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PDF - 60K, Linear correlation observed between tumor growth inhibition (% T/C) or tumor regression and the fraction of time over the in vivo S473P-Akt IC80 in different cancer cell line-derived tumor xenografts implanted in nude mice (represented as dots) and nude rats (represented as triangles) following NVP-BYL719 treatment (R2=0.77, p

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Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials. Mol Cancer Ther; 13(5); 1117–29. ©2014 AACR.

PDF file - 841K, Supplementary Figures S1-S6; legends for Supplementary Tables S1-S3; Supplementary Methods describing RNA interference, generation of Ba/F3 cell lines, site-directed mutagenesis, transient transfection, inhibition of cellular ATP production, proliferation assays, enzymatic assays with JAK2 JH1, purification of tyrosine phosporylated peptides and mass-spectrometry; Supplementary References.

PDF file - 11K, Activity of NVP-BBT594 in biochemical and cell-based assays. The IC50 (mean of two experiments) of NVP-BBT594 on the JAK2 kinase was determined in a biochemical kinase assay with the active enzyme. Half-maximal growth inhibition (GI50,) of NVP-BBT594 was determined in SET-2, CMK and K-562 cell lines (mean � SD, n = 4).

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PDF - 129K, Legends for Supplementary Figures 1 through 4 and Supplementary Tables 1 through 7. Supplementary Table 1. Determination of NVP-BYL719 in vitro effects on human protein kinases. Supplementary Table 2. Activity profile of NVP-BYL719 in the Invitrogen Kinase panel. Supplementary Table 3. Activity profile of NVP-BYL719 in the Ambit Kinase panel. Supplementary Table 4. NVP-BYL719 Kd (nmol/L) determination in the Ambit Kinase panel for the hits found to be inhibited >90%. Supplementary Table 5. NVP-BYL719 anti-tumor effects in diverse cancer cell line-derived xenograft models. Supplementary Table 6. CCLE cell lines responsive to NVP-BYL719. Supplementary Table 7 Anti-tumor effect of NVP-BYL719 in patient-derived xenograft models carrying PIK3CA genetic alterations.

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PDF - 71K, Rat1-myr-p110alpha (blue), beta (green) or delta (red) cells were treated with increasing concentrations of NVP-BYL719 for 30 minutes. Levels of S473P-Akt in cell extracts were quantified by Reverse Phase Protein Array as described in (18) and plotted as percentage of untreated control cells. The graph illustrates n=3 independent experiments. IC50s (plus/minus) SD and IC80s (plus/minus) SD of n=3 independent experiments are reported..

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PDF - 92K, NVP-BYL719 does not inhibit mTOR and PIKKs involved in DNA damage-repair processes. A. TSC1 -/- MEFs cells were grown in a 96-well format and treated for 1 h with increased concentrations of RAD001 or NVP-BYL719 (from 0.5 nmol/L to 10 ?mol/L in 1 third dilution steps) and immediately fixed. S235/236P-RPS6 levels were measured and IC50 determined with the Excel module XLfit. Background (no primary Ab incubated); BL, Baseline. B: TSC1 -/- MEFs cells were treated with increasing concentrations of NVP-BYL719 as indicated or RAD001 at 500 nmol/L or an equivalent DMSO concentration for 30 minutes. Levels of S235/236P-RPS6 and total RPS6 in protein- normalized lysates were detected by Western blot analyzis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. C: 24 h post seeding, A549 cells were treated at the same time with Actinomycin D (Act D) at a concentration of 5 ?mol/L (an agent used to induce DNA damage), and with increasing concentrations of NVP-BYL719 as indicated or with the vehicle control (DMSO) for 1 h. Levels of S15P-p53 and tubulin in protein-normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. D: 24 h post seeding, U2OS cells were pre-treated for 1 h with increased concentrations of NVP-BYL719 or KU55933 a specific small molecular mass inhibitor of ATM (Supplementary reference 1) at a concentration of 10 ?mol/L or with the vehicle control (DMSO). The cells were then irradiated with 15 Gy and re-incubated at 37 degrees C for 1 h and then lysed. Levels of S1981P-ATM in protein- normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL.

