Christine Orr

Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents

Although Akt is known as a survival kinase, inhibitors of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway do not always induce substantial apoptosis. We show that silencing Akt1 alone, or any combination of Akt isoforms, can suppress the growth of tumors established from phosphatase and tensin homologue–null human cancer cells. Although these findings indicate that Akt is essential for tumor maintenance, most tumors eventually rebound. Akt knockdown or inactivation with small molecule inhibitors did not induce significant apoptosis but rather markedly increased autophagy. Further treatment with the lysosomotropic agent chloroquine caused accumulation of abnormal autophagolysosomes and reactive oxygen species, leading to accelerated cell death in vitro and complete tumor remission in vivo. Cell death was also promoted when Akt inhibition was combined with the vacuolar H+–adenosine triphosphatase inhibitor bafilomycin A1 or with cathepsin inhibition. These results suggest that blocking lysosomal degradation can be detrimental to cancer cell survival when autophagy is activated, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PI3K–Akt pathway inhibition.

Identifying genotype-dependent efficacy of single and combined PI3K- and MAPK-pathway inhibition in cancer

In cancer, genetically activated proto-oncogenes often induce “upstream” dependency on the activity of the mutant oncoprotein. Therapeutic inhibition of these activated oncoproteins can induce massive apoptosis of tumor cells, leading to sometimes dramatic tumor regressions in patients. The PI3K and MAPK signaling pathways are central regulators of oncogenic transformation and tumor maintenance. We hypothesized that upstream dependency engages either one of these pathways preferentially to induce “downstream” dependency. Therefore, we analyzed whether downstream pathway dependency segregates by genetic aberrations upstream in lung cancer cell lines. Here, we show by systematically linking drug response to genomic aberrations in non-small-cell lung cancer, as well as in cell lines of other tumor types and in a series of in vivo cancer models, that tumors with genetically activated receptor tyrosine kinases depend on PI3K signaling, whereas tumors with mutations in the RAS/RAF axis depend on MAPK signaling. However, efficacy of downstream pathway inhibition was limited by release of negative feedback loops on the reciprocal pathway. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis and tumor shrinkage. Thus, by using a systematic chemo-genomics approach, we identify genetic lesions connected to PI3K and MAPK pathway activation and provide a rationale for combined inhibition of both pathways. Our findings may have implications for patient stratification in clinical trials.

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.

Intermittent Administration of MEK Inhibitor GDC-0973 plus PI3K Inhibitor GDC-0941 Triggers Robust Apoptosis and Tumor Growth Inhibition

Combinations of MAP/ERK kinase (MEK) and phosphoinositide 3-kinase (PI3K) inhibitors have shown promise in preclinical cancer models, leading to the initiation of clinical trials cotargeting these two key cancer signaling pathways. GDC-0973, a novel selective MEK inhibitor, and GDC-0941, a class I PI3K inhibitor, are in early stage clinical trials as both single agents and in combination. The discovery of these selective inhibitors has allowed investigation into the precise effects of combining inhibitors of two major signaling branches downstream of RAS. Here, we investigated multiple biomarkers in the mitogen-activated protein kinase (MAPK) and PI3K pathway to search for points of convergence that explain the increased apoptosis seen in combination. Using washout studies in vitro and alternate dosing schedules in mice, we showed that intermittent inhibition of the PI3K and MAPK pathway is sufficient for efficacy in BRAF and KRAS mutant cancer cells. The combination of GDC-0973 with the PI3K inhibitor GDC-0941 resulted in combination efficacy in vitro and in vivo via induction of biomarkers associated with apoptosis, including Bcl-2 family proapoptotic regulators. Therefore, these data suggest that continuous exposure of MEK and PI3K inhibitors in combination is not required for efficacy in preclinical cancer models and that sustained effects on downstream apoptosis biomarkers can be observed in response to intermittent dosing.

CRA-026440: a potent, broad-spectrum, hydroxamic histone deacetylase inhibitor with antiproliferative and antiangiogenic activity in vitro and in vivo

CRA-026440 is a novel, broad-spectrum, hydroxamic acid–based inhibitor of histone deacetylase (HDAC) that shows antitumor and antiangiogenic activities in vitro and in vivo preclinically. CRA-026440 inhibited pure recombinant isozymes HDAC1, HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-026440 resulted in the accumulation of acetylated histone and acetylated tubulin, leading to an inhibition of tumor cell growth and the induction of apoptosis. CRA-026440 inhibited ex vivo angiogenesis in a dose-dependent manner. CRA-026440 parenterally given to mice harboring HCT116 or U937 human tumor xenografts resulted in a statistically significant reduction in tumor growth. CRA-026440, when used in combination with Avastin, achieved greater preclinical efficacy in HCT 116 colorectal tumor model. Inhibition of tumor growth was accompanied by an increase in the acetylation of α-tubulin in peripheral blood mononuclear cells and an alteration in the expression of many genes in the tumors, including several involved in angiogenesis, apoptosis, and cell growth. These results reveal CRA-026440 to be a novel HDAC inhibitor with potent antitumor activity. [Mol Cancer Ther 2006;5(7):1693–701]

Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

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, [14C]GDC-0973 was well absorbed (fraction absorbed, 70–80%). The majority of [14C]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 KC50 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.

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 IC50 value for pERK inhibition in HCT116 tumours by G-573 was estimated to be 0.406 μM. The IC50 values for tumour growth inhibition in HCT116 and H2122 were estimated to be 3.43 and 2.56 μM, respectively. ED50 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.

Clinical responses to ERK inhibition in BRAF V600E -mutant colorectal cancer predicted using a computational model

Approximately 10% of colorectal cancers harbor BRAFV600E mutations, which constitutively activate the MAPK signaling pathway. We sought to determine whether ERK inhibitor (GDC-0994)-containing regimens may be of clinical benefit to these patients based on data from in vitro (cell line) and in vivo (cell- and patient-derived xenograft) studies of cetuximab (EGFR), vemurafenib (BRAF), cobimetinib (MEK), and GDC-0994 (ERK) combinations. Preclinical data was used to develop a mechanism-based computational model linking cell surface receptor (EGFR) activation, the MAPK signaling pathway, and tumor growth. Clinical predictions of anti-tumor activity were enabled by the use of tumor response data from three Phase 1 clinical trials testing combinations of EGFR, BRAF, and MEK inhibitors. Simulated responses to GDC-0994 monotherapy (overall response rate = 17%) accurately predicted results from a Phase 1 clinical trial regarding the number of responding patients (2/18) and the distribution of tumor size changes (“waterfall plot”). Prospective simulations were then used to evaluate potential drug combinations and predictive biomarkers for increasing responsiveness to MEK/ERK inhibitors in these patients.Systems pharmacology:Predicting efficacy of novel anti-cancer drugs in colorectal cancerWhile cancer drug development relies on experimental tumor models for testing, results observed in these systems often fail to translate clinically. Kirouac et al. demonstrate how computational systems modelling can help bridge this divide. Focusing on a class of colorectal cancers with poor prognosis (those with a mutant form of the BRAF oncogene) they develop a mathematical model linking drug exposure, via cellular signal transduction, to tumor growth. By triangulating experimental data from multiple cell lines and mouse models, with results from three clinical trials of related drugs, the model accurately predicted tumor shrinkage observed in a first-in-human study of GDC-0994, an ERK inhibitor. Simulations were then used to explore strategies for increasing the activity of this class of drugs (MAPK pathway inhibitors) via combinations, alternate dosing regimens, and predictive biomarkers to guide future clinical studies. Extended to other cancer types and drugs, the approach could streamline early clinical development.

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.