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AZD6738 and gemcitabine synergistically inhibit cell growth in a panel of PDAC cell lines

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra–S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra–S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo. In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell-cycle arrest, and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. In vivo, AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC. Mol Cancer Ther; 17(8); 1670–82. ©2018 AACR.

Genetic alterations likely to sensitize to ATR inhibition in the human PDAC cell lines

Supplementary Figure S1. Analytical characterization of INT016-ADCs; Supplementary Figure S2. Validation of B7-H4 antibodies for use in IHC; Supplementary Figure S3. Relationship between B7-H4 expression level and heterogeneity in the studied human triple-negative cohort; Supplementary Figure S4. Binding of antibody INT016 to HEK 293 cells stably expressing human, mouse, or cynomolgus monkey B7-H4; Supplementary Figure S5. Binding of antibody INT016 to human breast cancer cell lines and to HT29 cells stably expressing human B7-H4; Supplementary Figure S6. Internalization and lysosomal trafficking of INT016; Supplementary Figure S7. In vitro stability of INT016-ADCs in mouse and cynomolgus monkey serum; Supplementary Figure S8. In vitro cytotoxicity of INT016-ADCs in HT29 and HT29-huB7-H4 isogenic cell lines; Supplementary Figure S9. In vivo efficacy of INT016-ADCs in the MX-1 xenograft model; Supplementary Figure S10. Growth rate analysis of MX-1 in vivo efficacy study comparing INT016-ADCs from days 14–40.

Surface plasmon resonance; In vitro cytotoxicity assays; In vitro proliferation assays; Internalization assays; Subcellular localization microscopy; Immunoblotting; Statistical analysis

Supplementary Table S1. Primary antibodies used for immunoblotting; Supplementary Table S2. Characterization of ADCs evaluated in this study; Supplementary Table S3. B7-H4 expression in human normal tissue; Supplementary Table S4. Binding affinity of anti-B7-H4 antibody INT016 Fab to human, cynomolgus monkey, and mouse B7-H4, measured by surface plasmon resonance.

Purpose:We evaluated the activity of AZD8205, a B7-H4–directed antibody–drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models.Experimental Design:IHC and deep-learning–based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly–Gly–Phe–Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models.Results:Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala–PEG8–TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker–payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4–expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance.Conclusions:These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4–expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482).See related commentary by Pommier and Thomas, p. 991

Supplementary Data from ATR Inhibitor AZD6738 (Ceralasertib) Exerts Antitumor Activity as a Monotherapy and in Combination with Chemotherapy and the PARP Inhibitor Olaparib

AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2–M cell-cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication and homologous recombination repair. In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM pathway or that harbor putative drivers of replication stress such as CCNE1 amplification. This translated to in vivo antitumor activity, with tumor control requiring continuous dosing and free plasma exposures, which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimization of dose and schedules in vivo and showed superior antitumor activity at lower doses compared with that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3 to5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent. Significance: This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose–schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.

Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor approved for HER2+ metastatic breast and gastric cancer. Clinically, T-DXd has demonstrated antitumor activity in both HER2+ and HER2-low cancers. PARP performs a key role as a mediator in the resolution of topoisomerase1 cleavage complexes (TOP1cc) through recruitment of tyrosyl-DNA phosphodiesterase 1 (TDP1). We hypothesized that combination of T-DXd with the PARP1/2 inhibitor olaparib, will halt the resolution of TOP1cc and enhance the activity of T-DXd. Methods: To test the hypothesis, we evaluated the antiproliferative ability of the combination of T-DXd with olaparib in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. The combination was also evaluated in vivo in two non-HRD models, a HER2+ (KPL4) and HER2-low (JIMT1) cell line xenograft at 3mg/kg and 10mg/kg Q3W for T-DXd, as well as 100mg/kg BID of olaparib. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a human 2D in vitro bone marrow progenitor assay. Results: We found that the combination had enhanced in vitro cell killing activity over single agents in 8/27 of the models tested. The benefit was present in both Homologous Repair Deficient (HRD) as well as wild-type models, suggesting it does not depend on HRD (as defined by mutations in DNA damage repair genes). In vivo, the combination was more active than monotherapy of either compound in both KPL4 (28d TGI of 76% with T-DXd, olaparib 17% TGI, and T-DXd + olaparib 94.4% TGI; p=0.009) and JIMT1 (28d TGI of 80.8% with T-DXd, olaparib 52%, and T-DXd + Olaparib 88.1%; p=0.03). In the in vitro human bone marrow assay, the combination demonstrated modest enhancement over monotherapy activity (average Loewe Synergy Score of 2.5). To explore the ability to optimize therapeutic index, we tested alternative doses and schedules of the combination in vivo. Specifically, we tested whether lower doses of T-DXd or delayed administration of olaparib would provide greater activity. We found that combination of 3mg/kg of T-DXd with olaparib provided greater activity in KPL4 xenograft model than 3mg/kg T-DXd alone (69% TGI for combination vs. 35% TGI for monotherapy on d28), but this was no greater than high dose 10mg/kg monotherapy T-DXd (76% TGI on d28; p=0.19). Interestingly, 7-day delay of olaparib in combination with 10mg/kg T-DXd provided greater activity (91% TGI on d28) than monotherapy T-DXd or olaparib alone (76% TGI and 17% TGI on d28, respectively). Conclusions: These results suggest that T-DXd combined with olaparib is a potentially active combination in breast cancer, with preclinical activity demonstrated in HRD and non-HRD models. Citation Format: Theresa Proia, Yann Wallez, Azadeh Cheraghchi Bashi, Zena Wilson, Suzanne Randle, Mark Anderton, Danielle Carroll, Zeshaan Rasheed, J. Elizabeth Pease, Elisabetta Leo, Jerome Mettetal. Activity and tolerability of combination of trastuzumab deruxtecan with olaparib in preclinical HER2+ and HER2-low breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-18.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I (TOP1) inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. Despite its promising early clinical signals, drug resistance is a challenge that may emerge with time. The mechanisms of Dato-DXd resistance are currently unknown. Herein, we created and investigated novel models of Dato-DXd resistance to characterize resistance mechanisms and identify novel combinations to overcome Dato-DXd resistance. We used a cyclical dosing method to develop two Dato-DXd acquired resistant NCI-N87 gastric cancer cell line models from in vitro cells (N87-IVR) and in vivo tumors (N87-EVR). The N87-IVR and N87-EVR cells were found to be more than 40-fold resistant to Dato-DXd compared to the parental N87 cells. First, we assessed TROP2 levels, Dato-DXd binding and expression of drug efflux pumps known to have TOP1-inhibitor substrates between the parental and resistant cell populations. Minor changes in each of these were not sufficient to drive Dato-DXd resistance in the drug-tolerant models. Subsequently, we performed a proteogenomic analysis to identify which proteins and pathways were modulated in the resistant phenotype. Both the N87-IVR and N87-EVR cells were divergent from the parental N87 cells and, interestingly, they showed similar yet distinct profiles. Among other changes, our analysis revealed a dramatic loss of SLFN11, a putative DNA/RNA helicase, in both resistant models. Previously published data has shown that a combination with an ATR inhibitor (ATRi) can re-sensitize TOP1 inhibitor-resistant cells in SLFN11-low settings. Consistent with these data, we observed that the ATRi ceralasertib (AZD6738) in combination with Dato-DXd re-sensitizes the resistant NCI-N87 cells in vitro and in vivo. In addition to SLFN11 loss, we also identified a number of other modulations with the potential to drive resistance, e.g. downregulation of the caveolin internalization machinery and interferon response genes. These findings help us refine the mechanism of action of Dato-DXd and shed light on the potential mechanisms of Dato-DXd resistance that may emerge clinically. These data have been used to uncover new biomarkers of Dato-DXd sensitivity and can lead to the development of new clinical strategies to combat Dato-DXd resistance, e.g. the combination with ATRi. Citation Format: Anna Przybyla, Chetan Rane, Gopi Patel, Deepa Bhavsar, Shenghua Wen, David Clark, Yeoun Jin Kim, Alan Lau, Yann Wallez, Jay Mettetal, Wenyan Zhong, Matthew Sung. Leveraging novel Dato-DXd resistance models to inform biomarker discovery and rational combinations to combat drug resistance [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 LB227.

Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor. T-DXd is approved for patients with HER2+ metastatic breast cancer who have received two or more anti-HER2 based regimens as well as HER2+ gastric cancer, and has demonstrated antitumor activity in both HER2+ and HER2-low cancers clinically. Trapping of Topo1cc by DXd in targeted cells, induces replication stress (RS) and double strand breaks (DSB), activating a DNA Damage Repair (DDR) response. Therefore we aimed to investigate if combination of T-DXd with DDR inhibitors enhances efficacy and durability of response. Methods: We evaluated the antiproliferative effect of the combination of T-DXd with AZD6738 (ATR inhibitor), AZD1775 (WEE1 inhibitor), AZD1390 (ATM inhibitor), in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. Combinations were also evaluated in vivo in the HER2-high gastric NCI-N87 cell line xenograft. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a 2D in vitro human bone marrow progenitor assay. Results: We found that the combination with all DDRi significantly enhanced in vitro cell killing activity over single agents in many of the models tested (12/27 for AZD6738, 9/27 for AZD1775, 6/27 for AZD1390). This was not limited to models with DNA damage repair mutations (e.g. BRCA1, BRCA2, or ATM). Mechanistically, T-DXd activated PARP and the ATR, Wee1, ATM pathways, inducing cell cycle arrest. Combination of T-DXd with AZD6738 and AZD1775 inhibited the RS response (pATR-T1989, pChk1-S345) and cell cycle arrest (pCdc2-Y15), while AZD1390 inhibited the DSB response pathway (pATM-S1981, pRad50-S685, pKap1-S824, pChk2-T68). All DDR inhibitors exacerbated DNA damage (pRPA-S4/8, gH2AX) and cell death (cCasp3). In the in vitro human bone marrow (BM) assay, T-DXd had no synergistic interaction with AZD6738 and AZD1775 inhibitors, but synergy with AZD1390. In vivo, combination therapy with all DDRi tested achieved superior Tumor Growth Inhibition (TGI) compared to monotherapy alone. At day 41 in NCI-N87, 3mg/kg of T-DXd provided TGI of 74% while monotherapy arms of 25mg/kg BID AZD6738, 60mg/kg (5-days on/2-days off) AZD1775 or 10mg/kg AZD1390 provided 68%, 47%, 31% TGI respectively. The combinations of T-DXd with AZD6738, AZD1775 and AZD1390 provided TGI of >100% (19% regression), 90%, and 99% respectively. Conclusions: These results suggest that AZD6738, AZD1775, and AZD1390 all potentiate T-DXd activity in vitro and in vivo, and the combination interactions with AZD6738 and AZD1775 were much stronger in some tumor models over bone marrow cells in vitro. These data suggest further investigation of these combinations in clinical studies may be warranted. Citation Format: Yann Wallez, Theresa Proia, Azadeh Cheraghchi-Bashi-Astaneh, Ankur Karmokar, Zena Wilson, Suzanne Randle, Mark Anderton, Stephen Durant, Elisabetta Leo, Alan Lau, Mark O'Connor, Jerome Mettetal. Activity and tolerability of combinations of trastuzumab deruxtecan (T-DXd) with inhibitors of the DNA damage response in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5298.

Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor, approved for HER2+ metastatic breast cancer. Clinically, T-DXd has demonstrated antitumor activity in both HER2+ and HER2-low cancers. Due to the role of PARP1 in resolution of DNA damage induced by topoisomerase I trapping, we tested the combination of the next generation PARP1-selective inhibitor AZD5305 with T-DXd. Methods: We evaluated the antiproliferative ability of the combination of T-DXd with AZD5305 in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. The combination was also evaluated in vivo in two non-HRD HER2+ models, KPL4 (Breast) and NCI-N87 (Gastric) at doses of 3mg/kg and 10mg/kg Q3W for T-DXd combined with 0.01, 0.1, and 1 mg/kg QD of AZD5305. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a human 2D in vitro bone marrow progenitor assay. Results: We found that the combination had enhanced in vitro cell killing activity over single agents in 8/27 of the models tested. The benefit was present in both Homologous Recombination Deficient (HRD) as well as Homologous Recombination proficient, suggesting it does not depend on HRD (as defined by mutations in DNA damage repair genes). Mechanistically, T-DXd activated PARP and the combination of T-DXd with AZD5305 abrogated PARP1 auto-parylation, leading to enhanced DNA damage (gH2AX, pRPA-S4/8) and cell death (cCasp3). In vivo, the combination was well tolerated and more active than monotherapy of either compound in both KPL4 (at 30 days the growth inhibition was 95% at 10mg/kg T-DXd, 10% at 1mg/kg AZD5305, and 100% TGI with 97% regression with T-DXd + AZD5305) and NCI-N87 (at 41 days TGI of 74% with 10mg/kg T-DXd, 47% with 1mg/kg AZD5305, and 100% TGI with 40% regression for T-DXd + AZD5305; p100% TGI with 72% regression on day 30) vs. monotherapy T-DXd alone (95% TGI). Conclusions: These results suggest that T-DXd combined with the next generation PARP1 inhibitor AZD5305 is a potentially active combination, with preclinical activity demonstrated in HRD and HR proficient models. Further, the dose and scheduling may warrant exploration clinically to optimize therapeutic index. Citation Format: Yann Wallez, Theresa Proia, Elisabetta Leo, Laura Bradshaw, Zena Wilson, Joe’l Owusu, Azadeh Cheraghchi-Bashi-Astaneh, Anna Staniszewska, Mark O’Connor, Sabina Cosulich, Jerome Mettetal. Activity and tolerability of combination of trastuzumab deruxtecan with the next generation PARP1-selective inhibitor AZD5305 in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1142.