PDF - 37K, NVP-BYL719 was tested at 10 different concentrations (from 0.5 nmol/L to 10 μmol/L in 1 third dilution steps) against CLK2 (A) and LRRK2 (B). SelectScreenTM Kinase Profiling Service uses XLfit from IDBS to determine the IC50 values (IC50 CLK2=621 nmol/L; IC50 LRRK2=3'220 nmol/L). The dose response curve is curve fit to model number 205 (sigmoidal dose-response model).

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Supplementary Fig from Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity

Supplementary Material from Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945–55. ©2010 AACR.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials. [Mol Cancer Ther 2008;7(7):1–13 [Mol Cancer Ther 2008;7(7):1851–13]

Supplementary Data from Potent and Selective Inhibition of Polycythemia by the Quinoxaline JAK2 Inhibitor NVP-BSK805

The insulin-like growth factor-1 receptor (IGF1R) is a drug target for oncology, and many studies are ongoing to identify compounds that inhibit its tyrosine kinase activity. IGF1R is highly homologous to the insulin receptor (IR) and IGF1R inhibition might be beneficial for patients, while IR inhibition may lead to limiting toxicity. Therefore selectivity for IGF1R over IR is the aim for drug design in this context. A few compounds that selectively inhibit IGF1R over IR in cells have been identified, but none of them show the same levels of selectivity in enzymatic assays. To deter- mine whether this discrepancy is linked to the conditions used in the enzymatic assays, we have studied the interaction be- tween known IGF1R inhibitors (NVP-AEW541, OSI906, AG538, NVP-TAE226) and phosphorylated/unphosphorylated IGF1R/IR proteins with both biophysical (isothermal calorimetry and surface plasmon resonance) and enzymatic methods. In this report, we describe the results of this study and comment on the different degrees of selectivity IGF1R versus IR measured in biochemical and cellular assays. Finally, our study provides new information on the biochemical and mechanism of action of these small molecular weight IGF1R inhibitors.

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53–MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein–protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line.

Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional phosphoinositide 3-kinase (PI3K) inhibitors from different chemical classes with a different selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at least 50-fold selectivity against other protein kinases. The compound is also active against the most common somatic PI3Kα mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentration-dependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in patients with cancer. Mol Cancer Ther; 11(2); 317–28. ©2011 AACR.

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

The Janus kinase 2 (JAK2) is a drug target in particular because a missense mutation in this gene (V617F) has been identified in various human diseases. We report here the first kinetic study of the human full-length wild type and V617F JAK2 proteins and of their isolated kinase domain. The kinetic parameters of both full-length proteins are similar revealing that the mutation does not affect JAK2 catalytic activity suggesting that it has a more complex role in the regu- lation of JAK2 activity. Our study also shows that the domains located outside the kinase domain have little influence on JAK2 catalytical activity.

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials. Mol Cancer Ther; 13(5); 1117–29. ©2014 AACR.

Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor–induced activation loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop may or may not be elicited. Significance: This study shows that JAK inhibitors can lead to an increase of activation loop phosphorylation in a manner that is binding mode dependent. Our results highlight the need for detailed understanding of inhibitor mechanism of action, and that it may be possible to devise strategies that avoid target priming using alternative modes of inhibiting JAK kinase activity for the treatment of JAK-dependent diseases. Cancer Discov; 2(6); 512–23. © 2012 AACR. This article is highlighted in the In This Issue feature, p. 473

Disrupting the interaction between the p53 tumor suppressor and its regulator MDM2 is a promising therapeutic strategy in anticancer drug research. In our search for non peptide inhibitors of this protein–protein interaction, we have devised a ligand design concept exploiting the central position of Val 93 in the p53 binding pocket of MDM2. The design of molecules based on this concept has allowed us to rapidly identify compounds having a 3-imidazolyl indole core structure as the first representatives of a new class of potent inhibitors of the p53–MDM2 interaction.

Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors., Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.

