MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade.

Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy.

Antitumor efficacy and reduced toxicity using an anti-CD137 Probody therapeutic.

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation-related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor-dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers.

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle.

The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome-CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G2/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Quantitative phosphoproteomic analysis of the PI3K-regulated signaling network.

The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899).

Phosphoproteome analysis of the MAPK pathway reveals previously undetected feedback mechanisms.

The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 (http://proteomecentral.proteomexchange.org/dataset/PXD003908).

RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.

Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition.

Targeted therapeutics that block signal transduction through the RAS-RAF-MEK and PI3K-AKT-mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941-mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.

PAK1 mediates pancreatic cancer cell migration and resistance to MET inhibition.

Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper understanding of molecular drivers is needed to advance therapeutic options for patients. We report here that p21-activated kinase 1 (PAK1) is a central node in PDAC cells downstream of multiple growth factor signalling pathways, including hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition blocks signalling to cytoskeletal effectors and tumour cell motility driven by HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in clinical development. Given that even highly effective therapies have resistance mechanisms, we show that combination with PAK1 inhibition overcomes potential resistance mechanisms mediated either by activation of parallel growth factor pathways or by direct amplification of PAK1. Inhibition of PAK1 attenuated in vivo tumour growth and metastasis in a model of pancreatic adenocarcinoma. In human tissues, PAK1 is highly expressed in a proportion of PDACs (33% IHC score 2 or 3; n = 304) and its expression is significantly associated with MET positivity (p < 0.0001) and linked to a widespread metastatic pattern in patients (p = 0.067). Taken together, our results provide evidence for a functional role of MET/PAK1 signalling in pancreatic adenocarcinoma and support further characterization of therapeutic inhibitors in this indication.

Systems-wide analysis of K-Ras, Cdc42, and PAK4 signaling by quantitative phosphoproteomics.

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.

Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells.

p21-activated kinases (PAKs) are serine/threonine protein kinases that serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAK1 has been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion, and actin cytoskeleton organization. To better understand the role of PAK1 in tumorigenesis, PAK1 genomic copy number and expression were determined for a large panel of breast, lung, and head and neck tumors. PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer, and PAK1 protein expression was associated with lymph node metastasis. Breast cancer cells with PAK1 genomic amplification rapidly underwent apoptosis after inhibition of this kinase. Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous nonsmall cell lung carcinomas (NSCLCs), and selective PAK1 inhibition was associated with delayed cell-cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small-molecule inhibitors, and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked inhibitor of apoptosis efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in breast and squamous NSCLCs and a role for PAK1 in cellular survival and proliferation in these indications.

Discovery of thiazolobenzoxepin PI3-kinase inhibitors that spare the PI3-kinase ß isoform.

A series of suitable five-membered heterocyclic alternatives to thiophenes within a thienobenzoxepin class of PI3-kinase (PI3K) inhibitors was discovered. Specific thiazolobenzoxepin 8-substitution was identified that increased selectivity over PI3Kß. PI3Kß-sparing compound 27 (PI3Kß Ki,app/PI3Kα Ki,app=57) demonstrated dose-dependent knockdown of pAKT, pPRAS40 and pS6RP in vivo as well as differential effects in an in vitro proliferation cell line screen compared to pan PI3K inhibitor GDC-0941. A new structure-based hypothesis for reducing inhibition of the PI3K ß isoform while maintaining activity against α, δ and γ isoforms is presented.

Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

Preclinical disposition of GDC-0973 and prospective and retrospective analysis of human dose and efficacy predictions.

[3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone (GDC-0973) is a potent and highly selective inhibitor of mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase (ERK) 1/2 (MEK1/2), a MAPK kinase that activates ERK1/2. The objectives of these studies were to characterize the disposition of GDC-0973 in preclinical species and to determine the relationship of GDC-0973 plasma concentrations to efficacy in Colo205 mouse xenograft models. The clearance (CL) of GDC-0973 was moderate in mouse (33.5 ml · min(-1) · kg(-1)), rat (37.9 ± 7.2 ml · min(-1) · kg(-1)), and monkey (29.6 ± 8.5 ml · min(-1) · kg(-1)). CL in dog was low (5.5 ± 0.3 ml · min(-1) · kg(-1)). The volume of distribution across species was large, 6-fold to 15-fold body water; half-lives ranged from 4 to 13 h. Protein binding in mouse, rat, dog, monkey, and human was high, with percentage unbound, 1 to 6%. GDC-0973-related radioactivity was rapidly and extensively distributed to tissues; however, low concentrations were observed in the brain. In rats and dogs, [(14)C]GDC-0973 was well absorbed (fraction absorbed, 70-80%). The majority of [(14)C]GDC-0973-related radioactivity was recovered in the bile of rat (74-81%) and dog (65%). The CL and volume of distribution of GDC-0973 in human, predicted by allometry, was 2.9 ml · min(-1) · kg(-1) and 9.9 l/kg, respectively. The predicted half-life was 39 h. To characterize the relationship between plasma concentration of GDC-0973 and tumor growth inhibition, pharmacokinetic-pharmacodynamic modeling was applied using an indirect response model. The KC(50) value for tumor growth inhibition in Colo205 xenografts was estimated to be 0.389 µM, and the predicted clinical efficacious dose was ∼10 mg. Taken together, these data are useful in assessing the disposition of GDC-0973, and where available, comparisons with human data were made.

