Large-scale Pan-cancer Cell Line Screening Identifies Actionable and Effective Drug Combinations.

Oncology drug combinations can improve therapeutic responses and increase treatment options for patients. The number of possible combinations is vast and responses can be context-specific. Systematic screens can identify clinically relevant, actionable combinations in defined patient subtypes. We present data for 109 anticancer drug combinations from AstraZeneca's oncology small molecule portfolio screened in 755 pan-cancer cell lines. Combinations were screened in a 7 × 7 concentration matrix, with more than 4 million measurements of sensitivity, producing an exceptionally data-rich resource. We implement a new approach using combination Emax (viability effect) and highest single agent (HSA) to assess combination benefit. We designed a clinical translatability workflow to identify combinations with clearly defined patient populations, rationale for tolerability based on tumor type and combination-specific "emergent" biomarkers, and exposures relevant to clinical doses. We describe three actionable combinations in defined cancer types, confirmed in vitro and in vivo, with a focus on hematologic cancers and apoptotic targets. SIGNIFICANCE: We present the largest cancer drug combination screen published to date with 7 × 7 concentration response matrices for 109 combinations in more than 750 cell lines, complemented by multi-omics predictors of response and identification of "emergent" combination biomarkers. We prioritize hits to optimize clinical translatability, and experimentally validate novel combination hypotheses. This article is featured in Selected Articles from This Issue, p. 695.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

mTOR inhibition amplifies the anti-lymphoma effect of PI3Kß/δ blockage in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kß/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kß/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kß/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kß/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kß/δ signaling and thus identifies a previously unappreciated role of the PI3Kß isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kß/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.

AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kß/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors.

The PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kß inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kßi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kßi resistance through AKT. Conversely deletion of PIK3CA, ERBB2, ERBB3 increased PI3Kßi sensitivity while modulation of RRAGC, LAMTOR1, LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kßi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kß/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kßi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kßi/AKTi sensitive and resistant breast tumours.

Fragment-Based Design of a Potent MAT2a Inhibitor and in Vivo Evaluation in an MTAP Null Xenograft Model.

MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.

Drug mechanism-of-action discovery through the integration of pharmacological and CRISPR screens.

Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism-of-action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti-cancer drugs with genome-wide CRISPR loss-of-function screens in 484 cell lines to systematically investigate cellular drug mechanism-of-action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein-protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin-protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on-target and off-target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss-of-fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss-of-function screens can elucidate mechanism-of-action to advance drug development.

LSD1 inhibitors disrupt the GFI1 transcription repressor complex.

Pharmacologic inhibition of KDM1A (Lysine Demethylase 1A) induces differentiation in certain subtypes of acute myeloid leukemia. Our recent studies reveal this is dependent upon drug-induced disruption of the GFI1 (Growth Factor Independent 1) transcription repressor complex, leading to activation of enhancers distributed close to genes controlling monocytic lineage differentiation.

Combined Inhibition of PI3Kß and mTOR Inhibits Growth of PTEN-null Tumors.

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kß isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kß inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kß and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective in vivo controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers. In vitro, the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. In vivo biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kß inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. Mol Cancer Ther; 17(11); 2309-19. ©2018 AACR.

Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia.

Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1's histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes.

Inhibiting PI3Kß with AZD8186 Regulates Key Metabolic Pathways in PTEN-Null Tumors.

Purpose: PTEN-null tumors become dependent on the PI3Kß isoform and can be targeted by molecules such as the selective PI3Kß inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kß are poorly defined.Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models.Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kß also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis.Conclusions: This study identifies novel mechanistic biomarkers of PI3Kß inhibition in PTEN-null tumors supporting the concept that targeting PI3Kß may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584-95. ©2017 AACR.

Sensitivity to PI3K and AKT inhibitors is mediated by divergent molecular mechanisms in subtypes of DLBCL.

Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors.

