Griffin, James D.

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.

Abstract The impetus for kinase inhibition as a therapeutic endpoint in leukemia research has been driven largely by the success of imatinib mesylate in chronic myelogenous leukemia. However, while imatinib mesylate has been extremely effective in the treatment of chronic phase CML, resistant disease often emerges in both Philadelphia positive ALL and aggressive phase CML patients. Adaphostin is a tyrphostin inhibitor originally developed to target the bcr/abl kinase and demonstrates selectivity in CML patient isolates. Here we show that in vitro, adaphostin is cytotoxic in numerous models that are resistant to imatinib. Features of adaphostin action in bcr/abl containing cells include apoptosis induction via an oxidant dependent mechanism and degradation of bcr/abl protein. Cell lines containing point mutations in p210 bcr/abl which confer imatinib resistance exhibit intracellular peroxide production and DNA fragmentation in response to adaphostin. Bcr/abl degradation also occurs but can be dissociated from peroxide production and subsequent apoptosis signaling. Similar data has also been obtained in cell lines containing p190 bcr/abl. Studies extending these observations into mononuclear and polymorphonuclear cells from imatinib resistant CML patients and Ph+ ALL patients confirm the same mechanism in primary clinical isolates. Collectively, these data suggest that reactive oxygen species generation by adaphostin bypasses imatinib resistance in CML and Ph+ ALL. Further studies dissecting the mechanism of ROS generation by adaphostin in imatinib resistant cells may lead to the identification of new therapeutic targets.

Abstract The adenosine triphosphate binding-site–directed agent STI571 and the tyrphostin adaphostin are undergoing evaluation as bcr/abl kinase inhibitors. The current study compared the effects of these agents on the survival of K562 cells, bcr/abl-transduced FDC-P1 cells, and myeloid progenitors from patients with chronic myelogenous leukemia (CML) compared with healthy donors. Treatment of K562 cells with 10 μM adaphostin resulted in decreased p210bcr/ablpolypeptide levels in the first 6 hours, followed by caspase activation and accumulation of apoptotic cells in less than 12 hours. By 24 hours, 90% of the cells were apoptotic and unable to form colonies. In contrast, 20 μM STI571 caused rapid inhibition of bcr/abl autophosphorylation without p210bcr/abl degradation. Although this was followed by the inhibition of Stat5 phosphorylation and the down-regulation of Bcl-xL and Mcl-1, only 7% ± 3% and 25% ± 9% of cells were apoptotic at 16 and 24 hours, respectively. Instead, the cytotoxic effects of STI571 became more pronounced with prolonged exposure, with IC90values greater than 20 μM and 1.0 ± 0.6 μM after 24 and 48 hours, respectively. Consistent with these results, 24-hour adaphostin exposure inhibited CML granulocyte colony-forming units (CFU-G) (median IC50, 12 μM) but not normal CFU-G (median IC50, greater than 20 μM), whereas 24-hour STI571 treatment had no effect on CML or normal CFU-G. Additional experiments revealed that STI571-resistant K562 cells remained sensitive to adaphostin. Moreover, the combination of STI571 + adaphostin induced more cytotoxicity in K562 cells and in CML CFU-G than either agent alone did. Collectively, these results identify adaphostin as a mechanistically distinct CML-selective agent that retains activity in STI571-resistant cell lines.