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Aurora kinases play a key role in mitosis and are frequently overexpressed in a variety of tumor cells. Inhibition of aurora kinases results in mitotic arrest and death of cancer cells, and has been explored as an anticancer strategy. However, how aurora inhibition kills cancer cells is poorly understood. In this study, we found that inhibition of aurora kinases by siRNA or small-molecule inhibitors led to induction of p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, in colorectal cancer cells irrespective of p53 status. Deficiency in PUMA increased polyploidy, improved cell survival, and abrogated mitochondria-mediated apoptosis induced by aurora kinase inhibitors. In response to aurora kinase inhibition, PUMA was directly activated by p65 through the canonical NF-κB pathway following AKT inhibition. Furthermore, PUMA was necessary for the chemosensitization and in vivo antitumor effects of aurora kinase inhibitors in colon cancer cells. These results suggest that PUMA induction mediates the apoptotic response to mitotic arrest imposed by aurora kinase inhibition, and may be a useful indicator for the anticancer activity of aurora kinase inhibitors. Mol Cancer Ther; 13(5); 1298–308. ©2014 AACR.

PDF file - 364KB, Figure S1: p53-independent PUMA induction by the aurora kinase inhibitor VX-680 in colon cancer cells; Figure S2: PUMA mediates the apoptotic and anticancer activities of ZM-447439 and VX-680; Figure S3: Analysis of transcription factors and kinases involved in PUMA induction by aurora kinase inhibitors; Figure S4: Analysis of kinase pathways involved in PUMA induction by aurora kinase inhibitors.

PDF - 480KB, Figure S1. Levels of AKT, p-ERK and ERK change following 17AAG treatment; Figure S2. PUMA but not Bim is required for 17AAG-induced apoptosis in HCT 116 cells; Figure S3. PUMA is required for apoptosis induced by other Hsp90 inhibitors; Figure S4. p21 induction suppresses 17AAG-induced apoptosis; Figure S5. p53-KO HCT 116 cells are resistant to apoptosis induced by other Hsp90 inhibitors; Figure S6. 17AAG induces XBP1 splicing but XBP1 siRNA did not affect PUMA induction by 17AAG.

Oncogenic alterations in MET or anaplastic lymphoma kinase (ALK) have been identified in a variety of human cancers. Crizotinib (PF02341066) is a dual MET and ALK inhibitor and approved for the treatment of a subset of non–small cell lung carcinoma and in clinical development for other malignancies. Crizotinib can induce apoptosis in cancer cells, whereas the underlying mechanisms are not well understood. In this study, we found that crizotinib induces apoptosis in colon cancer cells through the BH3-only protein PUMA. In cells with wild-type p53, crizotinib induces rapid induction of PUMA and Bim accompanied by p53 stabilization and DNA damage response. The induction of PUMA and Bim is mediated largely by p53, and deficiency in PUMA or p53, but not Bim, blocks crizotinib-induced apoptosis. Interestingly, MET knockdown led to selective induction of PUMA, but not Bim or p53. Crizotinib also induced PUMA-dependent apoptosis in p53-deficient colon cancer cells and synergized with gefitinib or sorafenib to induce marked apoptosis via PUMA in colon cancer cells. Furthermore, PUMA deficiency suppressed apoptosis and therapeutic responses to crizotinib in xenograft models. These results establish a critical role of PUMA in mediating apoptotic responses of colon cancer cells to crizotinib and suggest that mechanisms of oncogenic addiction to MET/ALK-mediated survival may be cell type-specific. These findings have important implications for future clinical development of crizotinib. Mol Cancer Ther; 12(5); 777–86. ©2013 AACR.

