Combination Of PI3K Inhibitors Research Articles

Abstract A117: Evaluation of nab-sirolimus in combination with PI3K pathway inhibitors to overcome PI3K/mTOR resistance in PI3K-mutant breast cancer cell lines

Abstract Background: The PI3K-AKT-mTOR pathway is frequently activated in many cancer types and plays a central role in tumorigenesis. However, clinical use of therapies targeting this pathway is limited by compensatory vertical and horizontal feedback activation loops, which limit antitumor efficacy and lead to drug resistance. Combination therapy strategies employing simultaneous inhibition of multiple PI3K-AKT-mTOR pathway elements may overcome these resistance mechanisms and improve antitumor activity. nab-Sirolimus is a novel albumin-bound nanoparticle form of the mTOR inhibitor sirolimus that has significantly greater tumor accumulation, mTOR target suppression, and improved antitumor effects compared with conventional first generation mTOR inhibitors in preclinical models. Here, we analyzed the effects of nab-sirolimus combinations in PI3K-mutant breast cancer (BrCa) cells. Methods: PI3K mutated BrCa cell lines (MDA-MD-453, hormone receptor [HR]-; and MDA-MB-361, HR+) were incubated for 5 days with increasing concentrations of nab-sirolimus, the PI3K pathway inhibitors gedatolisib and alpelisib, or the AKT inhibitors miransertinib and capivasertib. The antiproliferative and cytotoxic effects of single agent and combination treatment were assessed using cell viability and cell death assays. Cell signaling was analyzed by western blot. Results: The antiproliferative effects of gedatolisib at low doses (2.5-10 nM) were enhanced by 61-71% in combination with nab-sirolimus (20 or 80 nM) in MDA-MB-453 cells. Limited cell death (4.2-9.6%) was observed in HR- MDA-MB-453 cells with low doses (2.5, 5, and 10 nM) of gedatolisib alone. In contrast, and despite the well-established cytostatic action of mTOR inhibitors, the addition of 20 and 80 nM nab-sirolimus led to high levels of cell death (up to 48.9%). Similar results were observed with alpelisib at 1 µM in combination with nab-sirolimus (20 or 80 nM), and also with capivasertib (1 µM) or miransertib (250 nM) combined with 80 nM nab-sirolimus. Western blot analysis demonstrated that gedatolisib or alpelisib alone each triggered upregulation of p4EBP1 whereas nab-sirolimus treatment alone activated a negative feedback loop through increased pAKT. Importantly, these compensatory feedback activation loops were both reversed by the combination of PI3K inhibitors and nab-sirolimus. Similar cell death and cell viability results were observed with nab-sirolimus plus PI3K inhibitors (gedatolisib or alpelisib) and AKT inhibitors (capivasertib or miransertib) in HR+ MDA-MB-361 cells. Conclusions: The antitumor effects of PI3K and AKT inhibitors were enhanced by the addition of the novel mTOR inhibitor nab-sirolimus. The improved effectiveness of the PI3K and mTOR inhibitor combination was due to the reciprocal overcoming of resistance mechanisms induced by single agent treatment. These data support a vertical PI3K pathway inhibition strategy using nab-sirolimus and PI3K/AKT inhibitors in PIK3CA-mutated BrCa, regardless of HR status. Citation Format: Sean Wallace, Khine Nyein Myint, Shihe Hou, Maria Zalath, Andrew Kwon, Brian McMorran, Igor Vivanco. Evaluation of nab-sirolimus in combination with PI3K pathway inhibitors to overcome PI3K/mTOR resistance in PI3K-mutant breast cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A117.

Pictilisib-Induced Resistance Is Mediated through FOXO1-Dependent Activation of Receptor Tyrosine Kinases in Mucinous Colorectal Adenocarcinoma Cells.

The phosphatidylinositol (PI3K)/AKT/mTOR axis represents an important therapeutic target to treat human cancers. A well-described downstream target of the PI3K pathway is the forkhead box O (FOXO) transcription factor family. FOXOs have been implicated in many cellular responses, including drug-induced resistance in cancer cells. However, FOXO-dependent acute phase resistance mediated by pictilisib, a potent small molecule PI3K inhibitor (PI3Ki), has not been studied. Here, we report that pictilisib-induced adaptive resistance is regulated by the FOXO-dependent rebound activity of receptor tyrosine kinases (RTKs) in mucinous colorectal adenocarcinoma (MCA) cells. The resistance mediated by PI3K inhibition involves the nuclear localization of FOXO and the altered expression of RTKs, including ErbB2, ErbB3, EphA7, EphA10, IR, and IGF-R1 in MCA cells. Further, in the presence of FOXO siRNA, the pictilisib-induced feedback activation of RTK regulators (pERK and pAKT) was altered in MCA cells. Interestingly, the combinational treatment of pictilisib (Pi3Ki) and FOXO1i (AS1842856) synergistically reduced MCA cell viability and increased apoptosis. These results demonstrate that pictilisib used as a single agent induces acute resistance, partly through FOXO1 inhibition. Therefore, overcoming PI3Ki single-agent adaptive resistance by rational design of FOXO1 and PI3K inhibitor combinations could significantly enhance the therapeutic efficacy of PI3K-targeting drugs in MCA cells.

