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To assess the implementation of an antihypertensive pathway order set to improve treatment of severe hypertension in pregnancy and the postpartum period in the inpatient setting.A multi-disciplinary task force created a hypertensive pathway order set and provided staff training. The order set allowed providers to initiate a treatment algorithm, which then gave nurses guidelines to recheck blood pressures and progressively increase short-acting antihypertensive dosage if needed. Pregnant and postpartum patients documented to have ≥2 consecutive severe range blood pressures in the year prior (2017) and the year after (2019) implementation of the pathway were included. Primary outcomes included whether any antihypertensive was given, whether it was given for all instances of severe hypertension, and time to antihypertensive administration.A total of 566 patients with severe hypertension were included-304 in the pre-implementation year and 262 in the post-implementation year. Significantly more patients received an antihypertensive at least once (67 % versus 80 %, p 0.01) and for all instances of severe hypertension (29 % versus 47 %, p 0.01) in the post-intervention cohort. There was a significant improvement in time to antihypertensive administration (24 versus 10 min, p 0.01).This study evaluates the efficacy of an antihypertensive intervention in the Southeast United States, which is particularly significant given the region's higher rates of hypertension and hypertension-related mortality. This study provides confirmatory evidence that implementation of a standardized order set along with measuring compliance and staff education is associated with improved treatment rates and time to treatment administration.

Supplementary Methods Supplementary Figure S1: Gö6976 selectively inhibits osteosarcoma cell growth through Chk1 inhibition in a p53-independent manner Supplementary Figure S2: Relationship between GDC-0425 sensitivity and Chk1 expression in cell lines derived from the indicated tissue types Supplementary Figure S3: Ras-MEK signaling is associated with Chk1 dependency Supplementary Figure S4: Feedback activation of Ras-MEK by Chk1 inhibition Supplementary Figure S5: BT-549 cells were treated with either GDC-0425 or cobimetinib alone or combination for 24 hr. Supplementary Figure S6: Synergistic anti-tumor growth activity by gemcitabine/GDC-0425 combination treatment in Chk1-dependent cancer cells.

PDF - 42KB, Bliss scores associated with combining each indicated agent with either GNE-783 or AZD-7762 in HT29 cells.

PDF - 43KB, In vivo potentiation of gemcitabine, SN-38, and TMZ wiht GNE-900.

GNE-900 potentiates the cytotoxicity of gemcitabine in HCT-116 isogenic variants, irrespective of NHEJ competence.

Kinase selectivity profile evaluated at 100 nM GNE-900

PDF file - 917 KB, Legends for Supplementary Figures: Supplementary Fig. S1: AKT1 and AKT3 are highly expressed in patient breast cancer tumors and AKT3 expression correlates with recurrence and ER negative status. Box and whisker plot of AKT isoforms expression in (A) normal versus breast carcinoma specimens, (B) tumors of different grade, and tumors with respect to (C) recurrence and (D) estrogen receptor status using Oncomine TCGA datasets. Supplementary Fig. S2: Tamoxifen induces cell proliferation in breast cancer cells with high levels of Akt. Venn diagrams show overlaps of differentially expressed genes (A) C, E and T treated parental MCF-7 versus Akt3 cells (MC:AC, ME:AE, and MT:AT). (B) C versus E treated parental MCF-7 (MC:ME) and T treated parental MCF-7 versus Akt overexpressing MCF-7 cells (MT:AT). Ingenuity pathway analysis was used to generate the network of differentially expressed genes (C) upon estrogen versus control treatment in parental MCF-7 cells and (D) upon tamoxifen treatment in parental MCF-7 versus Akt3 overexpressing cells. Genes shown in red are up- regulated, genes shown in green are down-regulated and central nodes are estradiol (C) and NFkB (D). Supplementary Fig. S3: Differential gene expression variations in breast cancer cells RT-qPCR confirmation analysis of (A) up-regulated and (B) down-regulated genes was examined in cells upon Akt overexpression. Fold change is shown relative to control treated parental MCF-7 cells. Supplementary Fig. S4: Altered gene expression in normal versus breast carcinoma specimens using the Oncomine database. Altered expression of (A) up-regulated and (B) down-regulated genes is shown. Fold changes (FC) of breast carcinoma were calculated relative to normal breast tissue and comparisons between groups were made by two-sample t-tests. Supplementary Fig. S5: (A) Knockdown efficiencies of siRRM2#1 and siRRM2#2 as compared to non-targeting control (NT C). (B) Western blots showing efficient knockdown of RRM2 protein in six breast cancer cell line. (C) Cellular proliferation between siRNA treatments were compared relative to NT C. (D) Knockdown efficiencies of siRRM2 remained high over seven days as validated by RT-qPCR. (E) Images were captured by phase contrast microscopy using a 4? objective at 20 h after removal of inserts for siRRM2 transfected and non-targeting control. siRRM2 significantly inhibited motility in Akt1 and Akt3 overexpressing MCF7 cells and MDA-MB-231 cells indicated by arrows.

