Compensatory Feedback Loops Research Articles

Des-gamma-carboxy prothrombin antagonizes the effects of Sorafenib on human hepatocellular carcinoma through activation of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways.

Despite significant progress, advanced hepatocellular carcinoma (HCC) remains an incurable disease, and the overall efficacy of targeted therapy by Sorafenib remains moderate. We hypothesized that DCP (des-gamma-carboxy prothrombin), a prothrombin precursor produced in HCC, might be one of the reasons linked to the low efficacy of Sorafenib. We evaluated the efficacy of Sorafenib in HLE and SK-Hep cells, both of which are known DCP-negative HCC cell lines. In the absence of DCP, Sorafenib effectively inhibited the growth of HCC and induced cancer cell apoptosis. In the presence of DCP, HCC was resistant to Sorafenib-induced inhibition and apoptosis, as determined by in vitro assays and in mice xenografted with HLE cells. Molecular analysis of HLE xenografted-nude mice showed that DCP activates the transduction of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR cascades. DCP might stimulate the formation of compensatory feedback loops in the intricately connected signaling pathways when kinases are targeted by Sorafenib. Our results indicate that DCP antagonizes the inhibitory effects of Sorafenib on HCC through activation of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR signaling pathways. Taken together, our findings define a DCP-mediated mechanism of inhibition of Sorafenib in HCC, which is critical for targeting therapy in advanced HCC.

A first in man, dose-finding study of the mTORC1/mTORC2 inhibitor OSI-027 in patients with advanced solid malignancies.

Background:The kinase activity of mTOR involves 2 multiprotein complexes, (mTORC1-mTORC2). Targeting mTORC1 with rapalogues induces compensatory feedback loops resulting in AKT/ERK activation, which may be abrogated by mTORC2 inhibition. A first-in-human trial evaluating tolerability, pharmacokinetics and pharmacodynamics of the dual TORC1/TORC2 inhibitor OSI-027 was conducted.Methods:Dose escalation was pursued for three schedules of administration (three consecutive days per week (S1), once a week (S2) and daily dosing (S3)), until dose-limiting toxicities (DLT) were identified. Expansion cohorts with paired tumour biopsies were initiated based on tolerability and pharmacodynamics.Results:One hundred and twenty eight patients with advanced cancer were enrolled. DLT consisted predominantly of fatigue, renal function disturbances and cardiac events. OSI-027 exposure was dose proportional, with Tmax within 4 h and a half-life of ∼14 h. Expansion cohorts were initiated for S1 and S2, as MTD for S3 was overall considered suboptimal. Target modulation in peripheral blood mononuclear cells were observed from 30 mg, but in tumour biopsies 120 mg QD were needed, which was a non-tolerable dose due to renal toxicity. No RECIST responses were recorded, with stable disease >6 months in six (5%) patients.Conclusions:OSI-027 inhibits mTORC1/2 in patients with advanced tumour s in a dose-dependent manner but doses above the tolerable levels in S1 and S3 are required for a sustained biological effect in tumour biopsies.

Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers.

Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

Abstract 4516: Evaluation of p70S6K/Akt inhibitor MSC2363318A in patient derived xenograft (PDX) models of breast cancer