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Aurora kinases play a key role in mitosis and are frequently overexpressed in a variety of tumor cells. Inhibition of aurora kinases results in mitotic arrest and death of cancer cells, and has been explored as an anticancer strategy. However, how aurora inhibition kills cancer cells is poorly understood. In this study, we found that inhibition of aurora kinases by siRNA or small-molecule inhibitors led to induction of p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, in colorectal cancer cells irrespective of p53 status. Deficiency in PUMA increased polyploidy, improved cell survival, and abrogated mitochondria-mediated apoptosis induced by aurora kinase inhibitors. In response to aurora kinase inhibition, PUMA was directly activated by p65 through the canonical NF-κB pathway following AKT inhibition. Furthermore, PUMA was necessary for the chemosensitization and in vivo antitumor effects of aurora kinase inhibitors in colon cancer cells. These results suggest that PUMA induction mediates the apoptotic response to mitotic arrest imposed by aurora kinase inhibition, and may be a useful indicator for the anticancer activity of aurora kinase inhibitors. Mol Cancer Ther; 13(5); 1298–308. ©2014 AACR.

Hsp90 is widely overexpressed in cancer cells and believed to be essential for the maintenance of malignant phenotypes. Targeting Hsp90 by small molecules has shown promise in solid and hematologic malignancies, which likely involves degradation of client oncoproteins in a cell-type–specific manner. In this study, we found that structurally unrelated Hsp90 inhibitors induce DNA damage and apoptosis via p53-dependent induction of PUMA, which indirectly triggers Bax activation and mitochondrial dysfunction in colon cancer cells. Deficiency in PUMA, BAX, or p53, at lesser extent, abrogated 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced apoptosis and mitochondrial dysfunction, and enhanced clonogenic cell survival. Furthermore, suppression of p53-dependent p21 induction or enhanced p53 activation synergized with 17-AAG to induce PUMA-dependent apoptosis. Finally, PUMA was found to mediate apoptotic and therapeutic responses to the 17-AAG analog 17-DMAG in xenografts. These results show an important role of the p53/PUMA/Bax axis in Hsp90 inhibitor–induced killing of p53 wild-type cells, and have important implications for their clinical applications. Mol Cancer Ther; 12(11); 2559–68. ©2013 AACR.

Aurora kinases play a key role in mitosis and are frequently overexpressed in a variety of tumor cells. Inhibition of aurora kinases results in mitotic arrest and death of cancer cells, and has been explored as an anti-cancer strategy. However, how aurora inhibition kills cancer cells is poorly understood. In this study, we found that inhibition of aurora kinases by siRNA or small-molecule inhibitors led to induction of p53-upregulated modulator of apoptosis (PUMA), a BH3-only BCL-2 family protein, in colorectal cancer cells irrespective of p53 status. Deficiency in PUMA increased polyploidy and abrogated mitochondria-mediated apoptosis induced by aurora kinase inhibitors. In response to aurora kinase inhibition, PUMA was directly activated by p65 through the canonical NF-kB pathway following AKT inhibition. Interestingly, aurora kinase inhibitors were found to preferentially kill KRAS-mutant colon cancer cells, which is associated with enhanced induction of PUMA and another BH3-only protein NOXA. Furthermore, PUMA was necessary for the chemo-sensitization and in vivo anti-tumor effects of aurora kinase inhibitors in colon cancer cells. These results suggest that PUMA induction mediates the apoptotic response to mitotic arrest imposed by aurora kinase inhibition, and may be a useful indicator for the anti-cancer activity of aurora kinase inhibitors. Citation Format: Jing Sun, Kyle Knickelbein, Kan He, Dongshi Chen, Jian Yu, Lin Zhang. Aurora kinase inhibitors require PUMA to induce apoptosis and preferentially kill KRAS-mutant colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2577. doi:10.1158/1538-7445.AM2015-2577

The mammalian target of rapamycin (mTOR) is commonly activated in colon cancer. mTOR complex 1 (mTORC1) is a major downstream target of the PI3K/ATK pathway and activates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. Rapamycin analogs Everolimus and Temsirolimus are non-ATP-competitive mTORC1 inhibitors, and suppress proliferation and tumor angiogenesis and invasion. We now show that apoptosis plays a key role in their anti-tumor activities in colon cancer cells and xenografts through the DR5, FADD and caspase-8 axis, and is strongly enhanced by TRAIL and 5-fluorouracil. The induction of DR5 by rapalogs is mediated by the ER stress regulator and transcription factor CHOP, but not the tumor suppressor p53, upon rapid and sustained inhibition of 4E-BP1 phosphorylation, and attenuated by eIF4E expression. ATP-competitive mTOR/PI3K inhibitors also promote DR5 induction and FADD-dependent apoptosis in colon cancer cells. These results establish activation of ER stress and the death receptor pathway as a novel anticancer mechanism of mTOR inhibitors. Citation Format: Kan He, Xingnan Zheng, Mei Li, Lin Zhang, Jian Yu. mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction upon 4E-BP1 dephosphorylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-040. doi:10.1158/1538-7445.AM2015-LB-040