PDF file - 184 KB, Legends for Supplementary Tables: Supplementary Table S1A: Primers used for real-time RT-PCR gene regulation and confirmation Supplementary Table S1B: Microarray data deposited into the Gene Expression Ominbus (GSE34291) using Agilent's platform GPL4133 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE34291) Supplementary Table S2: Genes modified upon hormonal treatment in MCF-7 and Akt3 overexpressing cells Supplementary Table S3: Ingenuity database gene networks associated with hormonal treatment and Akt over-expression Supplementary Table S4: Comparison of Fold change of selected genes by microarray analysis and RT-qPCR

Acquired tamoxifen resistance develops in the majority of hormone-responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biologic outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNA interference (RNAi)–mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated DNA damage and proapoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM inhibitor didox significantly reduced in vitro and in vivo growth. Thus, AKT-expressing breast cancer cells upregulate RRM2 expression, leading to increased DNA repair and protection from tamoxifen-induced apoptosis.Implications: These findings identify RRM2 as an AKT-regulated gene, which plays a role in tamoxifen resistance and may prove to be a novel target for effective diagnostic and preventative strategies. Mol Cancer Res; 12(3); 394–407. ©2013 AACR.

PDF - 66KB, A detailed description of the supplementary figures and tables.

Pharmacokinetics of GNE-900 in female CD-1 mice or male Sprague-Dawley rats.

Here we report that GNE-783, a novel checkpoint kinase-1 (CHK1) inhibitor, enhances the activity of gemcitabine by disabling the S- and G2 cell-cycle checkpoints following DNA damage. Using a focused library of 51 DNA-damaging agents, we undertook a systematic screen using three different cell lines to determine which chemotherapeutics have their activity enhanced when combined with GNE-783. We found that GNE-783 was most effective at enhancing activity of antimetabolite-based DNA-damaging agents; however, there was a surprisingly wide range of activity within each class of agents. We, next, selected six different therapeutic agents and screened these in combination with GNE-783 across a panel of cell lines. This revealed a preference for enhanced chemopotentiation of select agents within tumor types, as, for instance, GNE-783 preferentially enhanced the activity of temozolomide only in melanoma cell lines. Additionally, although p53 mutant status was important for the overall response to combinations with some agents; our data indicate that this alone was insufficient to predict synergy. We finally compared the ability of a structurally related CHK1 inhibitor, GNE-900, to enhance the in vivo activity of gemcitabine, CPT-11, and temozolomide in xenograft models. GNE-900 significantly enhanced activity of only gemcitabine in vivo, suggesting that strong chemopotentiation in vitro can translate into chemopotentiation in vivo. In conclusion, our results show that selection of an appropriate agent to combine with a CHK1 inhibitor needs to be carefully evaluated in the context of the genetic background and tumor type in which it will be used. Mol Cancer Ther; 12(11); 2285–95. ©2013 AACR.

PDF - 42KB, Comparison of bliss scores in p53 genetically matched HCT116 cell lines.

Representative data from a cell-based checkpoint abrogation assay.

PDF - 46KB, Bliss scores for each combination of agent and GNE-783 across a panel of cell lines.

PDF - 46KB, GNE-900 can potentiate the activity of gemcitabine, SN-38 and TMZ.

GNE-900 can potentiate the cytotoxicity of gemcitabine, SN-38 and pemetrexed.

Representative histograms from high content imaging studies.

PDF - 339KB, Profile of Bliss scores for each indicated agent in combination with GNE-783.

GNE-900 is more potent at chemopotentiation in p53 null HCT-116.

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Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G2–M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor. Mol Cancer Ther; 12(10); 1968–80. ©2013 AACR.