BackgroundChemotherapy and targeted agent anti-cancer efficacy is largely dependent on the proliferative state of tumours, as exemplified by agents that target DNA synthesis/replication or mitosis. As a result, cell cycle specificities of a number of cancer drugs are well known. However, they are yet to be described in a quantifiable manner.MethodsA scalable cell synchronisation protocol used to screen a library of 235 anti-cancer compounds exposed over six hours in G1 or S/G2 accumulated AsPC-1 cells to generate a cell cycle specificity (CCS) score.FindingsThe synchronisation method was associated with reduced method-related cytotoxicity compared to nocodazole, delivering sufficient cell cycle purity and cell numbers to run high-throughput drug library screens. Compounds were identified with G1 and S/G2-associated specificities that, overall, functionally matched with a compound's target/mechanism of action. This annotation was used to describe a synergistic schedule using the CDK4/6 inhibitor, palbociclib, prior to gemcitabine/AZD6738 as well as describe the correlation between the CCS score and published synergistic/antagonistic drug schedules.InterpretationThis is the first highly quantitative description of cell cycle-dependent drug sensitivities that utilised a tractable and tolerated method with potential uses outside the present study. Drug treatments such as those shown to be G1 or S/G2 associated may benefit from scheduling considerations such as after CDK4/6 inhibitors and being first in drug sequences respectively.FundingCancer Research UK (CRUK) Institute core grants C14303/A17197 and C9545/A29580. The Li Ka Shing Centre where this work was performed was generously funded by CK Hutchison Holdings Limited, the University of Cambridge, CRUK, The Atlantic Philanthropies and others.

Simple Summary Renal cell carcinoma (RCC) is the eighth leading malignancy in the world, accounting for 4% of all cancers with poor outcome when metastatic. Protein kinases are highly druggable proteins, which are often aberrantly activated in cancers. The aim of our study was to identify candidate targets for metastatic clear cell renal cell carcinoma therapy, using chemo-genomic-based high-throughput screening. We found that the combined inhibition of the CK2 and ATM kinases in renal tumor cells and patient-derived tumor samples induces synthetic lethality. Mechanistic investigations unveil that this drug combination triggers apoptosis through HIF-2α-(Hypoxic inducible factor HIF-2α) dependent reactive oxygen species (ROS) overproduction, giving a new option for patient care in metastatic RCC. Abstract Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

BackgroundPersonalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC.MethodsThrough kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo.ResultsComplete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts.ConclusionsATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra–S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra–S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo. In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell-cycle arrest, and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. In vivo, AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC. Mol Cancer Ther; 17(8); 1670–82. ©2018 AACR.

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard-of-care, but shows very limited anti-tumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra-S-phase DNA damage response (DDR), plays a central role in safeguarding cells from replication stress (RS) and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra-S-phase checkpoint activation and evaluated the anti-tumor potential of this combination in vitro and in vivo. In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell cycle arrest and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. In vivo, AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC.

Combination of cytotoxic therapy with emerging DNA damage response inhibitors (DDRi) has been limited by tolerability issues. However, the goal of most combination trials has been to administer DDRi with standard-of-care doses of chemotherapy. We hypothesized that mechanism-guided treatment scheduling could reduce the incidence of dose-limiting toxicities and enable tolerable multitherapeutic regimens. Integrative analyses of mathematical modeling and single-cell assays distinguished the synergy kinetics of WEE1 inhibitor (WEE1i) from CHEK1 inhibitor (CHK1i) by potency, spatiotemporal perturbation, and mitotic effects when combined with gemcitabine. These divergent properties collectively supported a triple-agent strategy, whereby a pulse of gemcitabine and CHK1i followed by WEE1i durably suppressed tumor cell growth. In xenografts, CHK1i exaggerated replication stress without mitotic CDK hyperactivation, enriching a geminin-positive subpopulation and intratumoral gemcitabine metabolite. Without overt toxicity, addition of WEE1i to low-dose gemcitabine and CHK1i was most effective in tumor control compared with single and double agents. Overall, our work provides quantitative insights into the mechanisms of DDRi chemosensitization, leading to the rational development of a tolerable multitherapeutic regimen. Significance: Multiple lines of mechanistic insight regarding DNA damage response inhibitors rationally guide the preclinical development of a tolerable multitherapeutic regimen. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/11/3054/F1.large.jpg. Cancer Res; 78(11); 3054–66. ©2018 AACR.

Structurally, protein kinase CK2 consists of two catalytic subunits (α and α') and two regulatory subunits (β), which play a critical role in targeting specific CK2 substrates. Compelling evidence shows the complexity of the CK2 cellular signaling network and supports the view that this enzyme is a key component of regulatory protein kinase networks that are involved in several aspects of cancer. CK2 both activates and suppresses the expression of a number of essential oncogenes and tumor suppressors, and its expression and activity are upregulated in blood tumors and virtually all solid tumors. The prognostic significance of CK2α expression in association with various clinicopathological parameters highlighted this kinase as an adverse prognostic marker in breast cancer. In addition, several recent studies reported its implication in the regulation of the epithelial-to-mesenchymal transition (EMT), an early step in cancer invasion and metastasis. In this review, we briefly overview the contribution of CK2 to several aspects of cancer and discuss how in mammary epithelial cells, the expression of its CK2β regulatory subunit plays a critical role in maintaining an epithelial phenotype through CK2-mediated control of key EMT-related transcription factors. Importantly, decreased CK2β expression in breast tumors is correlated with inefficient phosphorylation and nuclear translocation of Snail1 and Foxc2, ultimately leading to EMT induction. This review highlights the pivotal role played by CK2β in the mammary epithelial phenotype and discusses how a modest alteration in its expression may be sufficient to induce dramatic effects facilitating the early steps in tumor cell dissemination through the coordinated regulation of two key transcription factors.

