expressed SND1-BRAF fusion protein.

<p>(A) Western Immunoblot of total protein lysates identify a higher molecular weight band (arrowhead) recognized only by the anti-C-terminal BRAF antibody, and present exclusively in GTL16R1 and GTL16R3, consistent with a fusion event within BRAF. Additionally, the putative fusion BRAF is highly expressed and hyperphosphorylated compared to wild-type BRAF (arrow in BRAF panels). (B) 5′ RACE identified the nucleotide sequence of fusion junction spanning exon 16 of SND1 and exon 9 of BRAF. (C) Schematic representation of CNV amplification of the N-terminal portion of SND1 (mapping from exon 1 to 16) and the C-terminal protein of BRAF (mapping from exon 9 to exon 18).</p

The GTL16 gastric carcinoma cell line becomes resistant to c-Met inhibition after 4 months of continuous treatment with METi.

<p>(A) METi-resistant cells exhibit distinct squamous-like and rounded morphologies compared to GTL16 cells. 100X magnification. (B) Cell viability response curves to METi. METi inhibits GTL16 (blue) with an IC50 value of 10 nM, while it does not inhibit cell viability in clones GTL16R1 and GTL16R3 (red and green). Inhibition of cell viability is normalized relative to untreated control cells. (C) Cell viability response curves to crizotinib. Crizotinib inhibits GTL16 (blue) with an IC50 value of 3 nM. While IC50 values for GTL16R1 and GTL16R3 are >1 µM.</p

Higher ERK activity coincides with highly expressed SND1-BRAF fusion proteins in GTL16R1 and GTL16R3.

<p>(A) Western immunoblot of GTL16, GTL16R1 and GTL16R3 treated with inhibitors as single agent or combinations for 4 hr. (B) Densitometry measurement of the relative intensity of phospho ERK normalized to total ERK within each sample. (C) Densitometry measurement of the relative intensity of phospho AKT S473 normalized to total AKT within each sample.</p

A model highlighting the resistance mechanism in GTL16 GTL16R1 and GTL16R3.

<p>Highly expressed SND1-BRAF fusion protein signals through the MEK/ERK pathway and short-circuits c-Met inhibition.</p

Antiproliferative and antitumor activities of PF-04691502 through the modulation of the PI3K/mTOR pathway.

<p><i>A</i>. Representative dose-dependent responses of CSCs and differentiated cells after treatment with PF-04691502. Cell viability, as measured by CellTiter Glo® assay, is shown as the mean ± SEM (n = 5). <i>B</i>. Tumor growth inhibition induced by the treatment with PF-04691502 in SCID-bg mice in comparison with the vehicle controls. *P≤0.05; **P≤0.001 (two-way ANOVA analysis). <i>C.</i> Relative change in body weight of mice treated in the experiment (%). <i>D.</i> Western blot analysis of treated xenograft tumors. The expression levels of pAKT (S473), pERK (T202/T204), and the loading control α-tubulin are shown in four individual xenograft tumors in each treatment group. <i>E.</i> Quantitative data of the expression levels of pAKT (S473) relative to the loading controls (mean ± SEM; n = 4). F. CSCs were treated with PF-04691502 (502) at 0.1, 0.5 and 1 µM in vitro for 3, 24, and 48 hours. The changes in pAKT (S473) and pERK (T202/T204) were evaluated by Western Blot. GAPDH served as a loading control.</p

Culture of spheroid CSCs and identification of CD133+/EpCAM+ cells.

<p><i>A</i>, Schema illustrating the work flow of spheroid CSC generation and differentiated cell populations as an experimental system, including the transplantation of a patient tumor into NOD/SCID mice, propagation of CSCs from P₁ xenograft tumors (spheroid; 10× objective magnification), and differentiation of CSCs into adherent cells with epithelial morphology (differentiated; 20×). <i>B</i>. Flow cytometric analysis of the primary cells isolated from the original patient tumor. PI staining excludes the dead cells (a). APC- and PE-conjugated isotype controls are shown in (b). A population of CD133+/EpCAM+ cells was detected (c). <i>C</i>. FACS of CD133+/EpCAM+ colon CSCs from the primary cell population derived from P₁ xenograft tumors. Dead cells and murine cells were first excluded by PI staining and using an anti-mouse specific monoclonal antibody H-2k[d], respectively (a & b). CD133+/EpCAM+ and CD133−/EpCAM+ populations were gated according to the baselines of isotype controls and sorted (c). Finally, enrichment of both populations in the sorted samples was confirmed by flow cytometry (d & e). <i>D</i>. Expression of the CSC markers in a fraction of cultured spheroid CSCs (right panel). Left panel shows isotype controls.</p

Enhanced tumorigenic potential of CD133+/EpCAM+ CSCs, CSC differentiation and drug resistance.

<p><i>A</i>. Average tumor volumes in NOD/SCID mice inoculated with CD133+/EpCAM+ (3,400 cell/animal; putative CSCs) or CD133−/EpCAM+ (25,000 cell/animal; putative differentiated) cells after 12 weeks of implantation (mean±SEM; n = 5). <i>B</i>. Percentage of CD133+/EpCAM+ expressing cells in the CSC and differentiated cell populations (mean ± SEM, n = 3; unpaired, two-tailed student t-test, P<0.05). <i>C</i>. Representative results of cell proliferation rates and expression levels of cytokeratin in CSCs and differentiated cells. Cell numbers on Y axes are adjusted as a percentage of the maximum cell numbers analyzed. <i>D</i>. Representative data showing the <i>in vitro</i> drug sensitivity of CSCs and their differentiated progeny in response to oxaliplatin as assessed by CellTiter Glo® assay.</p