Representative data from enzyme assays evaluating the relative potency of GNE-900 against ChK1 or ChK2.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRAS

Purpose: Chk1 inhibition potentiates DNA-damaging chemotherapy by overriding cell-cycle arrest and genome repair. This phase I study evaluated the Chk1 inhibitor GDC-0425 given in combination with gemcitabine to patients with advanced solid tumors.Experimental Design: Patients received GDC-0425 alone for a 1-week lead-in followed by 21-day cycles of gemcitabine plus GDC-0425. Gemcitabine was initially administered at 750 mg/m2 (Arm A), then increased to 1,000 mg/m2 (Arm B), on days 1 and 8 in a 3 + 3 + 3 dose escalation to establish maximum tolerated dose (MTD). GDC-0425 was initially administered daily for three consecutive days; however, dosing was abbreviated to a single day on the basis of pharmacokinetics and tolerability. TP53 mutations were evaluated in archival tumor tissue. On-treatment tumor biopsies underwent pharmacodynamic biomarker analyses.Results: Forty patients were treated with GDC-0425. The MTD of GDC-0425 was 60 mg when administered approximately 24 hours after gemcitabine 1,000 mg/m2. Dose-limiting toxicities included thrombocytopenia (n = 5), neutropenia (n = 4), dyspnea, nausea, pyrexia, syncope, and increased alanine aminotransferase (n = 1 each). Common related adverse events were nausea (48%); anemia, neutropenia, vomiting (45% each); fatigue (43%); pyrexia (40%); and thrombocytopenia (35%). The GDC-0425 half-life was approximately 15 hours. There were two confirmed partial responses in patients with triple-negative breast cancer (TP53-mutated) and melanoma (n = 1 each) and one unconfirmed partial response in a patient with cancer of unknown primary origin.Conclusions: Chk1 inhibition with GDC-0425 in combination with gemcitabine was tolerated with manageable bone marrow suppression. The observed preliminary clinical activity warrants further investigation of this chemopotentiation strategy. Clin Cancer Res; 23(10); 2423–32. ©2016 AACR.

Supplemental Figure 3. Clinical trial tumor immunohistochemistry. Representative images from tumor biopsies were analyzed for pCDK1/2 and Ki-67expression. Representative images from a patient with NSCLC and unknown p53 mutation status were obtained 24 hours after administration of GDC-0425 alone (A, B), 48 hours after gemcitabine alone (C, D) and at a time point that was both 24 hours after GDC-0425 and 48 hours after gemcitabine (E, F). Quantitation of percent tumor cell Ki-67 positivity varied minimally between biopsies (G); pCDK1/2 increased from baseline after gemcitabine treatment, whereas the combination of GDC-0425 given 24 hours after gemcitabine suppressed the increase in pCDK1/2. Scale bar=40 µm.

Purpose: Non–small cell lung cancers (NSCLC) comprise multiple distinct biologic groups with different prognoses. For example, patients with epithelial-like tumors have a better prognosis and exhibit greater sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) pathway than patients with mesenchymal-like tumors. Here, we test the hypothesis that epithelial-like NSCLCs can be distinguished from mesenchymal-like NSCLCs on the basis of global DNA methylation patterns.Experimental Design: To determine whether phenotypic subsets of NSCLCs can be defined on the basis of their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative reverse transcriptase PCR and methylation-specific PCR in formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy.Results: We show that patterns of methylation divide NSCLCs into epithelial-like and mesenchymal-like subsets as defined by gene expression and that these signatures are similarly correlated in NSCLC cell lines and tumors. We identify multiple differentially methylated regions, including one in ERBB2 and one in ZEB2, whose methylation status is strongly associated with an epithelial phenotype in NSCLC cell lines, surgically resected tumors, and formalin-fixed biopsies from patients with NSCLC who went on to fail front-line chemotherapy.Conclusions: Our data show that patterns of DNA methylation can divide NSCLCs into two phenotypically distinct subtypes of tumors and provide proof of principle that differences in DNA methylation can be used as a platform for predictive biomarker discovery and development. Clin Cancer Res; 18(8); 2360–73. ©2012 AACR.

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Study design.

Supplemental Figure 2. Preclinical xenograft immunohistochemistry. HT-29 xenografts were analyzed for pCDK1/2 and Ki-67 expression. Representative images from 2 unique animals in each treatment group (n=5 animals per group) are shown. Ki-67 expression in surviving tumor cells is relatively consistent among treatment groups. pCDK1/2 expression increases from baseline (A, B) after gemcitabine treatment (I, J), whereas GDC-0425 alone (E, F) had little effect. In contrast, GDC-0425 given 16 hours after gemcitabine caused marked inhibition of the gemcitabine-induced expression of pCDK1/2. Scale bar=90 µm.