Abstract 2661: Novel pharmacological approaches of inhibiting the PI3K/AKT/mTOR signaling pathway in T-cell lymphoma (TCL)

Abstract Introduction: T-cell lymphoma (TCL) accounts for 15% of all non-Hodgkin’s lymphoma cases with most patients developing relapsed/refractory (R/R) disease. The management of R/R TCL is challenging and responses to second and beyond-line therapy are generally poor. Pre-clinical and clinical studies have identified the relevance of PI3K/AKT/mTOR signaling pathway in TCL biology. The combination of PI3K inhibitor (PI3Ki) Duvelisib and the HDACi Romidepsin improved the overall response rate (ORR) of R/R TCL to 50-55% in a phase I/II study (Horwitz et. al. ASH 2018, Lugano 2021). Evaluation of PI3Ki and HDACi combinations in TCL pre-clinical models is needed to guide the design of future clinical trials. Methods: A panel of TCL cells lines were chosen representing T-cell acute lymphoblastic leukemia, T-lymphoblastic lymphoma, cutaneous T-cell lymphoma. A panel of PI3Ki (Omipalisib, Umbralisib and Duvelisib) and HDACi (Entinostat and Romidepsin) were selected to determine which combination would be most synergistic. Entinostat, an oral HDACi, was chosen for its remarkable preclinical and clinical activity in other lymphoma subtypes. Umbralisib is a new PI3Kδ inhibitor which was recently approved for treatment of follicular lymphoma and marginal zone lymphoma. Cells were exposed to Omipalisib (3.125-100nM), Umbralisib (1-320uM), or Duvelisib (1-100uM) alone or in combination with Entinostat (0.08-10uM) or Romidepsin (0.25-6) for 24, 48 and 72 hrs. Cell viability was measured using the CellTiter-Glo viability assay. Half-maximal inhibitory concentration (IC50) at 72hrs was determined for each drug and coefficient of synergy was calculated using Calcusyn Software. Changes in cellular metabolism and cell cycle distribution after exposure to PI3Ki were determined using DiOC6 staining and Propidium Iodide staining respectively. Epigenetic changes following exposure to Entinostat or Romidepsin were investigated for possible mechanisms. Results: In vitro exposure of TCL cell lines to PI3Ki or HDACi resulted in dose-dependent cell death. The IC50 at 72 hrs was 30-500nM for Omipalisib, 9-35uM for Umbralisib, 1-35uM for Duvelisib, 0.2-2uM for Entinostat and for 1-4nM Romidepsin. All HDACi and Pi3Ki combinations demonstrated additive and/or synergistic activity. Duvelisib combined with Entinostat or Romidepsin exhibited the most synergy. Methylation profiling demonstrated genomic wide hypomethylation, specifically DNMT3A, in Romidepsin treated samples compared to Entinostat. Conclusions: PI3Ki and HDACi combinations show significant synergy in TCL preclinical models. DNMT3A hypomethylation following Romidepsin exposure is being explored as the basis of PI3Ki and HDACi synergy. If confirmed in clinical trials, these results may refine the tolerability of the regimen for patients treated with this very promising PI3Ki/HDACi combination. Citation Format: Gabrielle Hartman, Cory Mavis, Juan J. Gu, Amber White, Taylor Mandeville, Pallawi Torka, Paola Ghione, Francisco J. Hernandez-Ilizaliturri. Novel pharmacological approaches of inhibiting the PI3K/AKT/mTOR signaling pathway in T-cell lymphoma (TCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2661.

Abstract 411: Reversibility of tumor adaptations to PI3K inhibition decreases as a function of time

Abstract The PI3K pathway is a key regulator of metabolism, cell proliferation and migration and some of its components (e.g. PIK3CA, HER2 and PTEN) are frequently altered in cancer by genetic events that deregulate its output. However, inhibitors of components of the pathway have only modest antitumor effects and, in some instances, have resulted in “tumor flares” in patients upon cessation of therapy. Low efficacy has been attributed to toxicity and to adaptive resistance. Inhibition of the PI3K pathway has been associated with relief of feedback inhibition of receptor signaling (RTKs) by a variety of mechanisms including increased receptor expression and decreased expression of the PTEN phosphatase, a major negative regulator of the pathway. Now we show that changes in expressions of RTKs, PTEN, anti-apoptosis proteins like Mcl1 and transcription factors known to drive oncogenic and survival programs in tumors like c-Myc, c-Jun, FOXOs are induced upon continuous long-term PI3K inhibition (2 weeks and longer). These changes increase as a function of duration of inhibition and become less reversible with time in drug. Unbiased clustering of RNA seq of cells treated with PI3K inhibitors revealed clusters of gene expression changes based upon short or long-term inhibition. Correspondingly the mRNA of the RTKs and transcription factors change with similar kinetics as that of the proteins and become less reversible with duration of inhibition. Moreover, the persister cells treated with PI3K inhibitors for 2 weeks or longer grow faster than the treatment naive cells upon drug removal both in vitro and in vivo and the degree of reversibility of the increased proliferation rate is inversely proportional to the duration of inhibition. Persister cells treated with PI3K inhibitors for more than two weeks have higher levels of expression of a set of transcription factors, RTKs, Mcl1 and of pAKT after one week of drug wash-out and are less sensitive to re-inhibition of PI3K. Combined inhibition of PI3K with Mcl1 inhibitors lead to greater reduction of cell proliferation and increased cell death.This suggests that continuous long-term inhibition of PI3K leads to a chronic adaptive state driven by relief of negative feedback which becomes less reversible with increasing duration of inhibition. Cells undergoing long term inhibition have higher steady state levels of components of the pathway leading to increased cell growth upon drug removal as compared to untreated cells. The data suggests that induction of adaptive resistance is in part responsible for the continued persistence of tumor masses after the initial response to targeted therapy. We hypothesize that pulsatile targeted inhibition with short intervals off therapy could avoid development of chronic adaptation. Intermittent administration of PI3K inhibitor combinations that cause potent induction of cell death may result in improved therapeutic outcomes. Citation Format: Radha Mukherjee, Elisa de Stanchina, Sarat Chandarlapaty, Neal Rosen. Reversibility of tumor adaptations to PI3K inhibition decreases as a function of time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 411.