Identification of differential PI3K pathway target dependencies in T-cell acute lymphoblastic leukemia through a large cancer cell panel screen.

Selective phosphoinositide 3-kinase (PI3K)/AKT/mTOR inhibitors are currently under evaluation in clinical studies. To identify tumor types that are sensitive to PI3K pathway inhibitors we screened compounds targeting PI3Kα/δ (AZD8835), PI3Kß/δ (AZD8186), AKT (AZD5363) and mTORC1/2 (AZD2014) against a cancer cell line panel (971 cell lines). There was an enrichment of hematological malignancies that were sensitive to AKT and mTOR inhibition, with the greatest degree of sensitivity observed in T-cell acute lymphoblastic leukemia (T-ALL). We found that all NOTCH mutant T-ALL cell lines were sensitive to AKT and mTORC1/2 inhibitors, with only partial sensitivity to agents that target the PI3K α, ß or δ isoforms. Induction of apoptosis only occurred following AKTi treatment in cell lines with PTEN protein loss and high levels of active AKT. In summary, we have demonstrated that T-ALL cell lines show differential sensitivity to inhibition at different nodes in the PI3K/AKT/mTOR pathway and inhibiting AKT or mTOR may have a therapeutic benefit in this disease setting.

Frequent Derepression of the Mesenchymal Transcription Factor Gene FOXC1 in Acute Myeloid Leukemia.

Through in silico and other analyses, we identified FOXC1 as expressed in at least 20% of human AML cases, but not in normal hematopoietic populations. FOXC1 expression in AML was almost exclusively associated with expression of the HOXA/B locus. Functional experiments demonstrated that FOXC1 contributes to a block in monocyte/macrophage differentiation and enhances clonogenic potential. In in vivo analyses, FOXC1 collaborates with HOXA9 to accelerate significantly the onset of symptomatic leukemia. A FOXC1-repressed gene set identified in murine leukemia exhibited quantitative repression in human AML in accordance with FOXC1 expression, and FOXC1(high) human AML cases exhibited reduced morphologic monocytic differentiation and inferior survival. Thus, FOXC1 is frequently derepressed to functional effect in human AML.

Low PIP4K2B expression in human breast tumors correlates with reduced patient survival: A role for PIP4K2B in the regulation of E-cadherin expression.

Phosphatidylinositol-5-phosphate (PtdIns5P) 4-kinase ß (PIP4K2B) directly regulates the levels of two important phosphoinositide second messengers, PtdIns5P and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2]. PIP4K2B has been linked to the regulation of gene transcription, to TP53 and AKT activation, and to the regulation of cellular reactive oxygen accumulation. However, its role in human tumor development and on patient survival is not known. Here, we have interrogated the expression of PIP4K2B in a cohort (489) of patients with breast tumor using immunohistochemical staining and by a meta-analysis of gene expression profiles from 2,999 breast tumors, both with associated clinical outcome data. Low PIP4K2B expression was associated with increased tumor size, high Nottingham histological grade, Ki67 expression, and distant metastasis, whereas high PIP4K2B expression strongly associated with ERBB2 expression. Kaplan-Meier curves showed that both high and low PIP4K2B expression correlated with poorer patient survival compared with intermediate expression. In normal (MCF10A) and tumor (MCF7) breast epithelial cell lines, mimicking low PIP4K2B expression, using short hairpin RNA interference-mediated knockdown, led to a decrease in the transcription and expression of the tumor suppressor protein E-cadherin (CDH1). In MCF10A cells, knockdown of PIP4K2B enhanced TGF-ß-induced epithelial to mesenchymal transition (EMT), a process required during the development of metastasis. Analysis of gene expression datasets confirmed the association between low PIP4K2B and low CDH1expression. Decreased CDH1 expression and enhancement of TGF-ß-induced EMT by reduced PIP4K2B expression might, in part, explain the association between low PIP4K2B expression and poor patient survival.

CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1.

