Jingshan Tong

Vertical suppression of the EGFR pathway prevents onset of resistance in colorectal cancers

Molecular targeted drugs are clinically effective anti-cancer therapies. However, tumours treated with single agents usually develop resistance. Here we use colorectal cancer (CRC) as a model to study how the acquisition of resistance to EGFR-targeted therapies can be restrained. Pathway-oriented genetic screens reveal that CRC cells escape from EGFR blockade by downstream activation of RAS-MEK signalling. Following treatment of CRC cells with anti-EGFR, anti-MEK or the combination of the two drugs, we find that EGFR blockade alone triggers acquired resistance in weeks, while combinatorial treatment does not induce resistance. In patient-derived xenografts, EGFR-MEK combination prevents the development of resistance. We employ mathematical modelling to provide a quantitative understanding of the dynamics of response and resistance to these single and combination therapies. Mechanistically, we find that the EGFR-MEK Combo blockade triggers Bcl-2 and Mcl-1 downregulation and initiates apoptosis. These results provide the rationale for clinical trials aimed at preventing rather than intercepting resistance.

FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation

Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7-inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA or p53. These cells are defective in apoptosis owing to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug-resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target.

Mcl-1 Degradation Is Required for Targeted Therapeutics to Eradicate Colon Cancer Cells

The Bcl-2 family protein Mcl-1 is often degraded in cancer cells subjected to effective therapeutic treatment, and defective Mcl-1 degradation has been associated with intrinsic and acquired drug resistance. However, a causal relationship between Mcl-1 degradation and anticancer drug responses has not been directly established, especially in solid tumor cells where Mcl-1 inhibition alone is insufficient to trigger cell death. In this study, we present evidence that Mcl-1 participates directly in determining effective therapeutic responses in colon cancer cells. In this setting, Mcl-1 degradation was induced by a variety of multikinase inhibitor drugs, where it relied upon GSK3β phosphorylation and FBW7-dependent ubiquitination. Specific blockade by genetic knock-in (KI) abolished apoptotic responses and conferred resistance to kinase inhibitors. Mcl-1-KI also suppressed the antiangiogenic and anti-hypoxic effects of kinase inhibitors in the tumor microenvironment. Interestingly, these same inhibitors also induced the BH3-only Bcl-2 family protein PUMA, which is required for apoptosis. Degradation-resistant Mcl-1 bound and sequestered PUMA from other prosurvival proteins to maintain cell survival, which was abolished by small-molecule Mcl-1 inhibitors. Our findings establish a pivotal role for Mcl-1 degradation in the response of colon cancer cells to targeted therapeutics, and they provide a useful rational platform to develop Mcl-1-targeting agents that can overcome drug resistance. Cancer Res; 77(9); 2512-21. ©2017 AACR.

Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation

Bax is a proapoptotic Bcl-2 family member that has a central role in the initiation of mitochondria-dependent apoptosis. However, the mechanism of Bax activation during apoptosis remains unsettled. It is believed that the activation of Bax is mediated by either dissociation from prosurvival Bcl-2 family members, or direct association with BH3-only members. Several interaction sites on Bax that mediate its interactions with other Bcl-2 family members, as well as its proapoptotic activity, have been identified in previous studies by other groups. To rigorously investigate the functional role of these interaction sites, we knocked in their respective mutants using HCT116 colon cancer cells, in which apoptosis induced by several stimuli is strictly Bax-dependent. Bax-mediated apoptosis was intact upon knock-in (KI) of K21E and D33A, which were shown to block the interaction of Bax with BH3-only activators. Apoptosis was partially reduced by KI of D68R, which impairs the interaction of Bax with prosurvival members, and S184V, a constitutively mitochondria-targeting mutant. In contrast, apoptosis was largely suppressed by KI of L70A/D71A, which blocks homo-oligomerization of Bax and its binding to prosurvival Bcl-2 family proteins. Collectively, our results suggest that the activation of endogenous Bax in HCT116 cells is dependent on its homo-oligomerization sites, but not those previously shown to interact with BH3-only activators or prosurvival proteins only. We therefore postulate that critical interaction sites yet to be identified, or mechanisms other than protein-protein interactions, need to be pursued to delineate the mechanism of Bax activation during apoptosis.

BRAFV600E -dependent Mcl-1 stabilization leads to everolimus resistance in colon cancer cells

mTOR activation is commonly caused by oncogenic mutations in RAS/RAF/MAPK and PI3K/AKT pathways, and promotes cancer progression and therapeutic resistance. However, mTOR inhibitors show limited single agent efficacy in patients. mTOR inhibitors suppress tumor cell growth and angiogenesis, and have recently been shown to induce death receptor/FADD-dependent apoptosis in colon cancers. Using a panel of BRAF V600E and WT colorectal cancer cell lines and in vitro selected resistant culture, and xenograft models, we demonstrate here that BRAFV600E confers resistance to mTOR inhibitors. Everolimus treatment disrupts the S6K1-IRS-2/PI3K negative feedback loop, leading to BRAF V600E-dependent activation of ERK and Mcl-1 stabilization in colon cancer cells, which in turn blocks the crosstalk from the death receptor to mitochondria. Co-treatment with inhibitors to Mcl-1, PI3K, RAF or MEK restores mTOR inhibitor-induced apoptosis by antagonizing Mcl-1 or abrogating ERK activation in BRAFV600E cells. Our findings provide a rationale for genotype-guided patient stratification and potential drug combinations to prevent or mitigate undesired activation of survival pathways induced by mTOR inhibitors.