A high-throughput drug combination screen identifies an anti-glioma synergism between TH588 and PI3K inhibitors

BackgroundGlioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor. More than half of GBMs contain mutation(s) of PTEN/PI3K/AKT, making inhibitors targeting the PI3K pathway very attractive for clinical investigation. However, so far, PI3K/AKT/mTOR inhibitors have not achieved satisfactory therapeutic effects in clinical trials of GBM. In this study, we aimed to develop a high-throughput screening method for high-throughput identification of potential targeted agents that synergize with PI3K inhibitors in GBM.MethodsA Sensitivity Index (SI)-based drug combination screening method was established to evaluate the interactions between BKM120, a pan-PI3K inhibitor, and compounds from a library of 606 target-selective inhibitors. Proliferation, colony and 3D spheroid formation assays, western blotting, comet assay, γ-H2AX staining were used to evaluate the anti-glioma effects of the top-ranked candidates. The drug combination effects were analyzed by the Chou-Talalay method.ResultsSix compounds were successfully identified from the drug screen, including three previously reported compounds that cause synergistic antitumor effects with PI3K/mTOR inhibitors. TH588, an putative MTH1 inhibitor exhibited significant synergy with BKM120 in suppressing the proliferation, colony formation and 3D spheroid formation of GBM cells. Further investigation revealed that both DNA damage and apoptosis were markedly enhanced upon combination treatment with TH588 and BKM120. Finally, activation of PI3K or overexpression of AKT compromised the anti-glioma efficacy of TH588.ConclusionsThe screening method developed in this study demonstrated its usefulness in the rapid identification of synergistic drug combinations of PI3K inhibitors and targeted agents.

CUDC-907, a novel dual PI3K and HDAC inhibitor, in prostate cancer: Antitumour activity and molecular mechanism of action.

Targeting the androgen receptor (AR) signalling pathway remains the main therapeutic option for advanced prostate cancer. However, resistance to AR‐targeting inhibitors represents a great challenge, highlighting the need for new therapies. Activation of the PI3K/AKT pathway and increased expression of histone deacetylases (HDACs) are common aberrations in prostate cancer, suggesting that inhibition of such targets may be a viable therapeutic strategy for this patient population. Previous reports demonstrated that combination of PI3K inhibitors (PI3KIs) with histone deacetylase inhibitors (HDACIs) resulted in synergistic antitumour activities against preclinical models of prostate cancer. In this study, we demonstrate that the novel dual PI3K and HDAC inhibitor CUDC‐907 has promising antitumour activity against prostate cancer cell lines in vitro and castration‐resistant LuCaP 35CR patient‐derived xenograft (PDX) mouse model in vivo. CUDC‐907‐induced apoptosis was partially dependent on Mcl‐1, Bcl‐xL, Bim and c‐Myc. Further, down‐regulation of Wee1, CHK1, RRM1 and RRM2 contributed to CUDC‐907‐induced DNA damage and apoptosis. In the LuCaP 35CR PDX model, treatment with CUDC‐907 resulted in significant inhibition of tumour growth. These findings support the clinical development of CUDC‐907 for the treatment of prostate cancer.

Abstract LB-B03: PARP and PI3K inhibitor combination therapy eradicates c-MYC-driven murine prostate cancers via cGAS/STING pathway activation within tumor-associated macrophages

Abstract While immune checkpoint blockade (ICB) targeting CTLA-4 and PD-1/PD-L1 has shown remarkable success across a wide range of malignancies, the majority of prostate cancer (PC) patients do not demonstrate clinically meaningful responses to these therapies. Therefore, there is a critical unmet need to discover combinatorial therapeutic strategies that enhance immune-responsiveness in ICB-refractory cancers, such as PC. Here, we show that DNA damage induced by PARP inhibitor (PARPi), rucaparib, in combination with PI3K inhibitor (PI3Ki), buparlisib, resulted in STING activation within myeloid suppressive cells within the TME. Strikingly, this myeloid reprogramming induced by rucaparib/buparlisib combination was accompanied by enhanced T cell infiltration and tumor regression of myc-driven murine PCs in vivo. Furthermore, the anti-tumor mechanism of the rucaparib/buparlisib combination was non-tumor cell autonomous and immune-mediated, as the effect was abrogated in immunodeficient NOD/SCID mice. Critically, rucaparib/buparlisib-mediated tumor regression in myc-driven murine models was abrogated with concomitant clodronate treatment in vivo, which depletes macrophages. To unravel the mechanism for activation of macrophage-mediate anti-cancer immunity elicited by DNA damage, we performed co-culture assays, which revealed that STING pathway activation elicited within macrophages was mediated by double-stranded (ds) DNA-containing microvesicles (MVs) released from tumor cells treated with PARP inhibitors, coupled with c-GAS de-repression induced by PI3K inhibition within macrophages. Interestingly, ex vivo exosome reconstitution studies revealed that DNAse treatment of the MVs completely abolished the STING activation response elicited within macrophages, thus demonstrating that the DNA fragments on the surface of exosomes drives STING pathway activation within macrophages. Strikingly, the combination of rucaparib/buparlisib with immune checkpoint blockade enhanced macrophage and T cell activation/infiltration, resulting in durable tumor clearance. Taken together, these results demonstrate that the DNA damage elicited by PARPi/PI3Ki can reprogram the myeloid tumor microenvironment (TME) to enhance anti-tumor immunity, resulting in effective tumor eradication in ICB-refractory cancers. Preliminary immune profiling of paired metastatic biopsies from a combination co-clinical trial in castrate-resistant prostate cancer patients, showed decreased immunosuppression and T cell activation within the TME following 4 weeks of treatment with rucaparib/nivolumab, thus supporting the preclinical data described above. Citation Format: Priyanka Duttagupta, Kiranj Chaudagar, Sweta Sharma-Saha, Kaela Bynoe, Eileen Parkes, Akash Patnaik. PARP and PI3K inhibitor combination therapy eradicates c-MYC-driven murine prostate cancers via cGAS/STING pathway activation within tumor-associated macrophages [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B03. doi:10.1158/1535-7163.TARG-19-LB-B03

Abstract 675: Network modeling of drug resistance mechanisms and drug combinations in breast cancer