There is a lack of rapid cell-based assays that read out enzymatic inhibition of the histone demethylase LSD1 (lysine-specific demethylase 1). Through transcriptome analysis of human acute myeloid leukemia THP1 cells treated with a tranylcypromine-derivative inhibitor of LSD1 active in the low nanomolar range, we identified the cell surface marker CD86 as a sensitive surrogate biomarker of LSD1 inhibition. Within 24h of enzyme inhibition, there was substantial and dose-dependent up-regulation of CD86 expression, as detected by quantitative polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. Thus, the use of CD86 expression may facilitate screening of compounds with putative LSD1 inhibitory activities in cellular assays.

LSD1 inhibition: a therapeutic strategy in cancer?

INTRODUCTION: The role of epigenetic dysfunction in cancer is increasingly appreciated. This has raised the question as to whether enzymes that regulate the structure and function of chromatin might represent novel therapeutic targets. The histone demethylase LSD1 is one such candidate and novel, potent inhibitors are under development. AREAS COVERED: The literature on LSD1 (also known as KDM1A, AOF2, BHC110 or KIAA0601) was identified in Pubmed and is herein discussed. Areas covered include the structure and enzymatic activity of LSD1, its role in chromatin regulatory complexes, its functional roles in normal and malignant tissue, pharmacological inhibitors of its activity and their putative therapeutic roles. EXPERT OPINION: Pre-clinical data supporting a therapeutic role for LSD1 inhibitors are most encouraging in acute myeloid leukaemia, although optimal dosing strategies and beneficial combinations with other agents remain unclear. Studies making use of potent, selective LSD1 inhibitors active in the nanomolar range are required to establish therapeutic indications in other subtypes of haematological malignancy, and in solid tumours.

The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells.

Using a mouse model of human MLL-AF9 leukemia, we identified the lysine-specific demethylase KDM1A (LSD1 or AOF2) as an essential regulator of leukemia stem cell (LSC) potential. KDM1A acts at genomic loci bound by MLL-AF9 to sustain expression of the associated oncogenic program, thus preventing differentiation and apoptosis. In vitro and in vivo pharmacologic targeting of KDM1A using tranylcypromine analogs active in the nanomolar range phenocopied Kdm1a knockdown in both murine and primary human AML cells exhibiting MLL translocations. By contrast, the clonogenic and repopulating potential of normal hematopoietic stem and progenitor cells was spared. Our data establish KDM1A as a key effector of the differentiation block in MLL leukemia, which may be selectively targeted to therapeutic effect.

Quantitative analysis and modeling of glucocorticoid-controlled gene expression.

AIMS: Glucocorticoid hormones are used extensively in the clinic for the treatment of acute lymphoblastic leukemia. Despite intensive research, the molecular mechanisms of glucocorticoid receptor (GR)-mediated transcriptional events that lead to the induction of apoptosis of leukemia cells, as well as the causes for the development of resistance in leukemia patients, are not yet understood. It is thought that the B-cell lymphoma 2 family members that control apoptosis, including some of the GR target genes, may play an important role in deciding cell fate. In this report we have employed pathway modeling due to the recent discovery of its usefulness as a tool for improving understanding of the mechanisms of cellular signaling, and in discovering new therapeutic targets for the treatment of various diseases. MATERIALS & METHODS: Detailed kinetics of GR autoregulation, as well as the kinetics of expression of its target genes and proteins Bcl-xL, Bim, Bmf and GILZ in glucocorticoid responsive and resistant leukemia cell lines were carried out. Subsequently in order to obtain further insight into the molecular mechanisms of GR signaling in this pathway a dynamic model of the induction of these genes and proteins by GR was constructed. RESULTS: The simulations were in good agreement with the observed experimental data suggesting that Bim was induced between 6 and 10 h after the addition of the synthetic glucocorticoid dexamethasone, possibly through rapid glucocorticoid dependent modulation of an unknown factor. Simulations and experimental results also suggested that Bmf induction did not require novel protein synthesis, and is a potential direct GR target. CONCLUSION: This combination of experimental analysis and model development initiates a virtuous cycle enabling further data integration and model expansion, and constitutes a novel promising framework towards a global mechanistic understanding of GR function.