Abstract The PI3K pathway is involved in the regulation of cell growth, proliferation, metabolism and other functions. Aberrant signaling (PTEN loss of function, PIK3CA mutation, Akt amplification, etc.) from the PI3K pathway is observed in >50% of all tumors. Clinical evidence suggests that inhibiting the PI3K pathway is beneficial for the treatment of solid tumors and tumors of the hematopoietic system. Inhibition of mTOR via rapalogs has been shown to block a negative feedback loop, thereby leading to the activation of Akt. The activation of this Akt feedback loop has been suggested to potentially compromise the clinical efficacy of selective mTORC1 inhibitors such as temsirolimus and everolimus. Dual p70S6K/Akt inhibition may promote improved pathway inhibition and also block the negative consequences of Akt activation through the negative feedback loop. MSC2363318A is a highly selective, potent, adenosine triphosphate (ATP) competitive inhibitor of p70S6K, Akt1, and Akt3. In a cellular context, inhibition of p70S6K leads to potent inhibition of ribosomal protein S6 phosphorylation, while inhibition of Akt activity blocks the negative effects of a compensatory feedback loop. In addition, MSC2363318A exhibits potent anti-proliferative activity against many solid tumor cell lines in vitro, especially those with PI3K pathway genomic alterations. Further, MSC2363318A can also cross the blood-brain barrier (via pre-clinical studies in mice, rat, and dog), a unique characteristic that would allow for treating not only malignancies that are driven by PI3K pathway genomic alterations, but also indications with a high incidence of CNS metastases and primary malignancies of the central nervous system. Patient Derived Xenograft (PDX) models from breast cancers with a high prevalence of PI3K pathway genomic alterations; including, triple negative breast cancer and Her2+ breast cancer were evaluated. In addition, combinations with standard of care agents were then evaluated in these breast cancer PDX models. Results from these studies were correlated with PI3K pathway genomic modifications, and will be used to guide subsequent clinical studies. Citation Format: Bayard R. Huck, H Tian, Sakeena Syed, Jing Lin, Jianguo Ma, Anderson Clark, Remiguisz Kaleta, Andreas Machl, Erik Wilker, Marc Lecomte. Evaluation of p70S6K/Akt inhibitor MSC2363318A in patient derived xenograft (PDX) models of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4516. doi:10.1158/1538-7445.AM2014-4516

Clinical and Biologic Effects of the Angiopoietin 1/2 Neutralizing Peptibody Trebananib (AMG 386) in Acute Myeloid Leukemia Patients

Background Marrow angiogenesis plays an important role in the pathogenesis of acute myeloid leukemia (AML). Among the most prominent pro-angiogenic factors produced by AML cells are the angiopoietins (Ang-1/2), ligands of the Tie2 endothelial cell receptor. Elevated expression of Ang-2 is an independent prognostic factor for overall survival in AML. Trebananib (AMG 386) is a first-in-class neutralizing peptibody that provides potent and selective inhibition of angiopoietins by sequestering Ang1 and Ang2 and preventing their interaction with the Tie2 receptor. Purpose Here we evaluated the safety, tolerability, pharmacokinetic profile, and biologic effects of trebananib in de novo and relapsed/refractory AML patients considered to be poor candidates for standard induction chemotherapy. Methods Thirteen AML patients were enrolled in single agent arm A of this open-label, non-randomized phase Ib trial. ([ClinicalTrials.gov][1] ID: [NCT01555268][2], NCI-2011-02979). Drug was administered at two dose levels (15 and 30 mg/kg) via weekly intravenous infusion over 1 hour. Circulating angiogenic factor levels (Ang-2, VEGF-A/C/D, HGF, IL-8, FGF-1/2, G-CSF, PlGF) in the plasma were measured by multiplex flow cytometry prior to first dose, on cycle 1 day 29 (following 4 weekly doses of trebananib), and at the end of study. Results Median age was 76 years (range 53-82). Nine patients were male (69%). Two patients had de novo, 7 had refractory, and 4 had relapsed AML. One patient had undergone prior allogeneic stem cell transplantation. Aside from one possibly drug-related incident of grade 3 mucositis on the 15 mg/kg cohort, no other dose-limiting toxicities were reported. The most common toxicities were abdominal bloating, diarrhea, edema, and weight gain. Preliminary pharmacokinetic analysis of trebananib was dose-proportional. At the 15 and 30 mg/kg dose levels, median Cmaxand AUC on Day 1 were 193 and 473 ug/mL and 11,670 and 35,217 ug-hr/mL, respectively. No neutralizing antibodies to trebananib were detected. Among seven AML patients with consecutive plasma samples, therapy was associated with marked elevations of Ang-2 levels (from 15 to 336-fold higher than baseline) after 1-4 weeks of therapy (Table 1). Induction of Ang-2 did not differ significantly between the 15 mg/kg and 30 mg/kg cohorts. Ang-2 levels measured in bone marrow or plasma samples at the same time point were comparable. In contrast, levels of other angiogenic growth factors (VEGF-A/C/D, IL-8, G-CSF, HGF, FGF-1/2, PlGF) were not consistently altered by trebananib therapy. One AML patient (on the 15 mg/kg cohort) had a partial response with >50% decrease in marrow blasts. Two patients (15 mg/kg, 30 mg/kg) had stable disease over >1 cycle. Conclusions We report here the preliminary results of phase 1b study evaluating the safety, tolerability, pharmacokinetic profile, and biologic effects of trebananib in adult AML patients. Weekly infusions of trebananib administered at 15 and 30 mg/kg were well tolerated. Drug pharmacokinetics were similar to those previously reported in solid tumor patients. Trebananib resulted in substantial elevations in measurable circulating and marrow Ang-2 levels in AML patients, presumably due to a negative compensatory feedback loop resulting from potent in vivo Ang-1/2 inhibition. Arm B of this study (trebananib plus low dose cytarabine) is currently accruing. Further in depth studies examining the biological effects of trebananib on marrow angiogenesis and clinical response in AML patients are ongoing. Disclosures: Becker: Millenium: Research Funding. [1]: http://ClinicalTrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01555268&atom=%2Fbloodjournal%2F122%2F21%2F2701.atom