Objective: The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity. Methods: For contractility studies, a total of 57 trabeculae were isolated from two non- failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three α isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. Results: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p≪0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol·g−1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three α isoforms were expressed at the mRNA level in both normal and failing hearts. Conclusions: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition. Cardiovascular Research 1993; 27 :2229-2237

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945–55. ©2010 AACR.

Protein kinases can adopt multiple protein conformations depending on their activation status. Recently, in drug discovery, a paradigm shift has been initiated, moving from inhibition of fully activated, phosphorylated kinases to targeting the inactive, unphosphorylated forms. For identification and characterization of putative inhibitors, also interacting with the latent kinase conformation outside of the kinase domain, highly purified and homogeneous protein preparations of unphosphorylated kinases are essential. The kinetic parameters of nonphosphorylated kinases cannot be assessed easily by standard kinase enzyme assays as a result of their intrinsic autophosphorylation activity. Kinetic binding rate constants of inhibitor-protein interactions can be measured by biophysical means upon protein immobilization on chips. Protein immobilization can be achieved under mild conditions by binding biotinylated proteins to streptavidin-coated chips, exploiting the strong and highly specific streptavidin–biotin interaction. In the work reported here, the cytoplasmic domains of insulin receptor and insulin-like growth factor-1 receptor fused to a biotin ligase recognition sequence were coexpressed individually with the phosphatase YopH and the biotin-protein ligase BirA upon triple infection in insect cells. Tandem affinity purification yielded pure cytoplasmic kinase domains as judged by gel electrophoresis and HPLC. Liquid chromatography-mass spectrometry analysis showed the absence of any protein phosphorylation. Coexpression of BirA led to quantitative and site-specific biotinylation of the kinases, which had no influence on the catalytic activity of the kinases, as demonstrated by the identical phosphorylation pattern upon autoactivation and by enzymatic assay. This coexpression approach should be applicable to other protein kinases as well and should greatly facilitate the production of protein kinases in their phosphorylated and unphosphorylated state suitable for enzymatic and biophysical studies.

A series of novel benzoxazole derivatives has been designed and shown to exhibit attractive JAK2 inhibitory profiles in biochemical and cellular assays, capable of delivering compounds with favorable PK properties in rats. Synthesis and structure–activity relationship data are also provided.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials. [Mol Cancer Ther 2008;7(7):1–13 [Mol Cancer Ther 2008;7(7):1851–13]

Epidermal growth factor (EGF) has been reported to stimulate carbohydrate, amino acid, and electrolyte transport in the small intestine, but its effects on lipid transport are poorly documented. This study aimed to investigate EGF effects on fatty acid uptake and esterification in a human enterocyte cell line (Caco-2). EGF inhibited cell uptake of [14C]palmitate and markedly reduced its incorporation into triglycerides. In contrast, the incorporation in phospholipids was enhanced. To elucidate the mechanisms involved, key steps of lipid synthesis were investigated. The amount of intestinal fatty acid-binding protein (I-FABP), which is thought to be important for fatty acid absorption, and the activity of diacylglycerol acyltransferase (DGAT), an enzyme at the branch point of diacylglycerol utilization, were reduced. EGF effects on DGAT and on palmitate esterification occurred at 2–10 ng/ml, whereas effects on I-FABP and palmitate uptake occurred only at 10 ng/ml. This suggests that EGF inhibited palmitate uptake by reducing the I-FABP level and shifted its utilization from triglycerides to phospholipids by inhibiting DGAT. This increase in phospholipid synthesis might play a role in the restoration of enterocyte absorption function after intestinal mucosa injury.