The role of glucocorticoid receptor phosphorylation in Mcl-1 and NOXA gene expression.

BACKGROUND: The cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) mediated phosphorylation of glucocorticoid receptor (GR) exerts opposite effects on GR transcriptional activity and affects other posttranslational modifications within this protein. The major phosphorylation site of human GR targeted by MAPK family is the serine 226 and multiple kinase complexes phosphorylate receptor at the serine 211 residue. We hypothesize that GR posttranslational modifications are involved in the determination of the cellular fate in human lymphoblastic leukemia cells. We investigated whether UV signalling through alternative GR phosphorylation determined the cell type specificity of glucocorticoids (GCs) mediated apoptosis. RESULTS: We have identified putative Glucocorticoid Response Elements (GREs) within the promoter regulatory regions of the Bcl-2 family members NOXA and Mcl-1 indicating that they are direct GR transcriptional targets. These genes were differentially regulated in CEM-C7-14, CEM-C1-15 and A549 cells by glucocorticoids and JNK pathway. In addition, our results revealed that the S211 phosphorylation was dominant in CEM-C7-14, whereas the opposite was the case in CEM-C1-15 where prevalence of S226 GR phosphorylation was observed. Furthermore, multiple GR isoforms with cell line specific patterns were identified in CEM-C7-14 cells compared to CEM-C1-15 and A549 cell lines with the same antibodies. CONCLUSIONS: GR phosphorylation status kinetics, and site specificity as well as isoform variability differ in CEM-C7-14, CEM-C1-15, and A549 cells. The positive or negative response to GCs induced apoptosis in these cell lines is a consequence of the variable equilibrium of NOXA and Mcl-1 gene expression potentially mediated by alternatively phosphorylated GR, as well as the balance of MAPK/CDK pathways controlling GR phosphorylation pattern. Our results provide molecular base and valuable knowledge for improving the GC based therapies of leukaemia.

Cross talk of signaling pathways in the regulation of the glucocorticoid receptor function.

Several posttranslational modifications including phosphorylation have been detected on the glucocorticoid receptor (GR). However, the interdependence and combinatorial regulation of these modifications and their role in GR functions are poorly understood. We studied the effects of c-Jun N-terminal kinase (JNK)-dependent phosphorylation of GR on its sumoylation status and the impact that these modifications have on GR transcriptional activity. GR is targeted for phosphorylation at serine 246 (S246) by the JNK protein family in a rapid and transient manner. The levels of S246 phosphorylation of endogenous GR increased significantly in cells treated with UV radiation that activates JNK. S246 GR phosphorylation by JNK facilitated subsequent GR sumoylation at lysines 297 and 313. GR sumoylation increased with JNK activation and was inhibited in cells treated with JNK inhibitor. GR sumoylation in cells with activated JNK was mediated preferentially by small ubiquitin-like modifier (SUMO)2 rather than SUMO1. An increase in GR transcriptional activity was observed after inhibition of JNK or SUMO pathways and suppression of GR transcriptional activity after activation of both pathways in cells transfected with GR-responsive reporter genes. Endogenous GR transcriptional activity was inhibited on endogenous target genes IGF binding protein (IGFBP) and glucocorticoid-induced leucine zipper (GILZ) when JNK and SUMO pathways were induced individually or simultaneously. Activation of both of these signals inhibited GR-mediated regulation of human inhibitor of apoptosis gene (hIAP), whereas simultaneous activation had no effect. We conclude that phosphorylation aids GR sumoylation and that cross talk of JNK and SUMO pathways fine tune GR transcriptional activity in a target gene-specific manner, thereby modulating the hormonal response of cells exposed to stress.