UNDERSTANDING AND TACKLING THE TUMOR-IMMUNE CELL DIALOGUE DURING PROGRESSION AND THERAPEUTIC RESISTANCE

BACKGROUND: Cancers are heterogeneous entities in which tumor cell populations as well as distinct host cell constituents form a dynamic and interactive tumor community that is pivotal not only for the genesis and progression of a tumor but also for the tumor's ability to resist therapeutic elimination. METHODS: We have been using a combination of genetic screening and coculture systems, as well as genetic and pharmacological approaches to functional elicit paracrine factors from tumor and immune cell compartments that convey GBM progression and resistance. RESULTS: We found that GBM tumor cells dependent on their subtype send out specific signals to immune cells that in turn respond by secreting prosurvival and proinvasive factors. Disrupting the communication between innate immune cells and tumor cells in mesenchymal GBM by targeting PI3Kgamma in immune cells blocked the communication to tumor cells, promoted polarization and antitumor activity and inhibited tumor invasion and growth and sensitized therapeutic approaches. CONCLUSIONS: Leveraging from these results, it is our intention to identify appropriate combinatorial treatment modalities that interfere with these compensatory feedback loops and help to develop an adequate polytherapy that is tailored to more successfully impede therapeutic resistance and prolong survival in the distinct GBM subtypes. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention.

Mechanosensing through focal adhesion‐anchored intermediate filaments

Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.

Clinical and Biologic Effects Of The Angiopoietin 1/2 Neutralizing Peptibody, Trebananib (AMG 386), In Acute Myeloid Leukemia Patients