Most of the current oncology drug discovery and development work has shifted towards molecularly targeted therapies. A key focus has been on identifying inhibitors against components of pathways that drive tumor cell proliferation, survival, and metastasis such as the PI3K/mTOR pathway, reported to be implicated in many human cancers through various mechanisms, such as, somatic PIK3CA missense mutations that occur at high frequency. These mutations are largely point mutations predominantly clustered within three hotspots in the helical and kinase domains of p110α: E542K, E545K and H1047R, which represent about 80% of the mutations observed, and confirmed to be oncogenic gain-of-function mutations. Based on these findings cancer-specific mutants of p110α appear to be ideal targets for drug development and p110α specific inhibitors, such as NVP-BYL719, could then have potential anti-cancer activity without causing the potential side effects that could be expected from interference with other Class I PI3K isoforms or other members of the PIKK family. NVP-BYL719 is best described as a PI3Kalpha inhibitor as in biochemical assays, it inhibits p110α as well as p110α most common somatic mutations (IC50=5 nM) much more potently than p110α and ≤ and has weak or no activity against p110α, Vps34 and mTOR and is selective against a wide range of protein kinases (> 50-fold). The potency and selectivity profile of NVP-BYL719 is confirmed at the cellular level, is correlated with inhibition of various PI3K/Akt downstream signaling pathway components and is associated with anti-proliferation of breast cancer cell lines harboring PIK3CA mutations. In vivo, NVP-BYL719 shows dose and time-dependent inhibition of the PI3K/Akt pathway which correlates with compound exposure and is associated with good tolerability and significant dose-dependent anti-tumor efficacy in PIK3CA mutant tumor xenograft models in rodents. Moreover, plasma insulin levels are significantly increased while blood glucose levels remained normal at all tested doses and time points indicating on-target effects of NVP-BYL719 on the regulation of metabolism. Overall, as a PI3Kalpha inhibitor, NVP-BYL719 has good drug-like and pharmacological properties and presents all the characteristics required for its ongoing Phase I clinical development in adult patients with advanced solid malignancies whose tumors have an alteration of the PIK3CA gene. 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 3748. doi:1538-7445.AM2012-3748

PI3Ks are lipid kinases that control signaling pathways involved in cell proliferation, motility, cell death and cell invasion. Class I PI3K contains four isoforms, p110α, p110α, p110α and p110α which carry out non redundant signaling functions. The ≤ and ≤ isoforms are ubiquitously expressed, whereas the ≤ and ≤ isoforms are expressed primarily in lymphocytes and engaged in the regulation of immune responses. A gain of function in PI3K signaling is common to many types of human cancer, specifically mutations in PIK3CA which are found in more than 30% of various solid tumor types, including, breast, endometrium, bladder, colorectal cancers as well as lung cancers. As these mutations constitutively activate the lipid kinase activity of the protein, the cancer-specific mutants of p110α appear to be ideal therapeutic targets for anticancer drug development. p110α isoform specific low molecular weight inhibitors could be efficacious against tumors harboring p110alpha mutations and provide an improved safety profile compared to current pan-PI3K modulators. The 2-aminothiazole scaffold proved to be an excellent starting point for the development of potent PI3K inhibitors. Depending on the substitution pattern good PI3Kalpha selectivity can be achieved. Systematic modification of key residues and further optimization of the drug-like and PK properties have led to the identification of NVP-BYL719, a potent and selective PI3Kalpha inhibitor with a promising biological activity. SAR leading to the discovery of NVP-BYL719 and the structural basis for the PI3Kalpha selectivity will be discussed. 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 1922. doi:1538-7445.AM2012-1922