ERK is the effector kinase of the RAS-RAF-MEK-ERK signaling cascade, which promotes cell transformation and malignancy in many cancers and is thus a major drug target in oncology. Kinase inhibitors targeting RAF or MEK are already used for the treatment of certain cancers, such as melanoma. Although the initial response to these drugs can be dramatic, development of drug resistance is a major challenge, even with combination therapies targeting both RAF and MEK. Importantly, most resistance mechanisms still rely on activation of the downstream effector kinase ERK, making it a promising target for drug development efforts. Here, we report the design and structural/functional characterization of a set of bivalent ERK inhibitors that combine a small molecule inhibitor that binds to the ATP-binding pocket with a peptide that selectively binds to an ERK protein interaction surface, the D-site recruitment site (DRS). Our studies show that the lead bivalent inhibitor, SBP3, has markedly improved potency compared to the small molecule inhibitor alone. Unexpectedly, we found that SBP3 also binds to several ERK-related kinases that contain a DRS, highlighting the importance of experimentally verifying the predicted specificity of bivalent inhibitors. However, SBP3 does not target any other kinases belonging to the same CMGC branch of the kinome. Additionally, our modular click chemistry inhibitor design facilitates the generation of different combinations of small molecule inhibitors with ERK-targeting peptides.

Therapeutic targeting of the DNA damage response (DDR) has the potential to improve the poor survival outcomes of pancreatic ductal adenocarcinoma (PDAC). Many common genetic alterations in PDAC augment replication stress, which activates Ataxia-telangiectasia and Rad3-related kinase (ATR). We have demonstrated previously, efficacy of the ATR inhibitor (AZD6738) in PDAC models, particularly when combined with gemcitabine (gem) (Wallez et al, MCT, 2018). This combination strongly induces activation of Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), indicating the induction of double-strand breaks. It has been suggested that ATM-deficiency can sensitize cancer cells to ATR inhibitors. This study sought to assess the relevance of this finding to PDAC and to interrogate the distinct roles of ATM and DNA-PK in response to ATRi/gem in PDAC cell lines. The ATM inhibitor, AZD0156, sensitized human pancreatic cancer cell lines (MIA PaCa-2, HPAF-II, AsPC-1) to AZD6738, as determined by SRB assays (e.g. MIA PaCa-2; AZD6738 GI50 = 3.8 µM, versus 1.3 µM in the presence of 30 nM AZD0156) and in longer term colony forming assays (surviving fraction (SF) (1 µM AZD6738); 78 +/- 0.9%, SF (1 µM AZD6738 + 30nM AZD0156); 11 +/- 4.2%). However, ATM knockdown with siRNA did not sensitize to AZD6738, nor to the combination of AZD6738/gem. This suggests that the presence of kinase-inhibited ATM at sites of DNA damage is more deleterious to PDAC cells than deficiency of ATM protein expression. We established that AZD6738/gem could induce phosphorylation of the canonical targets of ATM (Chk2, KAP1) in MIA PaCa-2 when ATM was knocked down or inhibited. DNA-PK was also activated in response to exposure to ATRi/gem and we postulated that it was responsible for the Chk2/KAP1 activation. We then demonstrated that Chk2/KAP1 activation could be abrogated by DNA-PK inhibition with NU7441. Furthermore, in an ATM-null pancreatic cancer cell line, AZD6738/gem-induced KAP1 phosphorylation was also abrogated by inhibition of DNA-PK. Thus, DNA-PK appears to be responsible for the downstream activation of Chk2 and KAP1, induced by AZD6738/gem in PDAC cells. Therefore, as well as revealing that inhibition of ATM using the novel inhibitor AZD0156 is highly deleterious to ATR inhibited PDAC cells, we have identified a compensatory mechanism via DNA-PK, which may be relevant for maximizing the therapeutic potential of DDR inhibitors in PDAC. Since DNA-PK clearly plays a dominant role in the DDR signaling pathways, we are now assessing whether DNA-PK inhibition synergizes with AZD6738+/- gem. Citation Format: Charles R. Dunlop, Yann Wallez, Sandra Bernaldo de Quirós Fernández, Saadia A. Karim, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 271.