BackgroundMarrow angiogenesis plays an important role in the pathogenesis of acute myeloid leukemia (AML). Among the most prominent pro-angiogenic factors produced by AML cells are the angiopoietins (Ang-1/2), ligands of the Tie2 endothelial cell receptor. Elevated expression of Ang-2 is an independent prognostic factor for overall survival in AML. Trebananib (AMG 386) is a first-in-class neutralizing peptibody that provides potent and selective inhibition of angiopoietins by sequestering Ang1 and Ang2 and preventing their interaction with the Tie2 receptor. PurposeHere we evaluated the safety, tolerability, pharmacokinetic profile, and biologic effects of trebananib in de novo and relapsed/refractory AML patients considered to be poor candidates for standard induction chemotherapy. MethodsThirteen AML patients were enrolled in single agent arm A of this open-label, non-randomized phase Ib trial. (ClinicalTrials.gov ID: NCT01555268, NCI-2011-02979). Drug was administered at two dose levels (15 and 30 mg/kg) via weekly intravenous infusion over 1 hour. Circulating angiogenic factor levels (Ang-2, VEGF-A/C/D, HGF, IL-8, FGF-1/2, G-CSF, PlGF) in the plasma were measured by multiplex flow cytometry prior to first dose, on cycle 1 day 29 (following 4 weekly doses of trebananib), and at the end of study. ResultsMedian age was 76 years (range 53-82). Nine patients were male (69%). Two patients had de novo, 7 had refractory, and 4 had relapsed AML. One patient had undergone prior allogeneic stem cell transplantation. Aside from one possibly drug-related incident of grade 3 mucositis on the 15 mg/kg cohort, no other dose-limiting toxicities were reported. The most common toxicities were abdominal bloating, diarrhea, edema, and weight gain. Preliminary pharmacokinetic analysis of trebananib was dose-proportional. At the 15 and 30 mg/kg dose levels, median Cmaxand AUC on Day 1 were 193 and 473 ug/mL and 11,670 and 35,217 ug-hr/mL, respectively. No neutralizing antibodies to trebananib were detected. Among seven AML patients with consecutive plasma samples, therapy was associated with marked elevations of Ang-2 levels (from 15 to 336-fold higher than baseline) after 1-4 weeks of therapy (Table 1). Induction of Ang-2 did not differ significantly between the 15 mg/kg and 30 mg/kg cohorts. Ang-2 levels measured in bone marrow or plasma samples at the same time point were comparable. In contrast, levels of other angiogenic growth factors (VEGF-A/C/D, IL-8, G-CSF, HGF, FGF-1/2, PlGF) were not consistently altered by trebananib therapy. One AML patient (on the 15 mg/kg cohort) had a partial response with >50% decrease in marrow blasts. Two patients (15 mg/kg, 30 mg/kg) had stable disease over >1 cycle. ConclusionsWe report here the preliminary results of phase 1b study evaluating the safety, tolerability, pharmacokinetic profile, and biologic effects of trebananib in adult AML patients. Weekly infusions of trebananib administered at 15 and 30 mg/kg were well tolerated. Drug pharmacokinetics were similar to those previously reported in solid tumor patients. Trebananib resulted in substantial elevations in measurable circulating and marrow Ang-2 levels in AML patients, presumably due to a negative compensatory feedback loop resulting from potent in vivo Ang-1/2 inhibition. Arm B of this study (trebananib plus low dose cytarabine) is currently accruing. Further in depth studies examining the biological effects of trebananib on marrow angiogenesis and clinical response in AML patients are ongoing. Disclosures:Becker:Millenium: Research Funding.

Abstract A162: Identification of brain penetrant p70S6K/Akt inhibitor MSC2363318A.

Abstract The PI3K pathway is involved in the regulation of cell growth, proliferation, metabolism and other functions. Aberrant signaling (PTEN loss of function, PIK3CA mutation, Akt amplification, etc.) from the PI3K pathway is observed in >50% of all tumors. Clinical evidence suggests that inhibiting the PI3K pathway is beneficial for the treatment of solid tumors and tumors of the hematopoietic system. Inhibition of p70S6K via rapalogs has been shown to block a negative feedback loop, thereby leading to the activation of Akt. The activation of this Akt feedback loop has been suggested to potentially compromise the clinical efficacy of selective mTORC1 inhibitors such as temsirolimus and everolimus. Dual p70S6K/Akt inhibition may promote improved pathway inhibition and also block the negative consequences of Akt activation through the negative feedback loop. MSC2363318A is a highly kinase-selective, potent, adenosine triphosphate (ATP) competitive inhibitor of p70S6K, Akt1, and Akt3. In a cellular context, inhibition of p70S6K leads to potent inhibition of ribosomal protein S6 phosphorylation, while inhibition of Akt activity blocks the negative effects of a compensatory feedback loop. In addition, MSC2363318A exhibits potent anti-proliferative activity against many solid tumor cell lines in vitro, especially those with PI3K pathway genomic alterations. Further, MSC2363318A can also cross the blood-brain barrier, a unique characteristic that would allow for treating not only primary malignancies that are driven by PI3K pathway genomic alterations, but also indications with a high incidence of CNS metastases and primary malignancies of the central nervous system. Oral treatment of mice with MSC2363318A resulted in significant inhibition of tumor growth in several subcutaneous human cancer xenograft models representing breast, pancreatic, glioblastoma and ovarian cancers. Of note, a breast cancer model possessing a PTEN loss of function showed tumor regression upon treatment with MSC2363318A. In addition, MSC2363318A exhibited increased exposure in tumors as compared to plasma, resulting in sustained inhibition of S6K phosphorylation for up to 24 hours. Key preclinical activities are being completed and first in human clinical studies are scheduled to be initiated in 2013. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A162. Citation Format: Bayard R. Huck, Andreas Machl, Erik Wilker, Hui Tian, Sakeena Syed, Jing Lin, Jianguo Ma, Anderson Clark, Roseann Waterhouse, Remiguisz Kaleta, Alina Micu, Lynne Soughley, Long Li, Karin Groll, Sonja Kroesser, Frank Beier, Ursula Hering, Marc Lecomte, Katrin Wichert, Mauro D'Antonio, Paolo Di Eugenio. Identification of brain penetrant p70S6K/Akt inhibitor MSC2363318A. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A162.

