Low-dose triple drug combination targeting the PI3K/AKT/mTOR pathway and the MAPK pathway is an effective approach in ovarian clear cell carcinoma

Advanced stage ovarian clear cell carcinoma (OCCC) is poorly responsive to platinum-based chemotherapy and has an unfavorable prognosis. Previous studies revealed heterogeneous mutations in PI3K/AKT/mTOR and MAPK pathway nodules converging in mTORC1/2 activation. Here, we aimed to identify an effective low-dose combination of PI3K/AKT/mTOR pathway and MAPK pathway inhibitors simultaneously targeting key kinases in OCCC to preclude single-inhibitor initiated pathway rewiring and limit toxicity. Small molecule inhibitors of mTORC1/2, PI3K and MEK1/2 were combined at monotherapy IC20 doses in a panel of genetically diverse OCCC cell lines (n = 7) to determine an optimal low-dose combination. The IC20 dose triple combination reduced kinase activity in PI3K/AKT/mTOR and MAPK pathways, prevented single-inhibitor induced feedback mechanisms and inhibited short and long-term proliferation in all seven cell lines. Finally, this low-dose triple drug combination treatment significantly reduced tumor growth in two genetically characterized OCCC patient-derived xenograft (PDX) models without resulting in weight loss in these mice. The effectiveness and tolerability of this combined therapy in PDX models warrants clinical exploration of this treatment strategy for OCCC and might be applicable to other cancer types with a similar genetic background

Role of the cytosolic loop C2 and the C-terminus of YidC in ribosome binding and insertion activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions

ARID1A mutant ovarian clear cell carcinoma: A clear target for synthetic lethal strategies

SWI/SNF chromatin remodeling complexes play an important role in the epigenetic regulation of chromatin structure and gene transcription. Mutual exclusive subunits in the SWI/SNF complex include the DNA targeting members ARID1A and ARID1B as well as the ATPases SMARCA2 and SMARCA4. SWI/SNF complexes are mutated across many cancer types. The highest mutation incidence is found in ARID1A, primarily consisting of deleterious mutations. Current advances have reported synthetic lethal interactions with the loss of ARID1A in several cancer types. In this review, we discuss targets that are only important for tumor growth in an ARID1A mutant context. We focus on synthetic lethal strategies with ARID1A loss in ovarian clear cell carcinoma, a cancer with the highest ARID1A mutation incidence (46-57%). ARID1A directed lethal strategies that can be exploited clinically include targeting of the DNA repair proteins PARP and ATR, and the epigenetic factors EZH2, HDAC2, HDAC6 and BRD2

Role of the cytosolic loop C2 and the C-terminus of YidC in ribosome binding and insertion activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion and this process is initiated by the assembly of YidC:ribosome nascent chain (RNC) complexes at the inner leaflet of the lipid bilayer. The positively charged C-terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant as deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo, but did not influence ribosome binding or substrate insertion, while loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C-terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC:RNC complex, allowing for co-translational membrane insertion, while loop C1 may be involved in the downstream chaperone activity of YidC or in other protein:protein interactions

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

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

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

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

Elucidating the Native Architecture of the YidC:Ribosome Complex

<p>Membrane protein biogenesis in bacteria occurs via dedicated molecular systems SecYEG and YidC that function independently and in cooperation. YidC belongs to the universally conserved Oxal/Alb3/YidC family of membrane insertases and is believed to associate with translating ribosomes at the membrane surface. Here, we have examined the architecture of the YidC:ribosome complex formed upon YidC-mediated membrane protein insertion. Fluorescence correlation spectroscopy was employed to investigate the complex assembly under physiological conditions. A slightly acidic environment stimulates binding of detergent-solubilized YidC to ribosomes due to electrostatic interactions, while YidC acquires specificity for translating ribosomes at pH-neutral conditions. The nanodisc reconstitution of the YidC to embed it into a native phospholipid membrane environment strongly enhances the YidC: ribosome complex formation. A single copy of YidC suffices for the binding of translating ribosome both in detergent and at the lipid membrane interface, thus being the minimal functional unit. Data reveal molecular details on the insertase functioning and interactions and suggest a new structural model for the YidC:ribosome complex. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.</p>