Abstract Durable control of invasive solid tumors is thwarted by the lack of knowledge of effective drug combinations and of the acquired and intrinsic resistance mechanisms of drugs. In an effort to tackle this problem, the SU2C-NSF-TVF Drug Combination Convergence Team is using mechanistic models of cancer cell signaling based on therapeutic and cell line data in order to identify elements within cancer cells that might eventually be exploited through therapeutic combinations. Here we present a comprehensive mechanistic network model of signal transduction in ER+ PIK3CA-mutant breast cancer. Focusing on PI3K inhibitors, the model recapitulates known resistance mechanisms and predicts other possibilities for resistance: loss of RB1, FOXO3, P27, or PRAS40. To test these predictions, we analyzed genome-wide CRISPR screens of two breast cell lines in the presence of PI3K inhibitors (BYL719 and GDC0032) and found that the predicted genes (RB1, FOXO3, P27, and PRAS40) were significantly enriched in the screens. Some of these resistance genes (e.g. loss of RB1) were found to be cell-line specific and follow-up experiments in RB1-KO cells confirmed the cell-line-specific nature of PI3K-inhibitor resistance. The model also reveals known and novel combinatorial interventions that are more effective than PI3K inhibition alone. For example, the model predicts that the combination of PI3K inhibitors with inhibitors of anti-apoptotic protein MCL1 would be effective. Follow up experiments in cell lines using cell viability assays, cell death analyses, and dynamic BH3 profiling experiments to determine increases in apoptotic priming upon treatment confirmed that MCL1 inhibitors (S63845) enhance the effect of PI3K inhibitors (BYL719) and that this combinatorial effect is cell-line-specific, similarly to what was found in the resistance genes case. In conclusion, the model predicted drug resistance mechanisms and effective drug combinations, some of which were verified experimentally and found to be cell-line-specific. Next iterations of the model will incorporate the identified discrepancies and newly identified resistance mechanisms to drugs of clinical interest. Citation Format: Jorge Gómez Tejeda Zañudo, Pingping Mao, Joan Montero, Guotai Xu, Kailey J. Kowalski, Gabriela N. Johnson, José Baselga, Maurizio Scaltriti, Anthony G. Letai, Nikhil Wagle, Reka Albert. Network modeling of drug resistance mechanisms and drug combinations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 675.

Abstract 1962: Visualizing the efficacy of PI3K inhibitor combination treatment on tumor metabolism

Abstract Purpose: Many types of cancer exhibit an upregulation in glycolysis. Cancer therapies that target tumor metabolism include PI3K inhibitor combinations, which are currently in clinical trials. Real-time metabolic imaging can be possible using hyperpolarization, which is a signal enhancement technique for MRI that increases the signal by a factor of 10,000 or more. The purpose of this work was to determine whether the anti-metabolic effect of such therapies on preclinical breast cancer models could be visualized non-invasively by MRI of hyperpolarized 13C-pyruvate to lactate conversion. Methods: Experiments were carried out on a 9.4 T animal MRI scanner (Biospec 94/20, Bruker, Billerica MA), and all animal studies were done with IACUC approval. Two models of triple negative BRCA1 mutant breast cancer, either resistant (T1R1, n=4) or sensitive (T2S, n=4) to PARP inhibition, were implanted in the inguinal fat pad of mice. Animals with tumor sizes of approximately 10x10x10 mm3 were then imaged prior to and after four consecutive days of combination treatment with a PI3K inhibitor and PARP inhibitor. In order to visualize the interconversion of pyruvate to lactate in vivo, a bolus of hyperpolarized 13C pyruvate (Hypersense, Oxford Instruments, Oxfordshire UK) with concentration=100 mM and volume=200uL was administered to the mouse via tail vein. 13C MR images were acquired using echo-planar spectroscopic imaging (Flip angle=4 degrees; matrix size=16x16; resolution = 2.5mm; slice thickness=5mm; spectral points=512; spectral width=4kHz; repetitions=32) with a 13C radiofrequency transmit/receive surface coil (Bruker, MA) and overlaid on proton anatomical images obtained using a proton volume coil (78 mm diameter, Bruker, MA). Data were reconstructed (Mathematica, Wolfram) to obtain pixel by pixel time integrated pyruvate and lactate spectra for an axial slice. The area under the curve for both metabolites was calculated for all pixels and summed within the tumor to obtain a lactate to pyruvate ratio for each case. Statistical analysis was carried out using a paired t-test. Results: For the animals with T2S tumors, the lactate to pyruvate ratio reduced from 1.65 ± 0.44 to 0.84 ± 0.32 (mean ± std. dev.) after treatment with significance p = 0.0378. For animals with T1R1 tumors, the ratio before and after treatment was 0.91 ± 0.44 and 0.93 ± 0.20 (p = 0.9553). Conclusions: Noninvasive visualization of pyruvate to lactate interconversion can be achieved using 13C MRI, allowing for imaging the efficacy of treatments on cancer metabolism. Citation Format: Manushka Vaidya, Olmo Sonzogni, Cody Callahan, Jocelin Pinto, Gerburg M. Wulf, Aaron Grant. Visualizing the efficacy of PI3K inhibitor combination treatment on tumor metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1962.

JQ1 and PI3K inhibition synergistically reduce salivary adenoid cystic carcinoma malignancy by targeting the c-Myc and EGFR signaling pathways.