Synergistic Effects of Concurrent Blockade of PI3K and MEK Pathways in Pancreatic Cancer Preclinical Models

Patients with pancreatic cancer have dismal prognoses, and novel therapies are urgently needed. Mutations of the KRAS oncogene occur frequently in pancreatic cancer and represent an attractive target. Direct targeting of the predominant KRAS pathways have been challenging and research into therapeutic strategies have been now refocused on pathways downstream of KRAS, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK [MEK]). We hypothesized that concurrent inhibition of the PI3K and MEK pathways would result in synergistic antitumor activity, as it would circumvent the compensatory feedback loop between the two pathways. We investigated the combined effect of the PI3K inhibitor, GDC0941, and the MEK inhibitor, AZD6244, on cell viability, apoptosis and cell signaling in a panel of pancreatic cancer cell lines. An in vivo analysis was conducted on pancreatic cancer xenografts. While BxPC-3 (KRAS wild type) and MIA PaCa-2 (KRAS mutated) cell lines were sensitive to GDC0941 and AZD6244 as single agents, synergistic inhibition of tumor cell growth and induction of apoptosis were observed in both cell lines when the two drugs were combined. Interestingly, phosphorylation of the cap-dependent translational components, 4E-binding protein (p-4E-BP1) and S6 was found to be closely associated with sensitivity to GDC0941 and AZD6244. In BxPC-3 cell xenografts, survival differences were observed between the control and the AZD6244, GDC0941, and combination groups. Our study provides the rationale for concurrent targeting of the PI3K and MEK pathways, regardless of KRAS status, and suggests that phosphorylation of 4E-BP1and S6 can serve as a predictive biomarker for response to treatment.

The PTEN Long N-tail is intrinsically disordered: increased viability for PTEN therapy

Aberrant activation of the PI3K/Akt/mTOR pathway is observed in several cancers and hyper proliferative disorders. PTEN, a tumor suppressor gene, negatively regulates the PI3K/Akt/mTOR pathway. Inhibitors of various components of this pathway are currently being used for cancer therapy. However, the use of these small molecule inhibitors remains limited due to the presence of compensatory feedback loops within the pathway such that inhibition of one oncogenic molecule often results in the activation of another oncogenic molecule resulting in the development of chemoresistance. One novel strategy that has emerged as a means to circumvent the problem of feedback signaling is by activating tumor suppressor genes that abrogate oncogenic pathways and regress tumor growth. In this regard, a newly identified isoform of the PTEN protein shows promise for use in tumors with elevated PI3K/Akt/mTOR signaling. This isoform is a translational variant of PTEN, termed as PTEN Long, and has additional 173 amino acids at its N-terminus (N-173) than normal PTEN. The N-173 region is required for PTEN secretion and transport across the body. Given the potential of this N-173 region to act as a drug delivery system for PTEN, we herein analyze the structural properties of this region. This N-173 tail has a large intrinsically disordered region (IDR) and is composed of highly charged basic residues. Further, the region is enriched in potential linear binding motifs, protein-binding sites and post-translational modifications (PTMs) indicating its probable role in PTEN function and transport across cells. An extensive analysis of this region is warranted to better exploit its structural and biophysical peculiarities to drug discovery and drug delivery applications.

AT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial.

