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Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors.Significance:JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155.See related video: https://vimeo.com/720726054This article is highlighted in the In This Issue feature, p. 1397

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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PDF file - 43K, BEZ235 inhibits phosphorylation of PI3K/mTOR. Western blot showing phospho-AKT (Ser473) and phospho-S6 (Ser235/236) expression in serum-starved H460 and A549 cells treated with indicated doses of BEZ235 and stimulated with IGF-1 (100 ng/ml).

PDF file - 1.8MB, BEZ235 does not induce abnormal mitoses in irradiated H460 and A549 cells. Number of abnormal mitoses at 48 hours following indicated treatment. An average of 70 cells per group was counted to determine mean +/- SEM.

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, 300KB, A. Upon infection with viral particles obtained from the pBabe-puro-myr-Akt retroviral vectors, pools were selected upon addition of puromycin and maintained in culture for several weeks. Extracts from pools were generated for the indicated time of culture and analyzed by Western-blot for the expression of the exogenously expressed HA-tagged form of the dominant active myr-Akt protein. B. 2.5x106 MCF7-BP (upper panel) or MCF7-myr-Akt (bottom panel) were seeded in 10 cm dishes and exposed for 1 h to the indicated compounds at the indicated concentrations (in nM). Cells were then lyzed and cell extracts were analyzed by Western-blot for pathway inhibition as revealed by S473P-Akt levels.

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The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G2–M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α–dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 μmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor–bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K. Mol Cancer Ther; 11(8); 1747–57. ©2012 AACR.

PDF file - 2MB, IL-8, MIF and PAI-1 levels in supernatant from H460 and A549 cells after 96 hours treatment. Supernatant volumes were normalized to cell number and analyzed using cytokine antibody arrays.

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PDF file - 4.6MB, Representative immunofluorescence images of phospho-ATM (Ser1981) foci in H460 and A549 cells at 30 minutes after 10 Gy dose of radiation (x 60 magnification). Green, phospho-ATM foci. Blue, DAPI staining.

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PDF file, 53KB, A and B. 5x105 A2058 cells were seeded in 10 cm dishes and incubated for 24 h either with increasing amounts of BKM120 (A) or with 5 �M of either GDC-0941 (B, top panel) or BEZ235 (B, bottom panel). Cells were then fixed, prepared as described for quantification of the population in the different phases of the cell cycle by fluorescence-activated cell sorting. G1, S and G2/M distribution for control untreated cells are described in the mean text and in Figure 4A. The activities of BKM120 on the cell cycle were plotted along to the inhibitory effects on pAkt levels (A).

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PDF file - 7.4MB, TUNEL assay in H460 and A549 cells after 48 hours treatment. H460 cells irradiated with 20 Gy were used as a positive control.

PDF file - 9MB, Representative immunofluorescence images of gamma H2AX (Ser139) foci in H460 and A549 cells at indicated time points after 4 Gy radiation (x 60 magnification). Green, �H2AX foci. Blue, DAPI staining. Below, Number of gamma H2AX foci per cell. Data are mean +/- SEM from three independent experiments. ***, P

Supplementary Table 1 PDF file - 98K, p53 somatic point mutations in PTEN-null CRPC

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 file, 107KB, A. 2.5x106 Rat1-myr-p110a (left) or U87MG cells were seeded in 10 cm dishes and exposed for 6h either to BKM120 (5 �M) or to the DMSO control. Cells were then lyzed and cell extracts were analyzed by Western-blot for PI3K pathway inhibition and G2/M markers as revealed by S473P-Akt (upper panel) and Ser10P-Histone H3 (bottom panel) levels, respectively. B. U87MG tumor bearing mice were treated orally, once per day for 6 days with BKM120 at the indicated doses. Tumor volumes were callipered and plotted. ns: not significant.

PDF file - 4.8MB, Representative immunofluorescence images of phospho-DNA-PKcs (Thr2609) foci in H460 cells at 30 minutes after 10 Gy radiation (x 60 magnification). Green, phospho-DNA-PKcs foci. Blue, DAPI staining.