To investigate the therapeutic mechanism of the BRD4 inhibitor JQ1 in SACC-83 cells and explore strategies to enhance its therapeutic potential. SACC-83 cells were used in the experiment. Immunohistochemistry was used to assess BRD4 expression in SACC tissues and corresponding adjacent non-tumor tissues. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay. Flow cytometry was used to quantitate apoptosis. Levels of cleaved caspase-3, BRD4, c-Myc, pEGFR (γ-1173), and EGFR were determined by quantitative real-time PCR and Western blot. To study the role of EGFR in JQ1 resistance, we generated EGFR knockdown SACC-83 cells by siRNA transfection. Our study revealed that BRD4 was overexpressed and could be a treatment target in SACC. The BRD4 inhibitor JQ1 markedly inhibited c-Myc expression in SACC-83 cells, which produced modest therapeutic effects. Nevertheless, the EGFR pathway was strongly activated following JQ1 treatment, which led to JQ1 resistance. Combined JQ1 and PI3K inhibitor treatment effectively increased the therapeutic potential by inhibiting the EGFR and c-Myc signaling pathways in SACC-83 cells. Moreover, EGFR knockdown sensitized SACC-83 cells to JQ1. These data demonstrate that EGFR and c-Myc signaling synergistically drive SACC progression. The JQ1 and PI3K inhibitor combination exhibited a strong synergistic effect by suppressing c-Myc and EGFR in SACC-83 cells, identifying a novel rational combinatorial treatment. Moreover, EGFR expression influences the sensitivity of SACC-83 cells to JQ1, which is useful for planning treatment.

Abstract 5467: Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models

Abstract Introduction: RAS mutations are present in 30% of non-small cell lung cancer (NSCLC) and, despite activated MAPK signaling, MEK inhibitors have disappointed in clinical trials. MAPK pathway reactivation and activation of anti-apoptotic signaling via the PI3K/mTOR axis are major resistance mechanisms, among others. ERK inhibitors are believed to overcome some of the restrictions of MEK inhibitors, and combinations with PI3K/mTOR inhibitors could potentially increase therapeutic efficacy. Past trials of MEK and PI3K inhibitor combinations, however, showed that toxicities and side effects are likely to be expected also for the ERK-PI3K/mTOR inhibitor combination. Therefore, more specific and potent drugs as well as alternating dosing schedules may overcome toxicities and improve treatment outcome. Methods: We so far established five patient-derived RAS-mutant NSCLC cell lines (1/5 NRASQ61K, 1/5 KRASQ61K, 2/5 KRASG12C, 1/5 KRASG12D) and xenograft models (PDX), characterized them by next-generation sequencing (DFCI Oncopanel) and tested the antiproliferative (CellTiter-Glo® assay) and apoptosis-inducing (Caspase3/7 activity, IncuCyte® technology) efficacy of LY3214996 (ERK inhibitor) and LY3023414 (PI3K/mTOR inhibitor) in vitro. Effects on signal transduction were investigated by Western blot analysis. Results: The ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 exhibit single-agent antiproliferative and apoptosis-inducing activity in patient-derived RAS-mutant NSCLC cell lines (for LY3214996: 1/5 sensitive (IC50<1μM), 2/5 intermediate sensitive (IC50<5μM), 2/5 resistant (IC50>10μM); for LY3023414: all IC50 <3μM, 3/5 <1μM). Despite reactivation of MAPK signaling (pMEK) upstream of ERK during LY3214996 treatment, ERK downstream targets (cMyc, DUSP4, SPRY2) were strongly suppressed. In sensitive cell lines, LY3214996 induced PARP cleavage and Bim accumulation in a dose- and time-dependent manner. Sensitivities to ERK and PI3K/mTOR inhibition varied between cell lines, indicating different pathway dependencies for proliferation and cancer cell survival. Conclusions and Outlook: Both LY3214996 and LY3023414 have single-agent in vitro efficacy in patient-derived NSCLC models with different RAS mutations and co-mutational landscapes. Single-agent in vivo studies and tolerability studies of drug combinations are ongoing, and treatment predictors and intrinsic/adaptive resistance mechanisms will be investigated via RNAseq/GSEA and phospho-receptor tyrosine kinase arrays. Citation Format: Jens Köhler, Prafulla C. Gokhale, Jiaqi Li, Margaret K. Wilkens, Hong Tiv, Shripad V. Bhagwat, Greg Donoho, Patrick Brueck, Ramon V. Tiu, Amanda J. Redig, Emily S. Chambers, Atsuko Ogino, Jihyun Choi, Man Xu, Paul Kirschmeier, Pasi A. Jänne. Efficacy of the ERK inhibitor LY3214996 and the PI3K/mTOR inhibitor LY3023414 in patient-derived RAS-mutant NSCLC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5467.

Phase 1 Trial Testing Single Agent CUDC-907, a Novel, Oral Dual Inhibitor of Histone Deacetylase (HDAC) and PI3K: Initial Assessment of Patients with Relapsed or Refractory (RR) Diffuse Large B-Cell Lymphoma (DLBCL), Including Double Expressor (DE) Lymphoma