Members of the novel SH2-containing protein (NSP) and Crk-associated substrate (Cas) protein families form multidomain signaling platforms that mediate cell migration and invasion through a collection of distinct signaling motifs. Members of each family interact via their respective C-terminal domains, but the mechanism of this association has remained enigmatic. Here we present the crystal structures of the C-terminal domain from the NSP protein BCAR3 and the complex of NSP3 with p130Cas. BCAR3 adopts the Cdc25-homology fold of Ras GTPase exchange factors, but it has a 'closed' conformation incapable of enzymatic activity. The structure of the NSP3-p130Cas complex reveals that this closed conformation is instrumental for interaction of NSP proteins with a focal adhesion-targeting domain present in Cas proteins. This enzyme-to-adaptor conversion enables high-affinity, yet promiscuous, interactions between NSP and Cas proteins and represents an unprecedented mechanistic paradigm linking cellular signaling networks.

The marginal zone is a cellular niche bordering the marginal sinus of the spleen that contains specialized B-cell and macrophage subsets poised to capture bloodborne antigens. Marginal zone B cells are retained in this niche by integrin-mediated signaling induced by G protein-coupled receptors (GPCRs) and, likely, the B-cell receptor (BCR). Sphingosine-1-phosphate (S1P) signaling via the S1P family of GPCRs is known to be essential for B-cell localization in the marginal zone, but little is known about the downstream signaling events involved. Here, we demonstrate that the adaptor protein SHEP1 is required for marginal zone B-cell maturation. SHEP1 functions in concert with the scaffolding protein CasL, because we show that SHEP1 and CasL are constitutively associated in B cells. SHEP1 association is required for the BCR or S1P receptor(s) to induce the conversion of CasL into its serine/threonine hyperphosphorylated form, which is important for lymphocyte adhesion and motility. Thus, SHEP1 orchestrates marginal zone B-cell movement and retention as a key downstream effector of the BCR and S1P receptors.

Introduction The desmoplastic Tumour MicroEnvironment (TME) in Pancreatic Ductal Adenocarcinoma (PDA) plays a key role in drug delivery, metabolism and resistance; drug interventions in turn regulate tumour metabolism. Mass Spectrometry Imaging (MSI) techniques may provide information about regional tumour metabolic profile and drug delivery to enhance our understanding of TME heterogeneity and its impact on drug efficacy and resistance. Material and methods Multimodal MSI acquisition of the tissue distribution of gemcitabine, the ATR inhibitor AZD6738, their metabolites and the endogenous metabolome was performed in a KPC GEMM mouse model of PDA. Targeted analysis of the compounds and their metabolites were used to evaluate drug delivery. Small molecule quantitation including haem and metabolic markers such as ATP/ADP/AMP, were used together with H and E and staining for known molecular biomarkers including Pan-CK and αSMA to characterise tissue architecture. Untargeted analyses including statistical identification of discriminative or colocalised metabolites were used to identify de novo endogenous metabolite biomarkers and cellular phenotypes driving tumour heterogeneity. Results and discussions MSI revealed significant intratumoural heterogeneity of drug delivery and drug metabolism. Highest delivery of the parent compounds (AZD6738 and dFdC) were found to colocalise with haem in areas confirmed histologically to be necrotic and haemorrhagic. dFdC metabolism appeared related to TME metabolic heterogeneity. The active and inactive metabolites of gemcitabine (including dFdCTP and dFdU) demonstrated differential distribution, both from the parent compounds and each other in the tissue. Unsupervised clustering based segmentation and colocalisation analysis of the MSI metabolomic data enabled identification and characterisation of these distinct tissue regions based on similarities in their metabolic profile. Conclusion We have shown that MSI allows spatial resolution of drug delivery, metabolism and that MSI-based metabolomics analysis enables detection of greater tumour heterogeneity than visible by traditional pathology methods such as H and E. Combining this with biomarker information from Imaging Mass Cytometry (IMC) may enable identification of the cell types and phenotypes responsible for the differential metabolic effects observed with combination therapies. Innovative, information-rich technologies such as IMC and MSI may drive greater understanding of the impact of tumour heterogeneity on drug efficacy in vivo and ultimately in patients.

International audience; Because angiogenesis is a key step in a number of pathologic processes, including tumor growth and atherosclerosis, many research studies have investigated the regulatory signals active at various stages of vascular invasion. The differential activities of the endothelial junction protein vascular endothelial (VE)-cadherin reflect the versatile behavior of endothelial cells between vascular quiescence and angiogenesis. VE-cadherin function and signaling are deeply modified in proliferating cells, and this conversion is accompanied by phosphorylation of the protein on tyrosine residues and enhanced transcription of its gene. Recent advances in the complex interplay between protein tyrosine kinases and phosphatases regulating VE-cadherin phosphorylation and function are discussed in this review.