PDF file, 113KB, A. Effects of Paclitaxel and Nocodazole on Tubulin polymerization. Tubulin was mixed with either Paclitaxel (10 �M), Nocodazole (10 �M) or the DMSO control in the presence of GTP. The polymerization of monomeric tubulin into microtubule was started by transferring the reaction tubes from 4{degree sign}C to 37{degree sign}C, and monitored by the increase in absorbance (λ=340 nM) over a period of 60 min. B. Competition experiments of NVP-BKM120 with colchicine and podophyllotoxin by NMR spectroscopy. T1ρ relaxation of BKM120 in the presence of tubulin (50-fold excess of compound) remains unchanged after adding podophyllotoxin or colchicine, as emphasized by the drawn arrows. The spectra of the three compounds are shown in three colors at the bottom. C. Structures of GDC0941, BEZ235, Nocodazole and BKM120.

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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Supplementary Figures PDF file - 5047K, supplementary figures for opposing effects of androgen deprivation and targeted therapy on prostate cancer prevention

PDF file, 653KB, MDA-MB231 (left) and U87MG (right) cells grown on coverslips were treated for 24 h either with BKM120 (5 μM) or Nocodazole (100 nM) and the effects of compound treatment on microtubule dynamics and G2/M arrest was monitored by immuno-fluorescence staining of alpha-tubulin (microtubules), gamma tubulin (centrosomes) and DAPI (DNA). Pictures were taken with a 100X objective.

PDF file, 2791KB. A2058 (upper panels), MDA-MB231 (lower panels) and U87MG cells grown on coverslips were treated for 6 h, 6 h with a subsequent 18 h washout period or 24 h with either 5 μM BKM120 or 100 nM Nocodazole and the effects of compound treatment on microtubule dynamics and G2/M arrest was monitored by immuno-fluorescence staining of alpha-tubulin. Pictures were taken with a 40X objective.

PDF file - 171K, Induction of accelerated senescence by BEZ235 in irradiated cells is p53-dependent. A, Representative images of SA-�Gal activity cells at 48 hours after 10 Gy radiation (x 200 magnification). B, Western blot showing total p53 and p21 levels in p53-inducible H1299 cells exposed to indicated concentrations (�g/ml) of Ponasterone A (PoA) for 48 hours. C, Representative images of SA-�Gal activity cells in p53-inducible H1299 cells 48 hours after 10 Gy radiation (x 200 magnification). PoA (0.5 �g/ml) was added to growth medium 8 hours prior to irradiation and maintained in growth medium until cells were fixed and stained.

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Supplementary Data from Targeting Melanoma with Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitors

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 Figures S1-S4 from mTOR Inhibitor RAD001 (Everolimus) Has Antiangiogenic/Vascular Properties Distinct from a VEGFR Tyrosine Kinase Inhibitor

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Purpose: The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo.Experimental Design: The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model.Results: We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell line's sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts).Conclusion: Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment. Clin Cancer Res; 18(1); 184–95. ©2011 AACR.

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

Purpose: The growth and survival of acute myeloid leukemia (AML) cells are enhanced by the deregulation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR). Major efforts have thus been made to develop molecules targeting these activated pathways. The mTOR serine/threonine kinase belongs to two separate complexes: mTORC1 and mTORC2. The mTORC1 pathway is rapamycin sensitive and controls protein translation through the phosphorylation of 4E-BP1 in most models. In AML, however, the translation process is deregulated and rapamycin resistant. Furthermore, the activity of PI3K/Akt and mTOR is closely related, as mTORC2 activates the oncogenic kinase Akt. We therefore tested, in this study, the antileukemic activity of the dual PI3K/mTOR ATP-competitive inhibitor NVP-BEZ235 compound (Novartis).Experimental Design: The activity of NVP-BEZ235 was tested in primary AML samples (n = 21) and human leukemic cell lines. The different signaling pathways were analyzed by Western blotting. The cap-dependent mRNA translation was studied by 7-methyl-GTP pull-down experiments, polysomal analysis, and [3H]leucine incorporation assays. The antileukemic activity of NVP-BEZ235 was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation, and survival.Results: The NVP-BEZ235 compound was found to inhibit PI3K and mTORC1 signaling and also mTORC2 activity. Furthermore, NVP-BEZ235 fully inhibits the rapamycin-resistant phosphorylation of 4E-BP1, resulting in a marked inhibition of protein translation in AML cells. Hence, NVP-BEZ235 reduces the proliferation rate and induces an important apoptotic response in AML cells without affecting normal CD34+ survival.Conclusions: Our results clearly show the antileukemic efficiency of the NVP-BEZ235 compound, which therefore represents a promising option for future AML therapies. Clin Cancer Res; 16(22); 5424–35. ©2010 AACR.

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

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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]

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