CCR Translation for This Article from DNA Methylation Profiling Defines Clinically Relevant Biological Subsets of Non–Small Cell Lung Cancer

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PDF file - 6626K, Validation of IHC antibody specificity for LDHB, LDHA, MCT1, and MCT4

PDF file - 963K, Additional top targets from RNAi screen that are specifically required for triple negative breast cancer

XLSX file - 25K, RNAi optimization and screening conditions

XLSX file - 41KB, IC50 values for kinase inhibitors used in the study.

PDF file - 1635KB, Elevated ALDH1A1 activity in KATO II and GTL-16 DTPs (S1). ALDHhigh cells exhibit properties similar to CSCs (S2). Effect of DS on growth of parental cells and DTPs (S3). DS has opposite effects on pre- and post- KI-treated cells (S4). ALDH family members are expressed at higher level in DTPs (S5). Role of specific ALDH isoforms and cell density in drug tolerance drug (S6). Effect of the alternative ALDH inhibitor gossypol on drug-tolerant cells (S7). Increased mitochondrial respiration and ROS levels in KATO II ALDHhigh cells (S8). DS effect is not mediated by inhibition of efflux pumps or through proteasome inhibition (S9).

XLSX file - 59K, Genes that comprise the glycolysis and oxidative phosphorylation gene signatures

XLSX file - 42KB, Statistical analysis of xenograft tumor data.

PDF file - 663K, Glycolysis signature genes that correlate to LDHB expression in basal-like tumors

PDF file - 257K, Animal weight change and tumor volume of individual xenograft tumors

PDF file - 791K, mRNA expression profiles of lactate dehydrogenase enzymes in breast cancer subtypes

XLSX file - 31K, Results from the RNAi screen and identification of hits

The emergence of technological innovations has created the opportunity to envision new approaches to discover therapeutics at scale. We combined advances in high content microscopy with arrayed CRISPR genome editing techniques and machine learning (ML) to build a rigorously controlled dataset enabling exploration of biology and chemistry at scale. Phenotypes from millions of perturbations in multiple cell types were embedded in a unified representation space and leveraged to accelerate discovery and reverse translation, ultimately yielding novel biological insights, and optimizing the advancement of lead molecular series through structure-activity relationships (SAR). Here, we demonstrate the capability of our platform to discover potential cancer therapies with distinct mechanisms of action. First, we describe the identification of a novel compound series that potentiates the effects of immunotherapy in syngeneic mouse models, producing complete responses and immunological memory, while also limiting peripheral inflammation. Specific novel chemical entities (NCEs) caused robust CD45+ cell influx into the tumor microenvironment and significantly attenuated exhausted T cells and immunosuppressive macrophages, thereby enhancing anti-tumor immunity. Strikingly, the same NCEs suppressed peripheral inflammation while sustaining elevated levels of intra-tumoral proinflammatory cytokines. Second, we highlight a novel and differentiated strategy to potentiate PARP inhibitor response in homologous repair deficient (HRD) - negative or HR-proficient ovarian cancers. NCEs altered the expression of genes within the DNA damage repair (DDR) network and cell cycle checkpoints to synergize with PARP inhibition in vivo and re-sensitized a PARP-resistant patient-derived xenograft (PDX) model. Collectively, we believe future efforts on the industrialization and integration of various technological innovations across biology, chemistry, automation, data science, and engineering will ultimately modernize drug discovery and radically improve patient lives. Citation Format: Jenny Rudnick, Kiran Nadella, Chase Neumann, Shane Rowley, Ethan Gardner, Shadi Swaidani, Aimee Iberg, Lu Chen, Daria Beshnova, Aurora Blucher, Rebecca Sarto Basso, Malini Rajan, Kevin Fales, Ashraf Saeed, Christopher Bailey, Weston Judd, Chrissy Egbert, Joel Ellis, John Ansede, Pouya Hadipour, Kevin Jessing, Janet Paulsen, Paul Rearden, Vamshi Manda, Sashi Kasimsetty, Sashi Kasimsetty, Michael Hancock, Harish Shankaran, Bryan Ellis, Meenakshy Iyer, Carl Brooks, Ashish Bhandari, Chris Gibson, Irit Rappley, Laura Schaevitz, Imran Haque, Hayley Donnella, Michael Cuccarese, Marie Evangelista. A phenomics platform combining imaging and artificial intelligence for rapid validation and advancement of novel oncology targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB071.