Mutations in oncogenes, tumor suppressor and DNA damage response (DDR) mediator genes drive or permit malignant transformation but also increase endogenous replication stress. The serine/threonine kinase ATR plays a critical role in safeguarding genome integrity from such replication stress and several studies have demonstrated the increased reliance of cancer cells on ATR function. We investigated the therapeutic opportunities for the ATR inhibitor, AZD6738, in combination with DNA damaging or DDR-targeted agents, in the context of pancreatic ductal adenocarcinoma (PDAC). We evaluated four DNA-damaging agents (gemcitabine, 5-fluorouracil, oxaliplatin, SN38 (the active metabolite of irinotecan)) and three DDR-targeted agents (Wee1 inhibitor (AZD1775), Chk1 inhibitor (MK8776), PARP inhibitor (AZD2281)), each in combination with AZD6738 at multiple concentrations. Efficacy of these combinations was tested in growth inhibition assays in vitro, using a panel of cell lines in order to capture some of the genetic heterogeneity observed in PDAC: two human cell lines and four lines from the KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse. Synergistic growth inhibition was identified applying both Bliss Independence and Loewe models, using Combenefit software. All the KPC mouse cell lines were sensitive to AZD6738 as a single agent, with GI50 ranging from 346 to 566 nM. MIA PaCa-2 were sensitive to AZD6738, achieving >90% growth inhibition, with GI50 of 2.2 μM. PANC-1 cells were less sensitive, with GI50 21 μM and achieving only ~60% GI, at the highest concentration tested. PANC-1 cells are also less sensitive to gemcitabine than the other cell lines. Synergy was detected in most of the cell lines, with each of the seven drug combinations tested. The combinations of AZD6738 with gemcitabine and with oxaliplatin showed synergy in all cell lines tested. We next investigated scheduling of the gemcitabine/ATRi combination, at the specific GI50 concentrations for each cell line, using kinetic live-cell imaging assays. Concurrent treatment of gemcitabine/ATRi for 16h proved to be most effective, almost completely inhibiting cell growth for more than three days after washout. Sequential treatment (irrespective of the order) or shorter pulses (8h) were less effective. Maintaining ATRi after gemcitabine washout further enhanced growth inhibition for most cell lines. Mechanistically, ATRi impaired Chk1 activation (p-Ser345) and, in combination with gemcitabine, strongly potentiated DNA damage (gamma H2AX). Maintaining ATRi after gemcitabine washout helped to sustain the level of DNA damage. In vivo studies are underway to determine whether the gemcitabine/ATRi combination enhances efficacy compared to gemcitabine alone. The ATRi/oxaliplatin combination is also being investigated in vitro and in vivo using similar methods. Several genes have been described in the literature to increase the reliance on ATR functions when altered. Mining two published datasets (TCGA, 186 samples and UTSW, Nat. Commun. 2015, 109 samples) we have investigated the frequencies at which 21 of these genes were altered in human PDAC. Overall ~95% of PDAC samples exhibit at least one (9% only one, 28% two and 57% three or more) genetic alteration likely to sensitize to ATRi, potentially improving the therapeutic index of combination approaches. Thus, combinations including ATRi merit further evaluation as they have the potential to be effective in the treatment of patients with PDAC. Citation Format: Yann Wallez, Siang-Boon Koh, Venkata Sai Vivek Bhogadi, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The ATR inhibitor, AZD6738, synergizes with other DNA damage response inhibitors and genotoxic drugs in pancreatic ductal adenocarcinoma cell lines: Opportunities for new therapeutic combinations [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B19.

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3. BCAR1 and BCAR3 bind tightly to each other through their C-terminal domains, thus potentially connecting their associated signaling networks. However, recent studies using BCAR1 and BCAR3 interaction mutants concluded that association between the two proteins is not critical for many of their interrelated activities regulating breast cancer malignancy. We report that these previously used BCAR mutations fail to cause adequate loss-of-function of the complex. By using structure-based BCAR1 and BCAR3 mutants that lack the ability to interact, we show that BCAR3-induced antiestrogen resistance in MCF7 breast cancer cells critically depends on its ability to bind BCAR1. Interaction with BCAR3 increases the levels of phosphorylated BCAR1, ultimately potentiating BCAR1-dependent antiestrogen resistance. Furthermore, antiestrogen resistance in cells overexpressing BCAR1/BCAR3 correlates with increased ERK1/2 activity. Inhibiting ERK1/2 through overexpression of the regulatory protein PEA15 negates the resistance, revealing a key role for ERK1/2 in BCAR1/BCAR3-induced antiestrogen resistance. Reverse-phase protein array data show that PEA15 levels in invasive breast cancers correlate with patient survival, suggesting that PEA15 can override ERK1/2 activation by BCAR1/BCAR3 and other upstream regulators. We further uncovered that the BCAR3-related NSP3 can also promote antiestrogen resistance. Thus, strategies to disrupt BCAR1-BCAR3/NSP3 complexes and associated signaling networks could ultimately lead to new breast cancer therapies.