Abstract 62 Mutation within the kinase domain of tyrosine kinases is a common mechanisms of resistance to enzymatic inhibitors. Inhibitors of janus kinase 2 (JAK2) are under evaluation in patients with myeloproliferative neoplasms (MPNs), B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit CRLF2, and other tumors with constitutive JAK2 signaling. To identify resistance mutations in JAK2, we randomly mutagenized human JAK2 R683G, which is observed in approximately half of CRLF2-rearranged B-ALL. We transduced the mutagenized JAK2 cDNA library into murine Ba/F3 cells that express CRLF2. Expression of CRLF2 and JAK2 R683G confers IL3 independent growth in Ba/F3 cells. The transduced population was selected in the JAK2-selective inhibitor NVP-BVB808 in the absence of IL3. Multiple BVB808-resistant clones were recovered that harbored either E864K, Y931C or G935R mutations in JAK2. Alignment of homologous regions of the JAK2 kinase domain (JH1) with ABL1 demonstrated that the three mutations are located in regions homologous to imatinib resistance hotspots in ABL1. Codons Y931 and G935 are within the hinge region of the kinase domain. Based on structural modeling, Y931C is likely to inhibit substrate binding. E864K is located in the middle of b3 following the P-loop in the N-lobe and may modify the structure and flexibility of the preceding P-loop, thus destabilizing the conformation required for inhibitor binding. We expressed JAK2 V617F alleles harboring Y931C, G935R or E864K in Ba/F3-EPOR cells and exposed the cells to the JAK2 enzymatic inhibitors JAK inhibitor-1, NVP-BSK805, TG101348, tofacitinib (formerly tasocitnib), ruxolitinib (formerly INCB18424) and BVB808. All three mutations conferred 2- to >10-fold resistance against BVB808, NVP-BSK805, TG101348, ruxolitinib and JAK inhibitor-1. Y931C and E864K but not G935R conferred resistance to tofactinib. Modeling of G935R indicated that a 935R side-chain would occlude the hydrophobic channel of the ATP-binding pocket. As a consequence, this mutation would decrease the binding affinity of compounds occupying the hydrophobic channel like JAK inhibitor-1 or BSK805, but not affect the potency of tofactinib, which does not bind in this region. Mutation of G935 to arginine, histidine or glutamine reduced the inhibitory effects of JAK inhibitor-1, but not tofacitinib, on JAK2 kinase domain activity. None of the codon 935 mutations had significant effects on Km or Vmaxin vitro. BVB808 treatment partially reduced activation state-specific phosphorylation of STAT5 in Ba/F3-EPOR/JAK2 V617F cells but not in Ba/F3-EPOR/JAK2 V617F/G935R or G935H cells. JAK2 is a known client of HSP90, and HSP90 inhibitors promote the degradation of both wild-type and mutant JAK2. We hypothesized that resistance mutations within the JAK2 kinase domain would not affect JAK2 degradation induced by HSP90 inhibitors. We assayed the cytotoxicity of the resorcinylic isoxazole amide NVP-AUY922 and the benzoquinone ansamycin 17-AAG in Ba/F3 cells that express the erythropoietin receptor (EPOR) and JAK2 V617F, which is observed in more than half of MPNs. Mutation of JAK2 V617F to include E864K, Y931C or G935R did not affect sensitivity to either AUY922 or 17-AAG. In fact, AUY922 was more active against cells harboring G935R (GI50, 3.87 nM) or E864K (GI50, 6.14 nM) compared to cells with no resistance mutation (GI50, 14.7 nM; p Disclosures: Gaul: Novartis: Employment. Vangrevelinghe:Novartis: Employment. De Pover:Novartis: Employment. Regnier:Novartis: Employment. Erdmann:Novartis: Employment. Hofmann:Novartis: Employment. Eck:Novartis: Consultancy, Research Funding. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Radimerski:Novartis Pharma AG: Employment. Weinstock:Novartis: Consultancy, Research Funding.

Abstract 3777 Poster Board III-713 The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and from primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative disorders and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild type enzymes by novel small molecule inhibitors, which act in an ATP-competitive manner. The profile of a lead compound from this class will be presented that displays more than twenty-fold selectivity towards JAK2 within the JAK family, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, the inhibitor exhibited good oral bioavailability and is efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model as well as polycythemia and extramedullary erythropoiesis in mouse and rat models. Disclosures: Radimerski: Novartis: Employment, Equity Ownership. Baffert:Novartis: Employment. Regnier:Novartis: Employment. De Pover:Novartis: Employment. Pissot:Novartis: Employment. Gerspacher:Novartis: Employment. Brueggen:Novartis: Employment. Tavares:Novartis: Employment. Blasco:Novartis: Employment. Ledieu:Novartis: Employment. Nolan:Novartis: Employment. Ruetz:Novartis: Employment. Chene:Novartis: Employment. Erdmann:Novartis: Employment. Drueckes:Novartis: Employment. Furet:Novartis: Employment. Lang:Novartis: Employment. Trappe:Novartis: Employment. Vangrevelinghe:Novartis: Employment. Wartmann:Novartis: Employment. Hofmann:Novartis: Employment.