A Probody T Cell-Engaging Bispecific Antibody Targeting EGFR and CD3 Inhibits Colon Cancer Growth with Limited Toxicity.

T cell-engaging bispecific antibodies (TCB) are highly potent therapeutics that can recruit and activate cytotoxic T cells to stimulate an antitumor immune response. However, the development of TCBs against solid tumors has been limited by significant on-target toxicity to normal tissues. Probody therapeutics have been developed as a novel class of recombinant, protease-activated antibody prodrugs that are "masked" to reduce antigen binding in healthy tissues but can become conditionally unmasked by proteases that are preferentially active in the tumor microenvironment (TME). Here, we describe the preclinical efficacy and safety of CI107, a Probody TCB targeting EGFR and CD3. In vitro, the protease-activated, unmasked CI107 effectively bound EGFR and CD3 expressed on the surface of cells and induced T-cell activation, cytokine release, and cytotoxicity toward tumor cells. In contrast, dually masked CI107 displayed a >500-fold reduction in antigen binding and >15,000-fold reduction in cytotoxic activity. In vivo, CI107 potently induced dose-dependent tumor regression of established colon cancer xenografts in mice engrafted with human peripheral blood mononuclear cells. Furthermore, the MTD of CI107 in cynomolgus monkeys was more than 60-fold higher than that of the unmasked TCB, and much lower levels of toxicity were observed in animals receiving CI107. Therefore, by localizing activity to the TME and thus limiting toxicity to normal tissues, this Probody TCB demonstrates the potential to expand clinical opportunities for TCBs as effective anticancer therapies for solid tumor indications. SIGNIFICANCE: A conditionally active EGFR-CD3 T cell-engaging Probody therapeutic expands the safety window of bispecific antibodies while maintaining efficacy in preclinical solid tumor settings.

Nonclinical Efficacy and Safety of CX-2029, an Anti-CD71 Probody-Drug Conjugate.

Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.

Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti-PD-1/PD-L1 Agents.

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials.

Pharmacokinetic-pharmacodynamic modeling of tumor growth inhibition and biomarker modulation by the novel phosphatidylinositol 3-kinase inhibitor GDC-0941.

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation, by activation or transforming mutations of the p110alpha subunit, is associated with the development of many cancers. 2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of PI3K currently being evaluated in the clinic as an anticancer agent. The objectives of these studies were to characterize the relationships between GDC-0941 plasma concentrations and tumor reduction in MCF7.1 breast cancer xenografts and to evaluate the association between the tumor pharmacodynamic biomarker [phosphorylated (p) Akt and phosphorylated proline-rich Akt substrate of 40 kDa (pPRAS40)] responses and antitumor efficacy. MCF7.1 tumor-bearing mice were treated for up to 3 weeks with GDC-0941 at various doses (12.5-200 mg/kg) and dosing schedules (daily to weekly). An indirect response model fitted to tumor growth data indicated that the GDC-0941 plasma concentration required for tumor stasis was approximately 0.3 muM. The relationship between GDC-0941 plasma concentrations and inhibition of pAkt and pPRAS40 in tumor was also investigated after a single oral dose of 12.5, 50, or 150 mg/kg. An indirect response model was fitted to the inhibition of Akt and PRAS40 phosphorylation data and provided IC(50) estimates of 0.36 and 0.29 muM for pAkt and pPRAS40, respectively. The relationship between pAkt inhibition and tumor volume was further explored using an integrated pharmacokinetic biomarker tumor growth model, which showed that a pAkt inhibition of at least 30% was required to achieve stasis after GDC-0941 treatment of the MCF7.1 xenograft.

Preclinical disposition and pharmacokinetics-pharmacodynamic modeling of biomarker response and tumour growth inhibition in xenograft mouse models of G-573, a MEK inhibitor.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) pathway is a key signalling pathway that regulates cell proliferation. G-573 is an allosteric inhibitor of MEK that is both potent and selective. The objectives of these studies were to characterize the disposition of G-573 in preclinical species and to determine the relationship of G-573 plasma concentrations to pERK (phosphorylated ERK) and to tumour growth inhibition in HCT116 and H2122 mouse xenograft models. The clearance of G-573 was low in mouse (7.7 ml/min/kg), rat (2.24 ml/min/kg), dog (10 ml/min/kg), and cynomolgus monkey (0.754 ml/min/kg) while volumes of distribution (0.114-1.77 l/kg) was low to moderate, resulting in moderate half-lives across species (~2-9 h). Indirect response models were used to characterize the relationship between plasma concentration of G-573 to both pERK inhibition and tumour growth inhibition. The IC(50) value for pERK inhibition in HCT116 tumours by G-573 was estimated to be 0.406 µM. The IC(50) values for tumour growth inhibition in HCT116 and H2122 were estimated to be 3.43 and 2.56 µM, respectively. ED(50) estimates in HCT116 and H2122 mouse xenograft models were estimated to be ~4.6 and 1.9 mg/kg/day, respectively. The information from these studies provides useful information when characterizing candidates for potential further clinical testing.