Aurora kinases are a group of highly conserved protein kinases that play a key role in mitosis. It has been shown that aurora kinases are overexpressed in a variety of tumors, and their overexpression contributes to aneuploidy in cancer cells. Inhibitors of aurora kinases have been developed and showed promises in recent studies. However, the mechanisms by which aurora kinase inhibitors suppress tumor cell growth have remained unclear. In this study, we found that ZM-447439, an aurora B inhibitor that has been tested in a number of preclinical studies, suppressed colon cancer cell growth by inducing apoptosis through PUMA, a p53 downstream target and a BH3-only Bcl-2 family protein. Treatment with ZM-447439 strongly induced PUMA in a variety of colon cancer cells irrespective of their p53 status. The induction of PUMA by ZM-447439 is mediated by IκB-dependent activation of NF-κB, which directly binds to the PUMA promoter to activate its transcription. Deficiency in PUMA abrogated ZM-447439-induced apoptosis and caspase activation, and rendered ZM-447439 resistance in vitro and in vivo. Furthermore, chemosensitization by ZM-447439 was also found to be PUMA-dependent. Together, these results demonstrate a key role of PUMA-dependent apoptosis in mediating the therapeutic effect of aurora kinase inhibition. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to aurora kinase inhibitors. Citation Format: Jing Sun, Kan He, Jian Yu, Lin Zhang. Aurora kinase inhibition suppresses colon cancer cell growth through NF-κb-mediated PUMA induction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-97. doi:10.1158/1538-7445.AM2013-LB-97

Constitutive activation of pro-survival kinases has become a promising target of recently developed small molecules, while their biomarkers and resistance mechanisms remain largely unknown. In this study, we investigated the role of several BH3-only proteins PUMA, Bim and Noxa in apoptosis and growth suppression induced by different classes of targeted agents, including selective small molecule inhibitors to c-Met, IGFR, Hsp90, VEGFR and non selective kinases inhibitors in colon cancer cells and xenografts. Using isogenic colon cancer cells with targeted deletion in PUMA, Bim or Noxa, we demonstrated universal and persistent PUMA induction by all of these agents, which is mediated by either p53-dependent or -independent mechanisms. PUMA deficiency led to impaired apoptosis and caspase activation in colon cancer cells, and significantly blocked tumor cell apoptosis and therapeutic responses to these agents in xenografts. On the contrary, modulation of upstream signaling such as phosphorylation of PI3K/AKT was transient and involves diverse transcription factors. Interestingly, only a small subset of agents induced the expression of Bim or Noxa, or required Bim or Noxa for apoptosis induction. Moreover, small molecule BH3 mimetics, or enhanced PUMA levels resulted from another drug sensitized colon cancer cells to apoptosis induced by majority of these agents. Our results establish a critical role of the mitochondrial pathway and BH3-only proteins in mediating sensitivity of colon cancer cells to a wide variety of targeted-agents, and shed light on resistance mechanisms. In light of recent data on BH3-only proteins in therapy-induced apoptosis in other tumor types, our data suggest that modulations of selected BH3-only proteins, rather than their steady-state levels, might sever as potential biomarkers for predicting therapeutic responses in both treatment-naïve and pretreated patients, and provide a rationale for combination therapy in clinical trials. These novel preclinical findings can help guide future development and clinical use of targeted agents. Key words: kinase inhibitors; PUMA; Bim; Noxa; apoptosis; resistance mechanisms, colon cancer Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 932. doi:1538-7445.AM2012-932