(Na+ + K+)ATPase (ATP phosphohydrolase, EC 3.6.1.3.) has been prepared from human heart and guinea-pig heart, with respective specific activities of 10–15 μmol Pi · mg−1 · h−1 and of 25–30 μmol Pi · mg−1 · h−1. Residual Mg2+—ATPase activities were about 5 per cent. The parameters of (Na+ + K+)ATPase activity and of ouabain-interaction have been compared: (1) Half-maximal activity concentrations and Hill coefficients of Na+, K+, Mg2+ and ATP were similar for the two species. The apparent activation energies calculated from Arrhenius plots were also similar. A transition was observed at about 23°C. (2) Human heart (Na+ + K+)ATPase was 10 times more sensitive to ouabain-inhibition than that of guinea-pig. Hunter-Downs plots showed a competitive inhibition for K+ at low K+ concentration and noncompetitive inhibition at high concentration. (3) The Scatchard plot for [3H]ouabain binding was upward-concave with human heart and linear with guinea-pig heart. (4) The dissociation kinetics of [3H]ouabain from human preparations studied by an isotopic dilution technique indicated two classes of binding sites with kd of 0.058 min−1 and 0.0092 min−1. The dissociation kinetics with guinea-pig heart indicated one single class of binding sites with a kd of 0.43 min−1. (5) The time-course of 0.2 μM [3H]ouabain binding showed pseudo-first order association kinetics in man and in guinea-pig. ka for the two classes of binding sites in man were therefore similar, respectively equal to 3.4 × 106 min−1 · M−1 and to 3.7 × 106 min−1 · M−1 · ka for guinea-pig heart was equal to 2.3.106 min−1 · M−1. (6) In guinea-pig heart, KD c from Scatchard plot and from kd/ka ratio were equal to the inhibition constant Ki calculated from Hunter-Downs plot indicating that the binding sites were closely related to (Na+ + K+)ATPase inhibition. (7) In human heart, KD of the low affinity binding sites was close to Ki, whereas KD of the high affinity binding sites was several times lower. This suggests that only low affinity binding sites might be involved in (Na+ + K+)ATPase inhibition by ouabain.

The inhibition of guinea-pig heart (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) by calcium has been studied at pH 7.4, 6.8 and 6.4. 1. 1.A decrease in pH reduced the threshold inhibitory concentration of calcium and the calcium concentration producing an inhibition of 50% of the enzyme activity. 2. 2.Calcium reduced the apparent affinity of the enzyme for Na+, this effect occurred only at pH 7.4. 3. 3.Calcium increased the apparent affinity of the enzyme for K+, this effect was enhanced at acidic pH. 4. 4.Activation of the enzyme by Na+ for a constant Na+: K+ ratio has been studied at pH 7.4 and at pH 6.8 in the absence and in the presence of 3 · 10−4 M Ca2+; the results of this experiment indicate that Ca2+ effect at pH 7.4 was not influenced by Na+ − K+ competition and was probably due to a Na+ − Ca2+ interaction. 5. 5.At pH 7.4, the calcium inhibitory threshold concentration and the concentration producing 50% inhibition were reduced when Na+ was low; at pH 6.8, the calcium inhibition was not markedly modified by the change of Na+ concentration. 6. 6.The Ca2+-activated ATPase of myosin B which is related to the contractile behaviour of muscle and the Ca2+-ATPase of the sarcoplasmic reticulum which is related to the ability of this structure to accumulate calcium were activated in a range of calcium concentration producing an inhibition of (Na+ + K+)ATPase. The present results indicate that the increase by acidity of the (Na+ + K+)ATPase sensitivity to calcium might be due to a suppression of a Na+-Ca2+ interaction. On the basis of these observations, it is proposed that calcium might inhibit the Na+-pump during the repolarization phase of the action potential and that, by this effect, it might control cell excitability.