DLBCL, the most common lymphoid malignancy in adults, is an aggressive form of non-Hodgkin lymphoma (NHL) with significant heterogeneity in terms of gene expression, prognosis and response to treatment. Certain histone modifications and HDAC expression patterns have been implicated in the pathobiology of DLBCL and other cancers. Aberrant PTEN/PI3K/AKT signaling is also frequently observed in a variety of human cancers including DLBCL, where loss of PTEN activity and/or activating mutations in PI3K and AKT have been shown to reduce apoptosis and promote cell proliferation. Synergistic anti-tumor effects achieved with HDAC and PI3K inhibitor combinations in various DLBCL xenograft models have provided a strong rationale for testing a highly potent dual HDAC and PI3K inhibitor, CUDC-907. Significant anti-tumor effects have been observed across a wide range of DLBCL models exposed to CUDC-907, including the U2932 ABC DLBCL model that harbors BCL2 translocation and over-expresses cMYC and BCL6, and the WSU-DLCL-2 GCB DLBCL model that harbors EZH2 mutation. These disease mechanisms and preclinical data have led to testing of CUDC-907 in an ongoing Phase 1 trial in patients (pts) with various hematologic cancers.Sixty-three heavily pre-treated pts with lymphoma or multiple myeloma have received CUDC-907 in 21-day cycles according to once daily (QD), intermittent (BIW or TIW), or five days on/two days off (5/2) dosing schedules. CUDC-907 was escalated in 30 mg increments such that pts received 30-60 mg QD, 60-150 mg intermittently, or 60 mg 5/2. A dose expansion using the 60 mg 5/2 dose and schedule is ongoing. Thus far, CUDC-907 has demonstrated a manageable side effect profile and sustained clinical efficacy, particularly in the subset of pts with RR DLBCL.The most common treatment-related adverse events (AEs) were diarrhea (54%; 5% Grade ≥3), fatigue (37%; 3% Grade ≥3), nausea (22%) and thrombocytopenia (18%; 14% Grade ≥3), with dose limiting toxicities of hyperglycemia (QD and BIW schedules) and diarrhea (QD and TIW schedules). Side effects were reversible and manageable. Among 11/18 subjects with RR DLBCL who were evaluable for disease response, 6/11 (55%) achieved objective responses (2 CRs and 4 PRs); lasting a median of 119 days (range: 48 - 354+). Of 7 pts with sufficient tissue, 3 response-evaluable pts were found to over-express MYC and BCL2 by IHC, meeting criteria applied to "double-expressor" (DE) DLBCL. On CUDC-907 monotherapy, 2/3 confirmed pts with DE DLBCL have attained objective responses: 1 ongoing CR (followed by autologous stem cell transplant) and 1PR (lasting 132 days). The third response-evaluable pt with DE DLBCL has experienced lengthy stabilization of disease (171+ days).Signals emerging from preclinical and clinical studies suggest that pts with DLBCL, including those with particularly aggressive disease, may derive benefit from treatment with CUDC-907. Enrollment of pts with RR DLBCL is ongoing into the expansion phase of the Phase 1 trial that is testing CUDC-907 on the 60 mg 5/2 RP2D as monotherapy and in combination with rituximab. Tissue obtained from pts with RR DLBCL treated with CUDC-907 will be assessed for MYC, BCL2, and other genetic aberrations that will be correlated with clinical outcomes observed in the trial. A Phase 2 trial of CUDC-907 in RR NHL is currently being planned, with emphasis on MYC aberrations. DisclosuresYounes:Celgene: Honoraria; Incyte: Honoraria; Janssen: Honoraria; Seattle Genetics: Honoraria, Research Funding; Bayer: Honoraria; Bristol Meyer Squibb: Honoraria; Novartis: Research Funding; Sanofi-Aventis: Honoraria; Johnson and Johnson: Research Funding; Curis: Research Funding; Takeda Millenium: Honoraria. Berdeja:Acetylon: Research Funding; Onyx: Research Funding; Abbvie: Research Funding; Curis: Research Funding; Celgene: Research Funding; Takeda: Research Funding; MEI: Research Funding; Novartis: Research Funding; Janssen: Research Funding; BMS: Research Funding; Array: Research Funding. Flinn:Celgene Corporation: Research Funding. Clancy:Curis: Employment, Equity Ownership. Ma:Curis: Employment, Equity Ownership. Sun:Curis: Employment, Equity Ownership. Tian:Curis: Employment, Equity Ownership. Wang:Curis: Employment, Equity Ownership. Viner:Curis: Employment, Equity Ownership.

SP-050: Promising targets beyond EGFR

HNC represent a heterogeneous disease with regard to primary site, histology, molecular signature carcinogenesis and prognosis. EGFR represent a critical target for treatment approach both in tobacco/alcohol and HPV-induced HNC. A monoclonal antibody against EGFR (Cetuximab) has gained EMA and FDA approval to treat locally advanced HNC in combination with RT and in recurrent/metastatic setting in combination with platinum based CT. However cure rate and survival remain dismal. Therefore further drugs targeting molecular pathways associated with HNC are under evaluation. The specific targeting of new pathways (such as PI3K-AKT-mTOR pathway; JAK-STAT pathway; tumor suppressive miRNAs; Notch1 and 2; epithelial to mesenchymal transformation and immunitary check points) is needed to improve survival outcomes in HNSCC. Moreover second and third generation EGFR inhibitors have the ability to overcome Cetuximab resistance for example targeting the tumor-specific EGFR deletion mutant EGFR vIII. A promising approach is also to combine several target therapy to abrogate the cross-talk between various receptor TK cascades. Phase III are currently on going using combinations of PI3K inhibitors or mTOR inhibitors and chemotherapy in platinum resistant R/M HNC (6-8% of HNCs present PIK3CA mutations). Recently studies on JAK inhibitors showed their antitumor activity in combination with EGFR inhibitors. An emerging field of treatment is to act on immune system. HNC elicits T cell anergy in both peripheral and tumor infiltrating lymphocytes (TILs) In the tumor microenviroment a low level of antimuor cytokines and co-stimulatory mediators has been reported. On the other hand co-inhibitor signals are increased (cytotoxic T lymphocyte associated antigen 4 (CTLA4) and programmed death 1 (PD-1) Successful response depends on prevalence of co-stimulator and/or block of co-inhibitory signaling on immune system A new therapeutic paradigm is to use mAb against coinhibitory receptors CTLA4 and PD1. In particular objective responses with mAbs against PDL1 correlates with PDL! expression on tumour. Moreover as in the melanoma setting the use of antiPD1 AB (BMS936559) there is also growing enthusiasm for therapeutical vaccines against HPV16-18 and p53 respectively in HPV pos and neg disease

