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The CRTC1-MAML2 fusion is the major oncogenic driver in mucoepidermoid carcinoma
Zirong Chen, … , Frederic J. Kaye, Lizi Wu
Zirong Chen, … , Frederic J. Kaye, Lizi Wu
Published April 8, 2021
Citation Information: JCI Insight. 2021;6(7):e139497. https://doi.org/10.1172/jci.insight.139497.
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Research Article Oncology

The CRTC1-MAML2 fusion is the major oncogenic driver in mucoepidermoid carcinoma

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Abstract

No effective systemic treatment is available for patients with unresectable, recurrent, or metastatic mucoepidermoid carcinoma (MEC), the most common salivary gland malignancy. MEC is frequently associated with a t(11;19)(q14-21;p12-13) translocation that creates a CRTC1-MAML2 fusion gene. The CRTC1-MAML2 fusion exhibited transforming activity in vitro; however, whether it serves as an oncogenic driver for MEC establishment and maintenance in vivo remains unknown. Here, we show that doxycycline-induced CRTC1-MAML2 knockdown blocked the growth of established MEC xenografts, validating CRTC1-MAML2 as a therapeutic target. We further generated a conditional transgenic mouse model and observed that Cre-induced CRTC1-MAML2 expression caused 100% penetrant formation of salivary gland tumors resembling histological and molecular characteristics of human MEC. Molecular analysis of MEC tumors revealed altered p16-CDK4/6-RB pathway activity as a potential cooperating event in promoting CRTC1-MAML2–induced tumorigenesis. Cotargeting of aberrant p16-CDK4/6-RB signaling and CRTC1-MAML2 fusion–activated AREG/EGFR signaling with the respective CDK4/6 inhibitor Palbociclib and EGFR inhibitor Erlotinib produced enhanced antitumor responses in vitro and in vivo. Collectively, this study provides direct evidence for CRTC1-MAML2 as a key driver for MEC development and maintenance and identifies a potentially novel combination therapy with FDA-approved EGFR and CDK4/6 inhibitors as a potential viable strategy for patients with MEC.

Authors

Zirong Chen, Wei Ni, Jian-Liang Li, Shuibin Lin, Xin Zhou, Yuping Sun, Jennifer W. Li, Marino E. Leon, Maria D. Hurtado, Sergei Zolotukhin, Chen Liu, Jianrong Lu, James D. Griffin, Frederic J. Kaye, Lizi Wu

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Figure 1

Dox-inducible CRTC1-MAML2–targeting shRNAs decreased the growth of established human MEC xenografts by reducing cell proliferation and enhancing apoptosis.

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Dox-inducible CRTC1-MAML2–targeting shRNAs decreased the growth of estab...
(A) A diagram of the lentiviral-based vector (FH1tUTG) system for inducible CRTC1-MAML2 fusion–targeting shRNA expression, adapted from Herold et al. (34). (B and C) A stable H3118 MEC cell clone with CRTC1-MAML2 ishRNA (H3118-fusion ishRNA) was treated with 1 μg/mL Dox for 72 hours and analyzed for CRTC1-MAML2 fusion knockdown by Western blotting (B) and LINC00473 expression by qPCR (C). Data are mean ± SD. A 2-tailed t test was used to calculate the P values (*P < 0.05 and ***P < 0.001). (D and E) NOD.SCID mice were injected s.c. with 1 × 106 H3118-fusion ishRNA cells per mouse and provided with Dox or control diet starting on the day of tumor cell injection (Dox 1 cohort) (D) or when tumors reached approximately 50 mm3 (Dox 2 cohort) and approximately 100 mm3 (Dox 3 cohort) (E). Tumor volumes at various days after tumor cell implantation and the images of resected tumors at the endpoint were shown. (F and G) The expression levels of the CRTC1-MAML2 fusion and LINC00473 in Dox-treated versus control xenograft MEC tumors were analyzed by Western blotting (F) and qPCR (G), respectively. β-Actin was used as a protein loading control. (H–M) Cell proliferation and apoptosis were evaluated on 3 Dox-treated versus control MEC xenograft tumor sections by Ki-67 IHC (H and K), TUNEL staining (I and L), and cleaved caspase 3 IHC (J and M). The positively stained nuclei/cells were quantified in 6 randomly selected visual fields (5×, 1000 × 1000 pixels) using ImageJ. Scale bar: 100 μm (upper panels), 25 μm (lower panels). A 2-tailed t test was used for 2-group comparisons and 1-way ANOVA for multiple group comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

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