Sensitization of cancer cells to gemcitabine (Gem) has been shown with checkpoint kinase CHEK1 (CHK1) and WEE1 inhibitors. Our aim is to determine, using in vitro and in vivo models, the optimal regime for application of the triple combination in pancreatic cancer. First, we have investigated the dual combinations of Gem + either CHEK1 or WEE1 inhibitors in growth inhibition assays in vitro. Synergistic growth inhibition was identified using both Bliss Independence and Loewe models, in MIA PaCa-2 cells at 10 – 30 nM Gem + 300 – 1000 nM CHEK1 inhibitor MK8776; or 10 – 30 nM Gem + 30 – 100 nM WEE1 inhibitor MK1775. The synergistic concentrations were submaximal, i.e. below the single agent GI50 concentrations, which were 30 nM for Gem, 6 μM for MK8776, and 500 nM for MK1775. The Panc-1 cell line is more resistant to Gem as a single agent, but synergy was evident: in colony forming assays the dual combination of either 30 nM gem + 300 nM MK1775 or 30 nM Gem + 1 μM MK8776 inhibited colony formation by 97 +/- 1.6 % and 91 +/- 2% respectively. Single agent Gem inhibited colony formation by only 23 +/- 4 % and MK1775 or MK8776 did not inhibit colony formation at these concentrations. Next, we investigated scheduling of the Gem/CHEK1i/Wee1i triple combination, at the synergistic concentrations, with kinetic live-cell imaging assays. MIA PaCa-2 cells treated continuously with 10 nM Gem plus 1 μM MK8776 showed durable growth inhibition over 72 hours. However, if both drugs were washed off after 24 hours the Gem + CHEK1i-treated cells recovered and began to proliferate within 24 hours. Different schedules of the trio were tested, and the most durable growth inhibition (> 5 days) was obtained when Gem + MK8776 were washed off at 24 hours and replaced with 300 nM MK1775, whereas 300 nM MK1775 in the absence of Gem + MK8776 pretreatment did not significantly inhibit growth, compared to control. Different scheduling options were also tested in Panc-1 cells in colony forming assays, and again the most effective schedule was Gem + MK8776 for 24 hours, followed by MK1775. MIA PaCa-2 xenograft studies are now underway, initially with the dual combination, to be followed with the drug trio of simultaneous Gem + MK8776, followed by MK1775. To reduce the likelihood of toxicity, Gem doses lower than the typical “full” dose (100 mg/kg IP twice per week) were tested. We found that 25 mg/kg Gem twice in a day (8h apart), twice per week was not well tolerated even as a single agent, but both 25 and 50 mg/kg Gem once in a day, twice per week was tolerated when co-dosed with 25 mg/kg MK8776, and when this combination was followed 8 hours later by 60 mg/kg MK1775 (OG). A pharmacokinetic study revealed that plasma Gem (dFdC) concentrations were not altered by co-dosing Gem with MK8776, but intratumor dFdCTP (the active intracellular metabolite of Gem) was elevated at 1 – 8 hours after the 25 mg/kg Gem + MK8776 dose (AUC0 –last 49 +/- 7.9 hr.pmoles/mg tissue) compared to mice with 25 mg/kg Gem alone (AUC0-last 21 +/- 2.6 hr.pmoles/mg tissue). CHEK1 target inhibition was demonstrated in vivo, with abrogation of Gem-induced CHEK1 S296 autophosphorylation for at least 4h post Gem + MK8776 dosing, using Western blot analyses of tumor lysate. There was also increased γH2AX and pRPA32 S4/8 at 4 - 8 hours post-dose with Gem + MK8776 compared to Gem alone, indicating enhanced DNA damage. Pharmacodynamic and efficacy studies with the triple combination, will determine whether enhanced efficacy can be observed, when compared to full dose, single agent gemcitabine (100 mg/kg IP twice per week). Citation Format: Siang-Boon Koh, Frances M. Richards, Yann Wallez, Duncan I. Jodrell.{Authors}. Evaluation of scheduling for triple therapy gemcitabine/CHEK1 inhibitor/WEE1 inhibitor in pancreatic cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B62.

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion–targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas-Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.

International audience; Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes.

SUMMARY ERK1/2 kinases are the principal effectors of a central signaling cascade that converts extracellular stimuli into cell proliferation and migration responses and, when deregulated, can promote cell oncogenic transformation. The scaffolding protein PEA-15 is a death effector domain (DED) protein that directly interacts with ERK1/2 and affects ERK1/2 subcellular localization and phosphorylation. Here, to understand this ERK1/2 signaling complex, we have solved the crystal structures of PEA-15 bound to three different ERK2 phospho-conformers. The structures reveal that PEA-15 uses a bipartite binding mode, occupying two key docking sites of ERK2. Remarkably, PEA-15 can efficiently bind the ERK2 activation loop in the critical Thr-X-Tyr region in different phosphorylation states. PEA-15 binding triggers an extended allosteric conduit in dually phosphorylated ERK2, disrupting key features of active ERK2. At the same time PEA-15 binding protects ERK2 from dephosphorylation, thus setting the stage for immediate ERK activity upon its release from the PEA-15 inhibitory complex.