MUC1-C Is a Common Driver of Acquired Osimertinib Resistance in NSCLC

Abstract

Introduction: Osimertinib is an irreversible EGFR tyrosine kinase inhibitor approved for the first-line treatment of patients with metastatic NSCLC harboring EGFR exon 19 deletions or L858R mutations. Patients treated with osimertinib invariably develop acquired resistance by mechanisms involving additional EGFR mutations, MET amplification, and other pathways. There is no known involvement of the oncogenic MUC1-C protein in acquired osimertinib resistance.

Methods: H1975/EGFR (L858R/T790M) and patient-derived NSCLC cells with acquired osimertinib resistance were investigated for MUC1-C dependence in studies of EGFR pathway activation, clonogenicity, and self-renewal capacity.

Results: We reveal that MUC1-C is up-regulated in H1975 osimertinib drug-tolerant persister cells and is necessary for activation of the EGFR pathway. H1975 cells selected for stable osimertinib resistance (H1975-OR) and MGH700-2D cells isolated from a patient with acquired osimertinib resistance are found to be dependent on MUC1-C for induction of (1) phospho (p)-EGFR, p-ERK, and p-AKT, (2) EMT, and (3) the resistant phenotype. We report that MUC1-C is also required for p-EGFR, p-ERK, and p-AKT activation and self-renewal capacity in acquired osimertinib-resistant (1) MET-amplified MGH170-1D #2 cells and (2) MGH121 Res#2/EGFR (T790M/C797S) cells. Importantly, targeting MUC1-C in these diverse models reverses osimertinib resistance. In support of these results, high MUC1 mRNA and MUC1-C protein expression is associated with a poor prognosis for patients with EGFR-mutant NSCLCs.

Conclusions: Our findings reveal that MUC1-C is a common effector of osimertinib resistance and is a potential target for the treatment of osimertinib-resistant NSCLCs.

Keywords: EGFR; MUC1-C; NSCLC; Osimertinib; Resistance.

Figure 1.. MUC1-C contributes to the H1975 osimertinib DTP cell phenotype.

A. H1975 cells were treated with 1 μM osimertinib for 9 days to select for DTP cells (upper panel). H1975 and H1975-DTP cells treated with the indicated concentrations of osimertinib for 72 hours were analyzed for cell viability by Alamar Blue staining. The results (mean±SD of six determinations) are expressed as relative cell number (% control) compared to that for untreated cells (lower panel). Indicated are the calculated IC50 values. B. Lysates from parental H1975 cells, H1975 cells treated with 1 μM osimertinib for 24 hours, and H1975-DTP cells were immunoblotted with antibodies against the indicated proteins. C. Lysates from H1975-DTP cells expressing a tet-MUC1shRNA vector (H1975-DTP/tet-MUC1shRNA) treated with vehicle or DOX for 14 days were immunoblotted with antibodies against the indicated proteins. D. H1975-DTP/tet-MUC1-shRNA cells treated with vehicle or DOX for 14 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for untreated cells (assigned a value of 1)(right). E. The MUC1-C cytoplasmic domain (MUC1C/CD) is a 72 aa intrinsically disordered protein that includes a CQC sequence which is necessary for MUC1-C homodimerization. The GO-203 inhibitor blocks the CQC motif and thereby MUC1-C function. Highlighted are (i) tyrosine residues that are phosphorylated by the indicated RTKs, and (ii) pYGQLD, pYHPM and pYTNP motifs that function as binding sites for the SH2 domains of SHP2, PI3K and GRB2, respectively. Also highlighted are regions that interact with the ZEB1 and TWIST1 EMT-TFs. F. Lysates from H1975-DTP cells left untreated or treated with 3 μM GO-203 for 48 hours were immunoblotted with antibodies against the indicated proteins. G. H1975-DTP cells left untreated or treated with 3 μM GO-203 for 48 hours were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative absorbance compared to that for untreated cells (assigned a value of 1)(right).

Figure 2.. Osimertinib resistant H1975-OR cells are dependent on MUC1-C for activation of the EGFR pathway and colony formation.

A. H1975 cells were exposed to increasing osimertinib concentrations (0.01 to 2 μM) over 12 weeks to select osimertinib resistant H1975-OR cells (upper panel). H1975 and H1975-OR cells treated with the indicated concentrations of osimertinib for 72 hours were analyzed for cell viability by Alamar Blue staining. The results (mean±SD of six determinations) are expressed as relative cell number (% control) compared to that for untreated cells (lower panel). Indicated are the calculated IC50 values. B. Lysates from H1975-OR cells treated with vehicle or 1 μM osimertinib for 48 hours were immunoblotted with antibodies against the indicated proteins. C. Lysates from H1975-OR/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. D. Lysates from H1975-OR cells treated with 3 μM GO-203 for 72 hours were immunoblotted with antibodies against the indicated proteins. E. Chromatin fractions from H1975 and H1975-OR cells were immunoblotted with antibodies against the indicated proteins. F. Chromatin fractions from (i) H1975-OR/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left) and (ii) H1975-OR cells left untreated or treated with 3 μM GO-203 for 48 hours (right) were immunoblotted with antibodies against the indicated proteins. G. H1975-OR/tet-MUC1shRNA cells treated with vehicle or DOX for 10 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony number compared to that for untreated cells (assigned a value of 1)(right). H. H1975-OR cells left untreated or treated with 3 μM GO-203 for 7 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony number compared to that for untreated cells (assigned a value of 1) (right).

Figure 3.. MUC1-C confers osimertinib resistance in H1975-OR and MGH700–2D EMT phenotype cells.