Combination of PI3K and PDPK1 Inhibitors Is Highly Effective in Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL), is a distinct lymphoma subtype with an aggressive clinical course and a median survival of 3-5 years. New emerging strategies include especially inhibitors of the PI3K/Akt/mTOR pathway which is constitutively activated in MCL and plays a critical role in tumor growth and survival. In the present study we analysed four different PI3K-Inhibitors (IPI-145, CAL101, A66 and TGX221) targeting different isoforms (PI3Kδ/γ, PI3Kδ, PI3Kα and PI3Kβ) for their effectiveness in MCL.Methods: MCL cell lines (Z-138, Mino-1, Granta-519, Jeko-1, Rec-1, Maver-1) were exposed to different PI3K-Inhibitors (IPI-145, CAL101, A66 and TGX221) with or without murine feeder layer (M210B4). The effect of drugs was evaluated by cell count (trypan-blue staining), cell metabolism (WST-assay or ONE-GloTMLuciferase assay for luciferized MCL cell lines), cell cycle (FACS) and apoptosis (Annexin V PE/7-AAD staining). Subsequently, combinations of PI3K-inhibitors and other target molecules (PDPK1: BX912, OSU03012, GSK2334470) were analysed. Western blot and mRNA analyses were performed after exposure to various inhibitors of the PI3K/Akt/mTOR pathway and correlated to sensitivity of cell lines. Finally, efficiency of drug combinations were confirmed in primary patient samples.Results: The PI3K inhibitor IPI145 (8,2%-62,4%) was most effective in MCL cell lines followed by A66 (0%-41,7%), TGX221 (0%-33,8%) and CAL101 (0%-28,2%) suggesting a higher efficiency by inhibiting multiple isoforms of PI3K. In patient samples IPI145 revealed also the highest cytotoxicity followed by CAL101, TGX221 and A66 (72,4%; 67,9%; 67,1% and 43,7%, respectively). Comparing various combinations of PI3K-inhibitors in MCL cell lines the simultaneous inhibition of α (A66) and δ (CAL101) isoforms achieved the highest reduction of cell count (65,2%). However, combined inhibition of both of PI3K δ and PDPK1 (BX912), led to a more efficient G2 arrest and higher rate of apoptosis due to the simultaneous dephosphorylation of members of the mTOR- and MAPK-pathways. Accordingly, the observed cytotoxicity was even higher (59%-87%) than for dual inhibition of PI3K (23%-60%). Currently, Western blot analyses are being expanded to further identify the differential mechanisms of PI3K inhibitor combinations.In addition, the presence of a murine feeder layer enhanced the effect of PI3K inhibitors in MCL cells nidated into the feederlayer because of higher levels of Akt phosphorylation possibly due to interaction with the microenvironment. Accordingly, this effect was not detectable in the supernatant cells.Conclusion: Based on our comparative analysis of PI3K inhibitors targeting of a and d isoforms appears to be the most efficient mechanism in MCL. However, this impact could be further increased by simultaneous inhibition of PDPK1. Western blot results suggest that these results are due to the inhibition of complementary pathways. Interestingly, the murine feeder layer enhanced the effect of PI3K inhibitors emphasizing the role of the microenvironment in the molecular mechanisms of targeted strategies. DisclosuresDreyling:Gilead: Gilead: speaker’s honoraria Other.

Abstract A33: Enhancement of thymidine phosphorylase downregulation by erlotinib enhances cytotoxicity affected by tamoxifen in human non-small cell lung cancer cells

Abstract Tamoxifen is a triphenylethylene nonsteroidal antiestrogen used worldwide as an adjuvant chemotherapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen also could induce cytotoxicity in non-small cell lung cancer (NSCLC) cells have not been proved. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels in an AKT inactivation-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, a combination of PI3K inhibitors (LY294002 or wortmannin) further decreased the TP expression and cell viability induced by tamoxifen. Moreover, knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical use in the NSCLC therapy. Compared to each single treatment, the combining tamoxifen with erlotinib resulted in a synergistically inhibited the cell viability and cell growth, accompanied with the reduction of phospho-AKT, phosph-ERK1/2, and TP protein levels. An enhancement of AKT or ERK1/2 activation by transfecting the cancer cells with constitutively active AKT or MKK1/2 expression vectors significantly restored the two drugs combination-reduced TP protein levels as well as cell viability. Taken together, our results suggest that the down-modulation of AKT and ERK1/2-mediated TP expression by erlotinib represents a key factor in enhancing the cytotoxic effects of tamoxifen in NSCLC cells. Citation Format: Yun-Wei Lin, Hsien-Chun Chiu, Jhan-Jhang Syu, Yun-Ting Jian, Chien-Yu Chen. Enhancement of thymidine phosphorylase downregulation by erlotinib enhances cytotoxicity affected by tamoxifen in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A33. doi: 10.1158/1557-3125.RASONC14-A33

Abstract 4765: Drug synergies observed for antibody and toxin components of SAR3419 ADC contribute to overall conjugate efficacy and can be combination drug or tumor cell line dependent

Abstract SAR3419, a novel antibody-drug conjugate (ADC), is composed of a humanized monoclonal IgG1 anti-CD19 antibody (huB4) attached by a cleavable linker to DM4, a derivative of cytotoxic agent maytansine (an inhibitor of microtubule polymerization). CD19 expression is restricted to B-cells and treatment of CD19-positive human lymphoma cells with SAR3419 can result in cell cycle arrest followed by apoptosis. As traditional monotherapies can rarely treat cancer effectively, particularly in non-solid tumours, a combination high throughput screen (cHTS) was performed where SAR3419 was combined with two hundred potential enhancer compounds across a panel of twenty cell lines using a 6x6 combination dose response matrix. The enhancer compounds compose a library of approved, emerging oncology drugs, and well-defined molecular probes. Robust synergies were observed between SAR3419 and PI3K inhibitors and examined further. Target/pathway inhibitor redundancy for the class of compounds served to validate PI3K inhibition as important for combination synergy. Surprisingly, for several of the cell lines, the molecular mechanism important for SAR3419-dependent combination activity is distinct. For example, for the Daudi and REC-1 tumor cell lines, PI3K inhibitor synergy can be attributed to the toxin DM4. In contrast for SU-DHL4 and SU-DHL6 cell lines, little combination activity is observed with toxin alone while the conjugate is strongly synergistic. By examining three-way combination activities (PI3K inhibitor x DM4 x huB4) using high resolution matrices, we show that the anti-CD19 antibody huB4 contributes to the synergy in the SU-DHL6 cell line and linkage of DM4 to huB4 (SAR3419) shifts both the potency and maximum activity for PI3K inhibitor combinations. The results demonstrate that the antibody component of an ADC can contribute to combination activity for specific drug pairings in a drug and cell-type specific manner and has wide ranging implications with respect to combination drug design. Citation Format: Richard J. Rickles, Thomas P. Giordano, Shakira F. Cotard, Jill M. Grenier, Angela Romanelli, Ti Cai. Drug synergies observed for antibody and toxin components of SAR3419 ADC contribute to overall conjugate efficacy and can be combination drug or tumor cell line dependent. [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 4765. doi:10.1158/1538-7445.AM2014-4765