Abstract 928: The novel clinical candidate AT13148 is an oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity and demonstrates a mechanism of action distinct from AKT inhibitors

Abstract The AGC kinase AKT is a key component of the phosphatidylinositol 3-kinase (PI3K) pathway, which is frequently deregulated in cancer, making AKT a target of major therapeutic interest. However, PI3K signaling through both AKT-dependent and AKT-independent mechanisms involving other AGC kinases, such as p70S6K, PKA, SGK and ROCK, is important in a range of cancers. Hence, the pharmacological inhibition of these multiple AGC kinases may increase response rates and minimize clinical resistance compared with targeting AKT alone. The clinical drug candidate AT13148 is a multi-AGC kinase, ATP-competitive inhibitor, identified utilizing high-throughput X-ray crystallography and fragment-based lead discovery techniques. Screening of this oral small molecule against a panel of kinases at 10μM revealed >80% inhibition of the structurally related AGC kinases AKT, PKA, ROCK2, p70S6K, MSK, RSK1/2, and SGK. We demonstrate that AT13148 has antiproliferative activity in a range of in vitro models harboring relevant genetic abnormalities, including PTEN, KRAS, PIK3CA and HER2 aberrations. AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induction of apoptosis in both a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects both in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP competitive inhibitors of AKT, is not a therapeutically relevant reactivation step for this compound. We used gene expression microarray studies to characterize the underlying molecular mechanisms of action of AT13148 and the selective AKT inhibitor CCT128930, and observed the induction of upstream regulators including insulin receptor substrate-2 and PIK3IP1 due to compensatory feedback loops, consistent with blockade of AKT signaling. These studies also showed that AT13148 and CCT128930 have distinct molecular effects in cancer cells: AT13148 had a predominant effect on apoptosis genes and caused a greater apoptotic phenotype, while CCT128930 modulated genes in the network regulating cell cycle. This finding emphasizes the functional differences of AT13148 as a multi-AGC kinase inhibitor in contrast to a more AKT-selective inhibitor. In view of the potential mechanistic advantages detailed above, and the potent antitumor activity observed at well tolerated doses against established human tumor xenografts with clinically relevant genetic drivers, the clinical utility of such an AGC kinase inhibitor strategy will now be assessed in a first-in-human Phase I trial of AT13148. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 928. doi:1538-7445.AM2012-928

Pushing the Envelope in the mTOR Pathway: The Second Generation of Inhibitors

The phosphatidylinositol-3-kinase (PI3K)/mTOR pathway has been a major focus of attention for cancer researchers in the past decade. A preliminary and incomplete understanding of the molecular biology of this complex network has importantly conditioned not only the development of the first generation of mTOR inhibitors, but also the biomarker studies designed to identify the best responders to these agents. Most recently, research in this pathway has focused on the dual nature of mTOR that is integrated by the mTOR complex 1 and complex 2. These two complexes are formed and regulated by different proteins and are also driven by multiple different compensatory feedback loops. This deeper understanding has allowed the development of a promising second generation of inhibitors, which are able to block simultaneously both complexes due to their catalytic activity over mTOR. Moreover, some of them also exert an inhibitory effect over PI3K that is a key player in the feedback loops. This article reviews the newest insights in the signaling of the mTOR pathway and then focuses on the development of the new wave of mTOR inhibitors.

Targeting the Phosphoinositide-3 (PI3) Kinase Pathway in Breast Cancer

The phosphoinositide-3 kinase (PI3K) pathway has been identified as an important target in breast cancer research for a number of years, but is new to most clinicians responsible for the daily challenges of breast cancer management. In fact, the PI3K pathway is probably one of the most important pathways in cancer metabolism and growth. Mutations in the PI3K pathway are frequent in breast cancer, causing resistance to human epidermal growth factor receptor 2-targeted agents and, possibly, to hormonal agents as well. Available agents that affect the PI3K pathway include monoclonal antibodies and tyrosine kinase inhibitors, as well as PI3K inhibitors, Akt inhibitors, rapamycin analogs, and mammalian target of rapamycin (mTOR) catalytic inhibitors. Multiple PI3K inhibitors are currently under development, including pure PI3K inhibitors, compounds that block both PI3K and mTOR (dual inhibitors), pure catalytic mTOR inhibitors, and inhibitors that block Akt. It is likely that these agents will have to be given in combination with other signal inhibitors because anti-mTOR agents and PI3K inhibitors may result in the activation of compensatory feedback loops that would in turn result in decreased efficacy. This article reviews current data related to the PI3K pathway, its role in breast cancer, the frequency with which PI3K is aberrant in breast cancer, and the potential clinical implications of using agents that target the PI3K pathway.