A. Photomicrographs of H1975 and H1975-OR cells (left). Bar represents 50 μm. Lysates of H1975 and H1975-OR cells were immunoblotted with antibodies against the indicated proteins (right). B. H1975-OR cells treated with 3 μM GO-203 for 72 hours were immunoblotted with antibodies against the indicated proteins. C. H1975-OR cells were treated with 1, 2 and 3 μM GO-203 in the presence of the indicated concentrations of osimeritinib. Cell viability assessed by Alamar Blue staining was used to calculate the IC50 values of osimertinib at each concentration of GO-203. Values under the additive line were denoted as synergistic. D. Lysates from MGH700–2D/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. E. MGH700–2D cells left untreated or treated with 3 μM GO-203 for 72 hours were immunoblotted with antibodies against the indicated proteins. F. MGH700–2D cells left untreated or treated with 1 μM osimertinib for 14 days and 3 μM GO-203 for 14 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for untreated cells (assigned a value of 1)(right). G. MGH700–2D cells were treated with 1, 2 and 3 μM GO-203 in the presence of the indicated concentrations of osimeritnib. Cell viability assessed by Alamar Blue staining was used to calculate the IC50 values of osimertinib at each concentration of GO-203. Values under the additive line were denoted as synergistic.

Figure 4.. Dependence on MUC1-C in MET amplified and EGFR(C797S) mutated NSCLC cells.

A. Lysates from MGH170–1D #2/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. B. Lysates from MGH121 Res#2/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. C. Lysates from MGH170–1D #2 cells left untreated or treated with 2 μM GO-203 for 48 hours were immunoblotted with antibodies against the indicated proteins. D. Lysates from MGH121 Res#2 cells left untreated or treated with 3 μM GO-203 for 48 hours were immunoblotted with antibodies against the indicated proteins. E. MGH170–1D #2 cells left untreated, treated with 1 μM osimertinib for 14 days or 3 μM GO-203 for 14 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (upper). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for untreated cells (assigned a value of 1)(lower). F. MGH121 Res#2 cells left untreated, treated with 1 μM osimertinib for 14 days or 3 μM GO-203 for 14 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (upper). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for untreated cells (assigned a value of 1)(lower). G. MGH170–1D #2 cells were treated with 0.125, 0.25 and 0.5 μM GO-203 in the presence of the indicated concentrations of osimertinib. Cell viability assessed by Alamar Blue staining was used to calculate the IC50 values of osimertinib at each concentration of GO-203. H. MGH121 Res#2 cells were treated with 0.5, 1.0 and 2.0 μM GO-203 in the presence of the indicated concentrations of osimertinib. Cell viability assessed by Alamar Blue staining was used to calculate the IC50 values of osimertinib at each concentration of GO-203. I. Lysates from MGH170–1D #2 cells were precipitated with a control IgG or anti-SHP2. The precipitates were immunoblotted with anti-MUC1-C and anti-SHP2. H. Lysates from MGH170–1D #2/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left) and MGH170–1D #2 cells left untreated or treated with 3 μM GO-203 for 48 hours (right) were immunoblotted with antibodies against the indicated proteins.

Figure 5.. MUC1-C is essential for self-renewal capacity of osimertinib resistant NSCLC cells.

A-D. Representative images of tumorspheres for H1975-OR (A), MGH700–2D (B), MGH170–1D #2 (C), and MGH121 Res#2 (D) cells treated with vehicle or 5 μM GO-203 in the absence and presence of 300 nM osimertinib for 7 days. Bar represents 50 microns. Tumorsphere formation is expressed as the mean±SD of three independent replicates relative to that obtained for untreated cells. E and F. Six-week old nude mice were injected subcutaneously in the flank with 5 × 10⁶ MGH170–1D #2 cells. Mice pair-matched into four groups of 6 mice each when tumors reached 100–150 mm³ were treated with vehicle control (blue), osimertinib (orange), GO-203 (grey), and GO-203 plus osimertinib (yellow) for the indicated days. Tumor volumes are expressed as the mean±SEM for six mice (E). Lysates from MGH170–1D #2 tumors harvested on day 21 were immunoblotted with antibodies against the indicated proteins (F).

Figure 6.. High MUC1 expression in EGFR mutant NSCLCs associates with poor clinical outcomes.

A. Kaplan–Meier curves for overall survival (OS) after diagnosis of patients with EGFR-mutant NSCLCs expressing high (red) vs low (blue) MUC1 mRNA levels. B. Kaplan–Meier curves for OS from initiation of osimertinib treatment of patients with EGFR-mutant NSCLCs expressing high (red) vs low (blue) MUC1 mRNA levels. C. Representative images of high (upper) and low (lower) MUC1-C staining by IHC in surgically resected tumors from patients with primary EGFR-mutant NSCLCs. D. Kaplan–Meier curves for recurrence-free survival of patients with EGFR-mutant NSCLC expressing high (red) vs low (blue) MUC1-C protein levels. E. Kaplan–Meier curves for overall survival (OS) of patients with EGFR-mutant NSCLC expressing high (red) vs low (blue) MUC1-C protein levels. F. Schema depicting MUC1-C dependence of osimertinib resistant NSCLC cells for (i) activation of RTK, SHP2, ERK and AKT signaling pathways, and (ii) self-renewal capacity. MUC1-C forms complexes with EGFR, MET, AXL and other RTKs at the cell membrane and contributes to their downstream signaling pathways. In this way, the MUC1-C cytoplasmic domain functions as a scaffold for integrating downstream activation of SHP2, PI3K and other effectors. MUC1-C is also imported into the nucleus, where it interacts with TFs, including ZEB1 and TWIST1, and chromatin remodeling complexes that promote EMT in driving the CSC state, inflammatory memory and therapeutic resistance.