The Enhanced In Vivo Activity of the Combination of a MEK and a PI3K Inhibitor Correlates with [18F]-FLT PET in Human Colorectal Cancer Xenograft Tumour-Bearing Mice

Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3′-deoxy-3′-[18F]-fluorothymidine ([18F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [18F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [18F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [18F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.

Activity of the mTOR inhibitor RAD001, the dual mTOR and PI3‐kinase inhibitor BEZ235 and the PI3‐kinase inhibitor BKM120 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide with only few therapeutic options for patients with advanced disease. There is growing evidence indicating that activation of the PI3K/Akt/mTOR pathway plays an important role in HCC and therefore represents a promising target for novel therapeutic approaches. The aim of this study was to evaluate and compare the antitumour activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in vitro and in vivo. The antitumour effects of RAD001, BEZ235 and BKM120 were analysed in seven hepatoma cell lines as mono and combination therapy with Doxorubicin, Cisplatin, Irinotecan or 5-Flourouracil in vitro and in xenografts. Cell-cycle progression, apoptosis, and autophagy were analysed. Furthermore, effects on mitochondrial respiration and glycolysis were assessed. Treatment with RAD001, BEZ235 and BKM120 markedly reduced tumour cell viability. Combination of PI3K inhibitors with chemotherapy was most effective. RAD001, BEZ235 and BKM120 reduced tumour growth mainly by inhibiting cell-cycle progression rather than by inducing apoptosis. Interestingly, the antitumour effects were strongly associated with a reduction of mitochondrial respiration. BKM120, which exhibited the strongest antiproliferative effect, most strongly impaired oxidative phosphorylation compared with the other drugs. In this study, BKM120 showed the strongest antitumour activity. Our findings suggest impairment of mitochondrial function as a relevant mechanism of BKM120. Moreover, combination of PI3K and mTOR inhibitors with cytotoxic agents could be promising option for non-cirrhotic HCC patients.

PI3K inhibition potentiates Bcl‐2‐dependent apoptosis in renal carcinoma cells

Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3-K/Akt inhibitors is therefore of great interest. As PI3-K inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl-2 family inhibitor ABT-737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl-1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl-1 and Bim levels. Our results suggest that the combination of PI3-K inhibitors with BH3-mimetics may be a viable therapeutic strategy in RCC.

Abstract A44: 18F-FLT, a potential biomarker for predicting the synergistic combination of PI3K inhibitor BAY 80-6946* and MEK inhibitor BAY 86-9766.

Abstract Signaling pathways of particular interest in the cancer field are the RAS-RAF-MEK and PI3K-AKT pathways that act as parallel conduits to transduce signals involved in tumor cell proliferation, survival, and cell migration. Compared to targeting either pathway separately, simultaneous inhibition of these two key pathways has been considered to have greater therapeutic efficacy and broader applicability for the treatment of cancers. One of the challenges is the discovery of biomarkers that correlate with efficacy and synergy of PI3K and MEK combination therapy in early-stage clinical trials. To identify the best imaging tracer for monitoring the synergistic anti-tumor activity of BAY 80-6946* and BAY 86-9766, we first compared FDG and FLT uptake in vitro in 2 colorectal (CRC) and 2 non-small cell lung cancer (NSCLC) cell lines bearing either KRAS or KRAS plus PIK3CA mutations. Partial inhibition of FDG- and FLT-uptake (<50%) was observed in tumor cells treated with each agent, while synergistic and complete inhibition of FLT-uptake (>94%) was demonstrated in the combination group. Interestingly, the maximum inhibition of FDG-uptake was much weaker compared to FLT-uptake, indicating that FLT might be a better tracer for PI3K and MEK combination therapy. Further investigation iwas conducted in HCT116 CRC xenografts in mice and H460 NSCLC xenografts in rats. BAY 80-6946* was administered intraveneously (Q2D) at 14 mg/kg in mice and at 3 mg/kg in rats whereas BAY 86-9766 was dosed orally (QD) at 25 mg/kg in mice and at 15 mg/kg in rats. Tumor uptake of 18F-FLT was investigated by PET imaging as well as by quantitative biodistribution 1 day before and 1 day after treatment start. In both models, 18F-FLT tumor uptake was not significantly altered in animals treated with each single agent. However, one day after the treatment in the combination group, 18F-FLT tumor uptake was reduced from 8.50±0.99 %ID/g to 3.91±0.66 %ID/g (n=5, vs vehicle, p<0.05) in HCT116 bearing mice and from 0.91±0.27 %ID/g to 0.31±0.04 %ID/g (n=5, vs vehicle, p<0.05) in H460 bearing rats. Inhibition of 18F-FLT uptake in tumors ocurred before any significant reduction in tumor volume and correlates with tumor growth inhibition assessed by the tumor size at the later time points. Further detailed molecular mechanism on the synergistic inhibition of 18F-FLT uptake by combination of PI3K and MEK inhibitors will be presented. In conclusion, 18F-FLT might be a non-invasive PET imaging tracer that, when used serially ealy in treatment, may predict clinical efficacy and combination synergy of PI3K and MEK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A44.