First-in-human phase I study exploring three schedules of OSI-027, a novel small molecule TORC1/TORC2 inhibitor, in patients with advanced solid tumors and lymphoma.

3006 Background: The mTOR pathway regulates cell growth, proliferation and survival; and is a clinically validated target for cancer therapy. Current rapamycin analogs preferentially target TORC1, with therapeutic efficacy limited by compensatory feedback loops resulting in AKT and ERK activation. OSI-027 is an oral inhibitor of TORC1 and TORC2 kinase activity and may have additional clinical activity over TORC1 selective inhibitors. Methods: Patients (pts) with advanced solid tumors or lymphoma received escalating doses of OSI-027 in 3 schedules (S1: days (d) 1-3 q7d, S2: once weekly, and S3: continuous once daily) to determine safety, maximum tolerated dose, pharmacokinetics (PK) and preliminary antitumor activity. Pharmacodynamic (PD) effects on phosphorylation of 4E-BP1 at threonine 37/46, a rapamycin insensitive, mTOR-dependent phosphorylation site, were examined. Results: To date, 34 pts have been enrolled (13M/21F, median age 59 yrs) and 31 treated (S1 11 pts, S2 12 pts, S3 8 pts). S1 and S2 pts were dosed at 10, 15 and 20 mg, S3 pts at 5, 10 and 20 mg. Median number of weeks on study was 6 (range <1-34). Three DLTs have been reported: grade (G) 2 decreased LVEF (10mg S1), and 2 pts with G3 fatigue (15 mg S2 and 20 mg S3). Other drug-related toxicities included G3 nausea and vomiting (1), G3 pneumonia (1); G1/2 fatigue (4), nausea (2), diarrhea (1), anorexia (1), elevated creatinine (1), and reversible increase in QTc (1). Preliminary PK indicate exposure (AUC, Cmax) of OSI-027 increased with dose. The median Tmax and terminal T3/4 were 2-6 hrs and 6-17 hrs, respectively. Preliminary PD data indicate that substantial decreases in 4E-BP1 (T37/46) phosphorylation were observed in PBMCs from 13 of 23 pts following OSI-027 treatment. Eight pts have had SD lasting ≥ 12 wks (26% of pts treated, range 12-33 wks); tumor types were colorectal (3 pts), melanoma, neuroendocrine, endometrial, renal, and cervical cancer (1 pt each). Conclusions: OSI-027 is a potent TORC1/2 kinase inhibitor. Preliminary evidence of pharmacological activity has been observed. It is well tolerated at the doses and schedules tested to date. MTD has not been reached and dose escalation is ongoing. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration OSI OSI

Compensatory activation of Akt in response to mTOR and Raf inhibitors – A rationale for dual-targeted therapy approaches in neuroendocrine tumor disease

Several studies have established a link between aberrant PI(3)K–Akt–mTOR- and Ras–Raf–MEK–Erk1/2 signaling and neuroendocrine tumor disease. In this study, we comparatively investigate the antitumor potential of novel small-molecule inhibitors targeting mTOR (RAD001), mTOR/PI(3)K (NVP-BEZ235) and Raf (Raf265) on human NET cell lines of heterogeneous origin. All inhibitors induced potent antitumor effects which involved the induction of apoptosis and G0/G1 arrest. However, the dual mTOR/PI(3)K inhibitor NVP-BEZ235 was more efficient compared to the single mTOR inhibitor RAD001. Consistently, NVP-BEZ235 prevented the negative feedback activation of Akt as observed after treatment with RAD001. Raf265 inhibited Erk1/2 phosphorylation but strongly induced Akt phosphorylation and VEGF secretion, suggesting the existence of a compensatory feedback loop on PI3K-Akt signaling. Finally, combined treatment with RAD001 or NVP-BEZ235 and Raf265 was more efficient than single treatment with either kinase inhibitor. Together, our data provide a rationale for dual targeting of PI(3)K–Akt–mTOR- and Ras–Raf–MEK–Erk1/2 signaling in NET disease.

