PR72, a novel regulator of Wnt signaling required for Naked cuticle function

The Wnt signaling cascade is a central regulator of cell fate determination during embryonic development, whose deregulation contributes to oncogenesis. Naked cuticle is the first Wnt-induced antagonist found in this pathway, establishing a negative-feedback loop that limits the Wnt signal required for early segmentation. In addition, Naked cuticle is proposed to function as a switch, acting to restrict classical Wnt signaling and to activate a second Wnt signaling pathway that controls planar cell polarity during gastrulation movements in vertebrates. Little is known about the biochemical function of Naked cuticle or its regulation. Here we report that PR72, a Protein Phosphatase type 2A regulatory subunit of unknown function, interacts both physically and functionally with Naked cuticle. We show that PR72, like Naked cuticle, acts as a negative regulator of the classical Wnt signaling cascade, establishing PR72 as a novel regulator of the Wnt signaling pathway. Our data provide evidence that the inhibitory effect of Naked cuticle on Wnt signaling depends on the presence of PR72, both in mammalian cell culture and in Xenopus embryos. Moreover, PR72 is required during early embryonic development to regulate cell morphogenetic movements during body axis formation

E2F-7: a distinctive E2F family member with an unusual organization of DNA-binding domains

The E2F family of transcription factors play an important role in regulating cell cycle progression. We report here the characterization and functional properties of a new member of the human E2F family, referred to as E2F-7. E2F-7 has two separate DNA-binding domains, a feature that distinguishes E2F-7 from other mammalian E2F proteins, but resembling the organization of recently isolated E2F-like proteins from Arabidopsis. E2F-7 binds to DNA independently of a DP partner and delays cell cycle progression. Interestingly, E2F-7 modulates the transcription properties of other E2F proteins. A mutational analysis indicates that the integrity of both DNA-binding domains is required for cell cycle delay and transcriptional modulation. Biochemical results and protein modelling studies suggest that in binding to DNA interactions occur between the two DNA-binding domains, most probably as a homodimer, thereby mimicking the organization of an E2F/DP heterodimer. These structural and functional properties of E2F-7 imply a unique role in regulating cellular proliferation

A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer

A large-scale RNA interference screen to discover genes involved in trastuzumab resistance in breast cancer identified only PTEN as a modulator of drug sensitivity. Oncogenic mutants of PIK3CA (activator of the same pathway and frequently mutated in breast cancer) also conferred resistance to trastuzumab in cell culture. In a cohort of 55 breast cancer patients, activation of the PI3K pathway, as judged by the presence of oncogenic PIK3CA mutations or low PTEN expression, was associated with poor prognosis after trastuzumab therapy, and the combined analysis of PTEN and PIK3CA identified twice as many patients at increased risk for progression compared to PTEN alone. Thus, assessment of PI3K pathway activation may provide a biomarker to identify patients unlikely to respond to trastuzumab-based therap

ARID1A mutation sensitizes most ovarian clear cell carcinomas to BET inhibitors

Current treatment for advanced stage ovarian clear cell cancer is severely hampered by a lack of effective systemic therapy options, leading to a poor outlook for these patients. Sequencing studies revealed that ARID1A is mutated in over 50% of ovarian clear cell carcinomas. To search for a rational approach to target ovarian clear cell cancers with ARID1A mutations, we performed kinome-centered lethality screens in a large panel of ovarian clear cell carcinoma cell lines. Using the largest OCCC cell line panel established to date, we show here that BRD2 inhibition is predominantly lethal in ARID1A mutated ovarian clear cell cancer cells. Importantly, small molecule inhibitors of the BET (bromodomain and extra terminal domain) family of proteins, to which BRD2 belongs, specifically inhibit proliferation of ARID1A mutated cell lines, both in vitro and in ovarian clear cell cancer xenografts and patient-derived xenograft models. BET inhibitors cause a reduction in the expression of multiple SWI/SNF members including ARID1B, providing a potential explanation for the observed lethal interaction with ARID1A loss. Our data indicate that BET inhibition may represent a novel treatment strategy for a subset of ARID1A mutated ovarian clear cell carcinomas

NOTCH signaling limits the response of Low Grade Serous Ovarian Cancers to MEK inhibition

Low-grade serous ovarian cancer (LGSOC) is a rare subtype of epithelial ovarian cancer with high fatality rates in advanced stages due to its chemo-resistant properties. LGSOC is characterized by activation of MAPK signaling and recent clinical trials indicate that the MEK inhibitor (MEKi) trametinib may be a good treatment option for a subset of patients. Understanding MEKi resistance mechanisms and subsequent identification of rational drug combinations to suppress resistance may greatly improve LGSOC treatment strategies. Both gain-of-function and loss-of-function CRISPR-Cas9 genome-wide libraries were used to screen LGSOC cell lines to identify genes that modulate the response to MEKi. Overexpression of MAML2 and loss of MAP3K1 were identified, both leading to overexpression of the NOTCH target HES1, which has a causal role in this process as its knockdown reversed MEKi resistance. Interestingly, increased HES1 expression was also observed in selected spontaneous trametinib resistant clones, next to activating MAP2K1 (MEK1) mutations. Subsequent trametinib synthetic lethality screens identified SHOC2 downregulation as being synthetic lethal with MEKi. Targeting SHOC2 with pan-RAF inhibitors (pan-RAFi) in combination with MEKi was effective in parental LGSOC cell lines, in MEKi resistant derivatives, in primary ascites cultures from LGSOC patients and in LGSOC (cell line derived and patient-derived) xenograft mouse models. We found that the combination of pan-RAFi with MEKi downregulated HES1 levels in trametinib resistant cells, providing an explanation for the synergy that was observed. Combining MEKi with pan-RAFi may provide a promising treatment strategy for LGSOC patients, which warrants further clinical validation

Loss of ARID1A activates ANXA1, which serves as a predictive biomarker for trastuzumab resistance

Purpose: Despite the substantial progress in the development of targeted anticancer drugs, treatment failure due to primary or acquired resistance is still a major hurdle in the effective treatment of most advanced human cancers. Understanding these resistance mechanisms will be instrumental to improve personalized cancer treatment. Experimental Design: Genome-wide loss-of-function genetic screens were performed to identify genes implicated in resistance to HER2/PI3K/mTORtargeting agents in HER2+ breast cancer cell lines. Expression and adjuvant trastuzumab response data from the HER2+ breast cancer trials FinHer and Responsify were used to validate our findings in patient series. Results: We find that reduced ARID1A expression confers resistance to several drugs that inhibit the HER2/PI3K/mTOR signaling cascade at different levels. We demonstrate that ARID1A loss activates annexin A1 (ANXA1) expression, which is required for drug resistance through its activation of AKT. We find that the AKT inhibitor MK2206 restores sensitivity of ARID1A knockdown breast cancer cells to both the mTOR kinase inhibitor AZD8055 and trastuzumab. Consistent with these in vitro data, we find in two independent HER2+ breast cancer patient series that high ANXA1 expression is associated with resistance to adjuvant trastuzumab-based therapy. Conclusions: Our findings provide a rationale for why tumors accumulate ARID1A mutations and identify high ANXA1 expression as a predictive biomarker for trastuzumab-based treatment. Our findings also suggest strategies to treat breast cancers with elevated ANXA1 expression

Integrative Kinome Profiling Identifies mTORC1/2 Inhibition as Treatment Strategy in Ovarian Clear Cell Carcinoma

Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models.Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient-derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055.Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models.Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928-40. ©2018 AACR.status: publishe