Design of BET Inhibitor Bottlebrush Prodrugs with Superior Efficacy and Devoid of Systemic Toxicities.

Prodrugs engineered for preferential activation in diseased versus normal tissues offer immense potential to improve the therapeutic indexes (TIs) of preclinical and clinical-stage active pharmaceutical ingredients that either cannot be developed otherwise or whose efficacy or tolerability it is highly desirable to improve. Such approaches, however, often suffer from trial-and-error design, precluding predictive synthesis and optimization. Here, using bromodomain and extra-terminal (BET) protein inhibitors (BETi)-a class of epigenetic regulators with proven anticancer potential but clinical development hindered in large part by narrow TIs-we introduce a macromolecular prodrug platform that overcomes these challenges. Through tuning of traceless linkers appended to a "bottlebrush prodrug" scaffold, we demonstrate correlation of in vitro prodrug activation kinetics with in vivo tumor pharmacokinetics, enabling the predictive design of novel BETi prodrugs with enhanced antitumor efficacies and devoid of dose-limiting toxicities in a syngeneic triple-negative breast cancer murine model. This work may have immediate clinical implications, introducing a platform for predictive prodrug design and potentially overcoming hurdles in drug development.

Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality.

BACKGROUND: We have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features. RESULTS: Here we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue. CONCLUSIONS: In summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.

Genetic and pharmacological inhibition of PDK1 in cancer cells: characterization of a selective allosteric kinase inhibitor.

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

Phosphoproteomics of primary AML patient samples reveals rationale for AKT combination therapy and p53 context to overcome selinexor resistance.

Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.

Discovery of PDK1 kinase inhibitors with a novel mechanism of action by ultrahigh throughput screening.

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.

Publisher Correction: Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

In the version of this Article originally published, the author Peter Blume-Jensen was not denoted as a corresponding author; this has now been amended and the author's email address has been added. The 'Correspondence and requests for materials' statement was similarly affected and has now been updated with the author's initials 'P.B-J.'

Development and clinical validation of an in situ biopsy-based multimarker assay for risk stratification in prostate cancer.

PURPOSE: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. EXPERIMENTAL DESIGN: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish "favorable" versus "nonfavorable" pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). RESULTS: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on "false-negative" and "false-positive" rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non-GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95). CONCLUSIONS: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy.

RNA interference for the identification of disease-associated genes.

RNA interference (RNAi) has emerged as a novel cellular mechanism regulating gene expression at the post-transcriptional level and as a powerful tool to control gene function experimentally. Recent advances in the biology and application of RNAi include the definition of improved criteria for selecting effective small interfering RNA (siRNA) sequences, and the generation of vectors for the delivery of siRNAs and stable silencing of genes in mammalian cells, tissues and animals. High-throughput screening projects based on RNAi have been initiated to search for genes involved in basic biological processes and in complex pathological conditions such as cancer, autoimmunity and degenerative disorders. This research is helping to identify novel therapeutic targets for a range of diseases and may translate into novel clinical applications for RNAi.

Identification of direct target engagement biomarkers for kinase-targeted therapeutics.

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3'-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1(Ser410) and p-PDK1(Thr513) are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1(Ser241)), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.

PDK1 attenuation fails to prevent tumor formation in PTEN-deficient transgenic mouse models.

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.

Pathway-based identification of biomarkers for targeted therapeutics: personalized oncology with PI3K pathway inhibitors.

Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K-mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway-relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40(Thr246). Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)-deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40(Thr246) positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKT(Thr308), the phospho-PRAS40(Thr246) epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.