FBW7-Dependent Mcl-1 Degradation Mediates the Anticancer Effect of Hsp90 Inhibitors

Heat shock protein 90 (Hsp90) is widely overexpressed in cancer cells and necessary for maintenance of malignant phenotypes. Hsp90 inhibition induces tumor cell death through degradation of its client oncoproteins and has shown promises in preclinical studies. However, the mechanism by which Hsp90 inhibitors kill tumor cells is not well-understood. Biomarkers associated with differential sensitivity and resistance to Hsp90 inhibitors remain to be identified. In this study, we found that colorectal cancer cells containing inactivating mutations of FBW7, a tumor suppressor and E3 ubiquitin ligase, are intrinsically insensitive to Hsp90 inhibitors. The insensitive colorectal cancer cells lack degradation of Mcl-1, a prosurvival Bcl-2 family protein. Hsp90 inhibition promotes GSK3β-dependent phosphorylation of Mcl-1, which subsequently binds to FBW7 and undergoes ubiquitination and proteasomal degradation. Specifically blocking Mcl-1 phosphorylation by genetic knock-in abrogates its degradation and renders in vitro and in vivo resistance to Hsp90 inhibitors, which can be overcame by Mcl-1–selective small-molecule inhibitors. Collectively, our findings demonstrate a key role of GSK3β/FBW7-dependent Mcl-1 degradation in killing of colorectal cancer cells by Hsp90 inhibitors and suggest FBW7 mutational status as a biomarker for Hsp90-targeted therapy. Mol Cancer Ther; 16(9); 1979–88. ©2017 AACR.

PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors

Significance Necroptosis is a regulated form of necrotic cell death that is important in physiology and human diseases. However, the signaling process leading to eventual cell death in necroptosis remains unclear. We show that PUMA, a proapoptotic BH3-only Bcl-2 family member, is induced and plays a role in necroptotic death. PUMA induction enhances necroptotic signaling by promoting the release of mitochondrial DNA and activation of cytosolic DNA sensors. We provide genetic evidence for the functional role of PUMA in necroptosis-mediated developmental defects in mice. Our results demonstrate a previously unknown function of Bcl-2 family proteins and reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptosis in vitro and in vivo. Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo.

Restoring PUMA induction overcomes KRAS -mediated resistance to anti-EGFR antibodies in colorectal cancer

Intrinsic and acquired resistance to anti-EGFR antibody therapy, frequently mediated by a mutant or amplified KRAS oncogene, is a significant challenge in the treatment of colorectal cancer (CRC). However, the mechanism of KRAS-mediated therapeutic resistance is not well understood. In this study, we demonstrate that clinically used anti-EGFR antibodies, including cetuximab and panitumumab, induce killing of sensitive CRC cells through p73-dependent transcriptional activation of the pro-apoptotic Bcl-2 family protein PUMA. PUMA induction and p73 activation are abrogated in CRC cells with acquired resistance to anti-EGFR antibodies due to KRAS alterations. Inhibition of aurora kinases preferentially kills mutant KRAS CRC cells and overcomes KRAS-mediated resistance to anti-EGFR antibodies in vitro and in vivo by restoring PUMA induction. Our results suggest that PUMA plays a critical role in meditating the sensitivity of CRC cells to anti-EGFR antibodies, and that restoration of PUMA-mediated apoptosis is a promising approach to improve the efficacy of EGFR-targeted therapy.

Mcl-1 phosphorylation without degradation mediates sensitivity to HDAC inhibitors by liberating BH3-only proteins

Mcl-1, a prosurvival Bcl-2 family protein, is frequently overexpressed in cancer cells and plays a critical role in therapeutic resistance. It is well known that anticancer agents induce phosphorylation of Mcl-1, which promotes its binding to E3 ubiquitin ligases and subsequent proteasomal degradation and apoptosis. However, other functions of Mcl-1 phosphorylation in cancer cell death have not been well characterized. In this study, we show in colon cancer cells that histone deacetylase inhibitors (HDACi) induce GSK3β-dependent Mcl-1 phosphorylation, but not degradation or downregulation. The in vitro and in vivo anticancer effects of HDACi were dependent on Mcl-1 phosphorylation and were blocked by genetic knock-in of a Mcl-1 phosphorylation site mutant. Phosphorylation-dead Mcl-1 maintained cell survival by binding and sequestering BH3-only Bcl-2 family proteins PUMA, Bim, and Noxa, which were upregulated and necessary for apoptosis induction by HDACi. Resistance to HDACi mediated by phosphorylation-dead Mcl-1 was reversed by small-molecule Mcl-1 inhibitors that liberated BH3-only proteins. These results demonstrate a critical role of Mcl-1 phosphorylation in mediating HDACi sensitivity through a novel and degradation-independent mechanism. These results provide new mechanistic insights on how Mcl-1 maintains cancer cell survival and suggest that Mcl-1-targeting agents are broadly useful for overcoming therapeutic resistance in cancer cells.Significance: These findings present a novel degradation-independent function of Mcl-1 phosphorylation in anticancer therapy that could be useful for developing new Mcl-1-targeting agents to overcome therapeutic resistance. Cancer Res; 78(16); 4704-15. ©2018 AACR.