Compensatory responses to age-related decline in odor quality acuity: Cholinergic neuromodulation and olfactory enrichment

The perceptual differentiation of odors can be measured behaviorally using generalization gradients. The steepness of these gradients defines a form of olfactory acuity for odor quality that depends on neural circuitry within the olfactory bulb and is regulated by cholinergic activity therein as well as by associative learning. Using this system as a reduced model for age-related cognitive decline, we show that aged mice, while maintaining almost the same baseline behavioral performance as younger mice, are insensitive to the effects of acutely elevated acetylcholine, which sharpens generalization gradients in young adult mice. Moreover, older mice exhibit evidence of chronically elevated acetylcholine levels in the olfactory bulb, suggesting that their insensitivity to further elevated levels of acetylcholine may arise because the maximum capacity of the system to respond to acetylcholine has already been reached. We propose a model in which an underlying, age-related, progressive deficit is mitigated by a compensatory cholinergic feedback loop that acts to retard the behavioral effects of what would otherwise be a substantial age-related decline in olfactory plasticity. We also treated mice with 10-day regimens of olfactory environmental enrichment and/or repeated systemic injections of the acetylcholinesterase inhibitor physostigmine. Each treatment alone sharpened odor quality acuity, but administering both treatments together had no greater effect than either alone. Age was not a significant main effect in this study, suggesting that some capacity for acetylcholine-dependent plasticity is still present in aged mice despite their sharply reduced ability to respond to acute increases in acetylcholine levels. These results suggest a dynamical framework for understanding age-related decline in neural circuit processing in which the direct effects of aging can be mitigated, at least temporarily, by systemic compensatory responses. In particular, a decline in cholinergic efficacy can precede any breakdown in cholinergic production, which may help explain the limited effectiveness of cholinergic replacement therapies in combating cognitive decline.

Chronic Lymphocytic Leukemia and B and T Cells Differ in Their Response to Cyclic Nucleotide Phosphodiesterase Inhibitors

Phosphodiesterase (PDE)4 inhibitors, which activate cAMP signaling by reducing cAMP catabolism, are known to induce apoptosis in B lineage chronic lymphocytic leukemia (CLL) cells but not normal human T cells. The explanation for such differential sensitivity remains unknown. In this study, we report studies contrasting the response to PDE4 inhibitor treatment in CLL cells and normal human T and B cells. Affymetrix gene chip analysis in the three cell populations following treatment with the PDE4 inhibitor rolipram identified a set of up-regulated transcripts with unusually high fold changes in the CLL samples, several of which are likely part of compensatory negative feedback loops. The high fold changes were due to low basal transcript levels in CLL cells, suggesting that cAMP-mediated signaling may be unusually tightly regulated in this cell type. Rolipram treatment augmented cAMP levels and induced ATF-1/CREB serine 63/133 phosphorylation in both B lineage cell types but not T cells. As treatment with the broad-spectrum PDE inhibitor 3-isobutyl-1-methylxanthine induced T cell CREB phosphorylation, we tested a series of family-specific PDE inhibitors for their ability to mimic 3-isobutyl-1-methylxanthine-induced ATF-1/CREB phosphorylation. Whereas PDE3 inhibitors alone had no effect, the combination of PDE3 and PDE4 inhibitors induced ATF-1/CREB serine 63/133 phosphorylation in T cells. Consistent with this observation, PDE3B transcript and protein levels were low in CLL cells but easily detectable in T cells. Combined PDE3/4 inhibition did not induce T cell apoptosis, suggesting that cAMP-mediated signal transduction that leads to robust ATF-1/CREB serine 63/133 phosphorylation is not sufficient to induce apoptosis in this lymphoid lineage.